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Sample records for functional domains required

  1. Fibroblast migration on fibronectin requires three distinct functional domains.

    PubMed

    Clark, Richard A F; An, Jian-Qiang; Greiling, Doris; Khan, Azim; Schwarzbauer, Jean E

    2003-10-01

    Mesenchymal cell movement is normally constrained; however, fibronectin can provide a pathway for stromal cell migration during embryogenesis, morphogenesis, and wound healing. Cells can adhere to fibronectin via integrin and nonintegrin receptors, which bind multiple unique peptide sequences. Synthetic peptides and recombinant proteins were used to delineate the functional domains needed for human fibroblast migration over fibronectin. The 9th and 10th fibronectin type III repeats, which contain RGD and PHSRN synergy cell attachment sequences, support almost maximal fibroblast attachment, but not migration of primary dermal fibroblasts. Specific sequences within the heparin domain and the IIICS region are also required for migration. These findings predict and additional data confirm the necessity for the cooperation of multiple integrin and nonintegrin receptors for fibroblast migration on fibronectin. Such stringency of migration most likely imposes an immense constraint on normal mesenchymal cell mobility in unperturbed tissue. Loss of such restraint may be critical for the migration cancer cells through the extracellular matrix. PMID:14632184

  2. Uncertainty Analysis via Failure Domain Characterization: Polynomial Requirement Functions

    NASA Technical Reports Server (NTRS)

    Crespo, Luis G.; Munoz, Cesar A.; Narkawicz, Anthony J.; Kenny, Sean P.; Giesy, Daniel P.

    2011-01-01

    This paper proposes an uncertainty analysis framework based on the characterization of the uncertain parameter space. This characterization enables the identification of worst-case uncertainty combinations and the approximation of the failure and safe domains with a high level of accuracy. Because these approximations are comprised of subsets of readily computable probability, they enable the calculation of arbitrarily tight upper and lower bounds to the failure probability. A Bernstein expansion approach is used to size hyper-rectangular subsets while a sum of squares programming approach is used to size quasi-ellipsoidal subsets. These methods are applicable to requirement functions whose functional dependency on the uncertainty is a known polynomial. Some of the most prominent features of the methodology are the substantial desensitization of the calculations from the uncertainty model assumed (i.e., the probability distribution describing the uncertainty) as well as the accommodation for changes in such a model with a practically insignificant amount of computational effort.

  3. Uncertainty Analysis via Failure Domain Characterization: Unrestricted Requirement Functions

    NASA Technical Reports Server (NTRS)

    Crespo, Luis G.; Kenny, Sean P.; Giesy, Daniel P.

    2011-01-01

    This paper proposes an uncertainty analysis framework based on the characterization of the uncertain parameter space. This characterization enables the identification of worst-case uncertainty combinations and the approximation of the failure and safe domains with a high level of accuracy. Because these approximations are comprised of subsets of readily computable probability, they enable the calculation of arbitrarily tight upper and lower bounds to the failure probability. The methods developed herein, which are based on nonlinear constrained optimization, are applicable to requirement functions whose functional dependency on the uncertainty is arbitrary and whose explicit form may even be unknown. Some of the most prominent features of the methodology are the substantial desensitization of the calculations from the assumed uncertainty model (i.e., the probability distribution describing the uncertainty) as well as the accommodation for changes in such a model with a practically insignificant amount of computational effort.

  4. Centromere localization and function of Mis18 requires Yippee-like domain-mediated oligomerization.

    PubMed

    Subramanian, Lakxmi; Medina-Pritchard, Bethan; Barton, Rachael; Spiller, Frances; Kulasegaran-Shylini, Raghavendran; Radaviciute, Guoda; Allshire, Robin C; Arockia Jeyaprakash, A

    2016-04-01

    Mis18 is a key regulator responsible for the centromere localization of the CENP-A chaperone Scm3 in Schizosaccharomyces pombe and HJURP in humans, which establishes CENP-A chromatin that defines centromeres. The molecular and structural determinants of Mis18 centromere targeting remain elusive. Here, by combining structural, biochemical, and yeast genetic studies, we show that the oligomerization of S. pombe Mis18, mediated via its conserved N-terminal Yippee-like domain, is crucial for its centromere localization and function. The crystal structure of the N-terminal Yippee-like domain reveals a fold containing a cradle-shaped pocket that is implicated in protein/nucleic acid binding, which we show is required for Mis18 function. While the N-terminal Yippee-like domain forms a homodimer in vitro and in vivo, full-length Mis18, including the C-terminal α-helical domain, forms a homotetramer in vitro We also show that the Yippee-like domains of human Mis18α/Mis18β interact to form a heterodimer, implying a conserved structural theme for Mis18 regulation. PMID:26921242

  5. Identification of domains required for developmentally regulated SNARE function in Saccharomyces cerevisiae.

    PubMed Central

    Neiman, A M; Katz, L; Brennwald, P J

    2000-01-01

    Saccharomyces cerevisiae cells contain two homologues of the mammalian t-SNARE protein SNAP-25, encoded by the SEC9 and SPO20 genes. Although both gene products participate in post-Golgi vesicle fusion events, they cannot substitute for one another; Sec9p is active primarily in vegetative cells while Spo20p functions only during sporulation. We have investigated the basis for the developmental stage-specific differences in the function of these two proteins. Localization of the other plasma membrane SNARE subunits, Ssop and Sncp, in sporulating cells suggests that these proteins act in conjunction with Spo20p in the formation of the prospore membrane. In vitro binding studies demonstrate that, like Sec9p, Spo20p binds specifically to the t-SNARE Sso1p and, once bound to Sso1p, can complex with the v-SNARE Snc2p. Therefore, Sec9p and Spo20p interact with the same binding partners, but developmental conditions appear to favor the assembly of complexes with Spo20p in sporulating cells. Analysis of chimeric Sec9p/Spo20p molecules indicates that regions in both the SNAP-25 domain and the unique N terminus of Spo20p are required for activity during sporulation. Additionally, the N terminus of Spo20p is inhibitory in vegetative cells. Deletion studies indicate that activation and inhibition are separable functions of the Spo20p N terminus. Our results reveal an additional layer of regulation of the SNARE complex, which is necessary only in sporulating cells. PMID:10924463

  6. BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition.

    PubMed

    Gabellini, Chiara; De Luca, Teresa; Trisciuoglio, Daniela; Desideri, Marianna; Di Martile, Marta; Passeri, Daniela; Candiloro, Antonio; Biffoni, Mauro; Rizzo, Maria Giulia; Orlandi, Augusto; Del Bufalo, Donatella

    2013-11-01

    Beyond its classical role as apoptosis inhibitor, bcl-2 protein promotes tumor angiogenesis and the removal of N-terminal bcl-2 homology (BH4) domain abrogates bcl-2-induced hypoxia-inducible factor 1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in hypoxic cancer cells. Using M14 human melanoma cell line and its derivative clones stably overexpressing bcl-2 wild-type or deleted of its BH4 domain, we found that conditioned media (CM) from cells expressing BH4-deleted bcl-2 protein showed a reduced capability to increase in vitro human endothelial cells proliferation and differentiation, and in vivo neovascularization compared with CM from cells overexpressing wild-type bcl-2. Moreover, xenografts derived from cells expressing bcl-2 lacking BH4 domain showed a reduction of metastatic potential compared with tumors derived from wild-type bcl-2 transfectants injection. Stably expressing the Flag-tagged N-terminal sequence of bcl-2 protein, encompassing BH4 domain, we found that this domain is sufficient to enhance the proangiogenic HIF-1/VEGF axis under hypoxic condition. Indeed, lacking of BH4 domain abolishes the interaction between bcl-2 and HIF-1α proteins and the capability of exogenous bcl-2 protein to localize in the nucleus. Moreover, when endoplasmic reticulum-targeted bcl-2 protein is overexpressed in cells, this protein lost the capability to synergize with hypoxia to induce the proangiogenic HIF-1/VEGF axis as shown by wild-type bcl-2 protein. These results demonstrate that BH4 domain of bcl-2 is required for the ability of this protein to increase tumor angiogenesis and progression and indicate that bcl-2 nuclear localization may be required for bcl-2-mediated induction of HIF-1/VEGF axis. PMID:23836782

  7. Function of the MAPK scaffold protein, Ste5, requires a cryptic PH domain

    PubMed Central

    Garrenton, Lindsay S.; Young, Susan L.; Thorner, Jeremy

    2006-01-01

    Ste5, the prototypic mitogen-activated protein kinase (MAPK) scaffold protein, associates with plasma membrane-tethered Gβγ freed upon pheromone receptor occupancy, thereby initiating downstream signaling. We demonstrate that this interaction and membrane binding of an N-terminal amphipathic α-helix (PM motif) are not sufficient for Ste5 action. Rather, Ste5 contains a pleckstrin-homology (PH) domain (residues 388–518) that is essential for its membrane recruitment and function. Altering residues (R407S K411S) equivalent to those that mediate phosphoinositide binding in other PH domains abolishes Ste5 function. The isolated PH domain, but not a R407S K411S derivative, binds phosphoinositides in vitro. Ste5(R407S K411S) is expressed normally, retains Gβγ and Ste11 binding, and oligomerizes, yet is not recruited to the membrane in response to pheromone. Artificial membrane tethering of Ste5(R407S K411S) restores signaling. R407S K411S loss-of-function mutations abrogate the constitutive activity of gain-of-function Ste5 alleles, including one (P44L) that increases membrane affinity of the PM motif. Thus, the PH domain is essential for stable membrane recruitment of Ste5, and this association is critical for initiation of downstream signaling because it allows Ste5-bound Ste11 (MAPKKK) to be activated by membrane-bound Ste20 (MAPKKKK). PMID:16847350

  8. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  9. Membrane association of the CD3ε signaling domain is required for optimal T cell development and function1

    PubMed Central

    Bettini, Matthew L.; Guy, Clifford; Dash, Pradyot; Vignali, Kate M.; Hamm, David E.; Dobbins, Jessica; Gagnon, Etienne; Thomas, Paul G.; Wucherpfennig, Kai W.; Vignali, Dario A.A.

    2014-01-01

    The T cell receptor (TCR):CD3 complex transduces signals that are critical for optimal T cell development and adaptive immunity. In resting T cells, the CD3ε cytoplasmic tail associates with the plasma membrane via a proximal basic-rich stretch (BRS). Here we show that mice lacking a functional CD3ε-BRS exhibited substantial reductions in thymic cellularity and limited CD4−CD8− double negative-3 (DN3) to DN4 thymocyte transition, due to enhanced DN4 TCR signaling resulting in increased cell death and TCR downregulation in all subsequent populations. Furthermore, positive, but not negative, T cell selection was affected in mice lacking a functional CD3ε-BRS, which led to limited peripheral T cell function and substantially reduced responsiveness to influenza infection. Collectively, these results indicate membrane association of the CD3ε signaling domain is required for optimal thymocyte development and peripheral T cell function. PMID:24899501

  10. Ribonucleotide reduction - horizontal transfer of a required function spans all three domains

    PubMed Central

    2010-01-01

    Background Ribonucleotide reduction is the only de novo pathway for synthesis of deoxyribonucleotides, the building blocks of DNA. The reaction is catalysed by ribonucleotide reductases (RNRs), an ancient enzyme family comprised of three classes. Each class has distinct operational constraints, and are broadly distributed across organisms from all three domains, though few class I RNRs have been identified in archaeal genomes, and classes II and III likewise appear rare across eukaryotes. In this study, we examine whether this distribution is best explained by presence of all three classes in the Last Universal Common Ancestor (LUCA), or by horizontal gene transfer (HGT) of RNR genes. We also examine to what extent environmental factors may have impacted the distribution of RNR classes. Results Our phylogenies show that the Last Eukaryotic Common Ancestor (LECA) possessed a class I RNR, but that the eukaryotic class I enzymes are not directly descended from class I RNRs in Archaea. Instead, our results indicate that archaeal class I RNR genes have been independently transferred from bacteria on two occasions. While LECA possessed a class I RNR, our trees indicate that this is ultimately bacterial in origin. We also find convincing evidence that eukaryotic class I RNR has been transferred to the Bacteroidetes, providing a stunning example of HGT from eukaryotes back to Bacteria. Based on our phylogenies and available genetic and genomic evidence, class II and III RNRs in eukaryotes also appear to have been transferred from Bacteria, with subsequent within-domain transfer between distantly-related eukaryotes. Under the three-domains hypothesis the RNR present in the last common ancestor of Archaea and eukaryotes appears, through a process of elimination, to have been a dimeric class II RNR, though limited sampling of eukaryotes precludes a firm conclusion as the data may be equally well accounted for by HGT. Conclusions Horizontal gene transfer has clearly played an

  11. Structural and functional domains of the myb oncogene: requirements for nuclear transport, myeloid transformation, and colony formation.

    PubMed Central

    Ibanez, C E; Lipsick, J S

    1988-01-01

    The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in vivo and transforms only myeloid cells in vitro. Its product, p48v-myb, is a nuclear protein of unknown function. To determine structure-function relationships for this protein, we constructed a series of deletion mutants of v-myb, expressed them in retroviral vectors, and studied their biochemical and biological properties. We used these mutants to identify two separate domains of p48v-myb which had distinct roles in its accumulation in the cell nucleus. We showed that the viral sequences which normally encode both termini of p48v-myb were dispensible for transformation. In contrast, both copies of the highly conserved v-myb amino-terminal repeat were required for transformation. We also identified a carboxyl-terminal domain of p48v-myb which was required for the growth of v-myb-transformed myeloblasts in soft agar but not for morphological transformation. Images PMID:2835503

  12. The C-Terminal Domain of Yeast PCNA Is Required for Physical And Functional Interactions With Cdc9 DNA Ligase

    SciTech Connect

    Vijayakumar, S.; Chapados, B.R.; Schmidt, K.H.; Kolodner, R.D.; Tainer, J.A.; Tomkinson, A.E.

    2007-07-13

    There is compelling evidence that proliferating cell nuclear antigen (PCNA), a DNA sliding clamp, co-ordinates the processing and joining of Okazaki fragments during eukaryotic DNA replication. However, a detailed mechanistic understanding of functional PCNA:ligase I interactions has been incomplete. Here we present the co-crystal structure of yeast PCNA with a peptide encompassing the conserved PCNA interaction motif of Cdc9, yeast DNA ligase I. The Cdc9 peptide contacts both the inter-domain connector loop (IDCL) and residues near the C-terminus of PCNA. Complementary mutational and biochemical results demonstrate that these two interaction interfaces are required for complex formation both in the absence of DNA and when PCNA is topologically linked to DNA. Similar to the functionally homologous human proteins, yeast RFC interacts with and inhibits Cdc9 DNA ligase whereas the addition of PCNA alleviates inhibition by RFC. Here we show that the ability of PCNA to overcome RFC-mediated inhibition of Cdc9 is dependent upon both the IDCL and the C-terminal interaction interfaces of PCNA. Together these results demonstrate the functional significance of the {beta}-zipper structure formed between the C-terminal domain of PCNA and Cdc9 and reveal differences in the interactions of FEN-1 and Cdc9 with the two PCNA interfaces that may contribute to the coordinated, sequential action of these enzymes.

  13. Requirements analysis, domain knowledge, and design

    NASA Technical Reports Server (NTRS)

    Potts, Colin

    1988-01-01

    Two improvements to current requirements analysis practices are suggested: domain modeling, and the systematic application of analysis heuristics. Domain modeling is the representation of relevant application knowledge prior to requirements specification. Artificial intelligence techniques may eventually be applicable for domain modeling. In the short term, however, restricted domain modeling techniques, such as that in JSD, will still be of practical benefit. Analysis heuristics are standard patterns of reasoning about the requirements. They usually generate questions of clarification or issues relating to completeness. Analysis heuristics can be represented and therefore systematically applied in an issue-based framework. This is illustrated by an issue-based analysis of JSD's domain modeling and functional specification heuristics. They are discussed in the context of the preliminary design of simple embedded systems.

  14. Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

    SciTech Connect

    Kim, Seong K. Kim, Seongman; Dai Gan; Zhang Yunfei; Ahn, Byung C.; O'Callaghan, Dennis J.

    2011-09-01

    The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain. - Highlights: > We examine the functional domains of IR2P that mediates negative regulation. > IR2P inhibits at the transcriptional level. > DNA-binding mutant or TFIIB-binding mutant fails to inhibit. > C-terminal aa 707 to 1116 are required for full inhibition. > Inhibition requires the DNA-binding domain, TFIIB-binding domain, and C-terminus.

  15. Zinc finger domain of Su(Hw) protein is required for the formation of functional Su(Hw)-dependent insulator complex.

    PubMed

    Golovnin, A K; Molodina, V V; Georgiev, P G; Melnikova, L S

    2016-07-01

    This study is devoted to clarifying the role of Mod(mdg4)-67.2 and Su(Hw) proteins in the interaction between Su(Hw)-dependent insulator complexes and identifying the specific domains of the Su(Hw) protein required for insulation or mutual neutralization of insulators. Using genetic techniques and experiments in yeast two-hybrid system, we have demonstrated that the zinc finger domain of the Su(Hw) protein is involved in forming a functional insulator complex and cannot be replaced with the DNA-binding domain of the GAL4 protein. PMID:27599504

  16. Phenotypic Complementation Establishes Requirements for Specific POU Domain and Generic Transactivation Function of Oct-3/4 in Embryonic Stem Cells

    PubMed Central

    Niwa, Hitoshi; Masui, Shinji; Chambers, Ian; Smith, Austin G.; Miyazaki, Jun-ichi

    2002-01-01

    Transcription factors of the POU family govern cell fate through combinatorial interactions with coactivators and corepressors. The POU factor Oct-3/4 can define differentiation, dedifferentation, or self-renewal of pluripotent embryonic stem (ES) cells in a sensitive, dose-dependent manner (H. Niwa, J.-I. Miyazali, and A. G. Smith, Nat. Genet. 24:372-376, 2000). Here we have developed a complementation assay based on the ability of Oct-3/4 transgenes to rescue self-renewal in conditionally null ES cells and used this to define which domains of Oct-3/4 are required to sustain the undifferentiated stem cell phenotype. Surprisingly, we found that molecules lacking either the N-terminal or C-terminal transactivation domain, though not both, can effectively replace full-length Oct-3/4. Furthermore, a fusion of the heterologous transactivation domain of Oct-2 to the Oct-3/4 POU domain can also sustain self-renewal. Thus, the unique function of Oct-3/4 in ES cell propagation resides in combination of the specific POU domain with a generic proline-rich transactivation domain. Interestingly, however, Oct-3/4 target gene expression elicited by the N- and C-terminal transactivation domains is not identical, indicating that at least one class of genes activated by Oct-3/4 is not required for ES cell propagation. PMID:11839818

  17. The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist

    SciTech Connect

    Madauss, Kevin P.; Burkhart, William A.; Consler, Thomas G.; Cowan, David J.; Gottschalk, William K.; Miller, Aaron B; Short, Steven A.; Tran, Thuy B.; Williams, Shawn P.

    2009-06-15

    Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain-CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined -helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.

  18. The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist

    SciTech Connect

    Madauss, Kevin P.; Burkhart, William A.; Consler, Thomas G.; Cowan, David J.; Gottschalk, William K.; Miller, Aaron B.; Short, Steven A.; Tran, Thuy B.; Williams, Shawn P.

    2009-05-01

    The use of biophysical assays permitted the identification of a specific human ACC2 carboxyl transferase (CT) domain mutant that binds inhibitors and crystallizes in their presence. This mutant led to determination of the human ACC2 CT domain–CP-640186 complex crystal structure, which revealed differences in the inhibitor conformation from the yeast protein complex that are caused by differing residues in the binding pocket. Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain–CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined α-helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.

  19. A Conserved Functional Domain of Drosophila Coracle Is Required for Localization at the Septate Junction and Has Membrane-organizing Activity

    PubMed Central

    Ward IV, Robert E.; Lamb, Rebecca S.; Fehon, Richard G.

    1998-01-01

    The protein 4.1 superfamily is comprised of a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins. Coracle, a Drosophila protein 4.1 homologue, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells. Using in vitro mutagenesis, we demonstrate that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos. Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. Furthermore, the localization of Coracle and the transmembrane protein Neurexin to the septate junction display an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members. PMID:9508778

  20. STAS Domain Structure and Function

    PubMed Central

    Sharma, Alok K.; Rigby, Alan C.; Alper, Seth L.

    2011-01-01

    Pendrin shares with nearly all SLC26/SulP anion transporters a carboxy-terminal cytoplasmic segment organized around a Sulfate Transporter and Anti-Sigma factor antagonist (STAS) domain. STAS domains of divergent amino acid sequence exhibit a conserved fold of 4 β strands interspersed among 5 α helices. The first STAS domain proteins studied were single-domain anti-sigma factor antagonists (anti-anti-σ). These anti-anti-σ indirectly stimulate bacterial RNA polymerase by inactivating inhibitory anti-σ kinases, liberating σ factors to direct specific transcription of target genes or operons. Some STAS domains are nucleotide-binding phosphoproteins or nucleotidases. Others are interaction/transduction modules within multidomain sensors of light, oxygen and other gasotransmitters, cyclic nucleotides, inositol phosphates, and G proteins. Additional multidomain STAS protein sequences suggest functions in sensing, metabolism, or transport of nutrients such as sugars, amino acids, lipids, anions, vitamins, or hydrocarbons. Still other multidomain STAS polypeptides include histidine and serine/threonine kinase domains and ligand-activated transcription factor domains. SulP/SLC26 STAS domains and adjacent sequences interact with other transporters, cytoskeletal scaffolds, and with enzymes metabolizing transported anion substrates, forming putative metabolons. STAS domains are central to membrane targeting of many SulP/SLC26 anion transporters, and STAS domain mutations are associated with at least three human recessive diseases. This review summarizes STAS domain structure and function. PMID:22116355

  1. An auxiliary peptide required for the function of two activation domains in upstream stimulatory factor 2 (USF2) transcription factor.

    PubMed

    Gourdon, L; Lefrançois-Martinez, A M; Viollet, B; Martinez, A; Kahn, A; Raymondjean, M

    1997-04-01

    Ubiquitous upstream stimulatory factors (USF1, USF2a and USF2b) are members of the basic-helix-loop-helix-leucine-zipper family of transcription factors that have been shown to be involved in the transcriptional response of the L-type pyruvate kinase (L-PK) gene to glucose. To understand the mechanisms of action of the USF2 isoforms, we initiated a series of co-transfection assays with deletion mutants and Ga14-USF2 fusions. The transactivating efficiency of the different native and mutant factors was determined at similar DNA binding activity. We found that: (i) exons 3- and 5-encoded regions are activation domains, (ii) a modulator domain encoded by exon 4 could be necessary to their additive action, (iii) a hexapeptide encoded by the first 5' codons of exon 6 is indispensable for transmitting activation due to both exon 3- and exon 5-encoded domains to the transcriptional machinery. Therefore, USF2 presents a modular structure and mediates transcriptional activation thanks to two non-autonomous activation domains dependent on an auxiliary peptide for expressing their activating potential. PMID:9680311

  2. Characterization of the osmoprotectant transporter OpuC from Pseudomonas syringae and demonstration that cystathionine-beta-synthase domains are required for its osmoregulatory function.

    PubMed

    Chen, Chiliang; Beattie, Gwyn A

    2007-10-01

    The plant pathogen Pseudomonas syringae may cope with osmotic stress on plants, in part, by importing osmoprotective compounds. In this study, we found that P. syringae pv. tomato strain DC3000 was distinct from most bacterial species in deriving greater osmoprotection from exogenous choline than from glycine betaine. This superior osmoprotection was correlated with a higher capacity for uptake of choline than for uptake of glycine betaine. Of four putative osmoregulatory ABC transporters in DC3000, one, designated OpuC, functioned as the primary or sole transporter for glycine betaine and as one of multiple transporters for choline under high osmolarity. Surprisingly, the homolog of the well-characterized ProU transporter from Escherichia coli and Salmonella enterica serovar Typhimurium did not function in osmoprotection. The P. syringae pv. tomato OpuC transporter was more closely related to the Bacillus subtilis and Listeria monocytogenes OpuC transporters than to known osmoprotectant transporters in gram-negative bacteria based on sequence similarity and genetic arrangement. The P. syringae pv. tomato OpuC transporter had a high affinity for glycine betaine, a low affinity for choline, and a broad substrate specificity that included acetylcholine, carnitine, and proline betaine. Tandem cystathionine-beta-synthase (CBS) domains in the ATP-binding component of OpuC were required for transporter function. The presence of these CBS domains was correlated with osmoregulatory function among the putative transporters examined in DC3000 and was found to be predictive of functional osmoregulatory transporters in other pseudomonads. These results provide the first functional evaluation of an osmoprotectant transporter in a Pseudomonas species and demonstrate the usefulness of the CBS domains as predictors of osmoregulatory activity. PMID:17660277

  3. Functional domain walls in multiferroics

    NASA Astrophysics Data System (ADS)

    Meier, Dennis

    2015-11-01

    During the last decade a wide variety of novel and fascinating correlation phenomena has been discovered at domain walls in multiferroic bulk systems, ranging from unusual electronic conductance to inseparably entangled spin and charge degrees of freedom. The domain walls represent quasi-2D functional objects that can be induced, positioned, and erased on demand, bearing considerable technological potential for future nanoelectronics. Most of the challenges that remain to be solved before turning related device paradigms into reality, however, still fall in the field of fundamental condensed matter physics and materials science. In this topical review seminal experimental findings gained on electric and magnetic domain walls in multiferroic bulk materials are addressed. A special focus is put on the physical properties that emerge at so-called charged domain walls and the added functionality that arises from coexisting magnetic order. The research presented in this review highlights that we are just entering a whole new world of intriguing nanoscale physics that is yet to be explored in all its details. The goal is to draw attention to the persistent challenges and identify future key directions for the research on functional domain walls in multiferroics.

  4. Functional domain walls in multiferroics.

    PubMed

    Meier, Dennis

    2015-11-25

    During the last decade a wide variety of novel and fascinating correlation phenomena has been discovered at domain walls in multiferroic bulk systems, ranging from unusual electronic conductance to inseparably entangled spin and charge degrees of freedom. The domain walls represent quasi-2D functional objects that can be induced, positioned, and erased on demand, bearing considerable technological potential for future nanoelectronics. Most of the challenges that remain to be solved before turning related device paradigms into reality, however, still fall in the field of fundamental condensed matter physics and materials science. In this topical review seminal experimental findings gained on electric and magnetic domain walls in multiferroic bulk materials are addressed. A special focus is put on the physical properties that emerge at so-called charged domain walls and the added functionality that arises from coexisting magnetic order. The research presented in this review highlights that we are just entering a whole new world of intriguing nanoscale physics that is yet to be explored in all its details. The goal is to draw attention to the persistent challenges and identify future key directions for the research on functional domain walls in multiferroics. PMID:26523728

  5. The coiled-coil domain containing protein CCDC151 is required for the function of IFT-dependent motile cilia in animals.

    PubMed

    Jerber, Julie; Baas, Dominique; Soulavie, Fabien; Chhin, Brigitte; Cortier, Elisabeth; Vesque, Christine; Thomas, Joëlle; Durand, Bénédicte

    2014-02-01

    Cilia are evolutionarily conserved organelles endowed with essential physiological and developmental functions. In humans, disruption of cilia motility or signaling leads to complex pleiotropic genetic disorders called ciliopathies. Cilia motility requires the assembly of multi-subunit motile components such as dynein arms, but mechanisms underlying their assembly pathway and transport into the axoneme are still largely unknown. We identified a previously uncharacterized coiled-coil domain containing protein CCDC151, which is evolutionarily conserved in motile ciliated species and shares ancient features with the outer dynein arm-docking complex 2 of Chlamydomonas. In Drosophila, we show that CG14127/CCDC151 is associated with motile intraflagellar transport (IFT)-dependent cilia and required for geotaxis behavior of adult flies. In zebrafish, Ccdc151 is expressed in tissues with motile cilia, and morpholino-induced depletion of Ccdc151 leads to left-right asymmetry defects and kidney cysts. We demonstrate that Ccdc151 is required for proper motile function of cilia in the Kupffer's vesicle and in the pronephros by controlling dynein arm assembly, showing that Ccdc151 is a novel player in the control of IFT-dependent dynein arm assembly in animals. However, we observed that CCDC151 is also implicated in other cellular functions in vertebrates. In zebrafish, ccdc151 is involved in proper orientation of cell divisions in the pronephros and genetically interacts with prickle1 in this process. Furthermore, knockdown experiments in mammalian cells demonstrate that CCDC151 is implicated in the regulation of primary cilium length. Hence, CCDC151 is required for motile cilia function in animals but has acquired additional non-motile functions in vertebrates. PMID:24067530

  6. The N-Terminal Extension Domain of the C. elegans Half-Molecule ABC Transporter, HMT-1, Is Required for Protein-Protein Interactions and Function

    PubMed Central

    Kim, Sungjin; Selote, Devarshi S.; Vatamaniuk, Olena K.

    2010-01-01

    Background Members of the HMT-1 (heavy metal tolerance factor 1) subfamily of the ATP-binding cassette (ABC) transporter superfamily detoxify heavy metals and have unique topology: they are half-molecule ABC transporters that, in addition to a single transmembrane domain (TMD1) and a single nucleotide-binding domain (NBD1), possess a hydrophobic NH2-terminal extension (NTE). These structural features distinguish HMTs from other ABC transporters in different species including Drosophila and humans. Functional ABC transporters, however, are comprised of at least four-domains (two TMDs and two NDBs) formed from either a single polypeptide or by the association of two or four separate subunits. Whether HMTs act as oligomers and what role the NTE domain plays in their function have not been determined. Methodology/Principal Findings In this study, we examined the oligomeric status of Caenorhabditis elegans HMT-1 and the functional significance of its NTE using gel-filtration chromatography in combination with the mating-based split-ubiquitin yeast two-hybrid system (mbSUS) and functional in vivo assays. We found that HMT-1 exists in a protein complex in C. elegans. Studies in S. cerevisiae showed that HMT-1 at a minimum homodimerizes and that oligomerization is essential for HMT-1 to confer cadmium tolerance. We also established that the NTE domain plays an important structural and functional role: it is essential for HMT-1 oligomerization and Cd-detoxification function. However, the NTE itself was not sufficient for oligomerization suggesting that multiple structural features of HMT-1 must associate to form a functional transporter. Conclusions The prominence of heavy metals as environmental toxins and the remarkable conservation of HMT-1 structural architecture and function in different species reinforce the value of continued studies of HMT-1 in model systems for identifying functional domains in HMT1 of humans. PMID:20886084

  7. Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I: OUTER MEMBRANE INSERTION, TRIMERIZATION, AND PORIN FUNCTION REQUIRE A C-TERMINAL β-BARREL DOMAIN.

    PubMed

    Anand, Arvind; LeDoyt, Morgan; Karanian, Carson; Luthra, Amit; Koszelak-Rosenblum, Mary; Malkowski, Michael G; Puthenveetil, Robbins; Vinogradova, Olga; Radolf, Justin D

    2015-05-01

    We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprC(N) and TprC(C)) orthologous to regions in the major outer sheath protein (MOSP(N) and MOSP(C)) of Treponema denticola and that TprC(C) is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSP(C)-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSP(N)-like domains are tethered within the periplasm. TprF, which does not contain a MOSP(C)-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSP(N) and MOSP(C)-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSP(N)-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP. PMID:25805501

  8. Structure-function analysis of the GmRIC1 signal peptide and CLE domain required for nodulation control in soybean.

    PubMed

    Reid, Dugald E; Li, Dongxue; Ferguson, Brett J; Gresshoff, Peter M

    2013-04-01

    Legumes control the nitrogen-fixing root nodule symbiosis in response to external and internal stimuli, such as nitrate, and via systemic autoregulation of nodulation (AON). Overexpression of the CLV3/ESR-related (CLE) pre-propeptide-encoding genes GmNIC1 (nitrate-induced and acting locally) and GmRIC1 (Bradyrhizobium-induced and acting systemically) suppresses soybean nodulation dependent on the activity of the nodulation autoregulation receptor kinase (GmNARK). This nodule inhibition response was used to assess the relative importance of key structural components within and around the CLE domain sequences of these genes. Using a site-directed mutagenesis approach, mutants were produced at each amino acid within the CLE domain (RLAPEGPDPHHN) of GmRIC1. This approach identified the Arg1, Ala3, Pro4, Gly6, Pro7, Asp8, His11, and Asn12 residues as critical to GmRIC1 nodulation suppression activity (NSA). In contrast, none of the mutations in conserved residues outside of the CLE domain showed compromised NSA. Chimeric genes derived from combinations of GmRIC1 and GmNIC1 domains were used to determine the role of each pre-propeptide domain in NSA differences that exist between the two peptides. It was found that the transit peptide and CLE peptide regions of GmRIC1 significantly enhanced activity of GmNIC1. In contrast, the comparable GmNIC1 domains reduced the NSA of GmRIC1. Identification of these critical residues and domains provides a better understanding of how these hormone-like peptides function in plant development and regulation. PMID:23386683

  9. Identification of domains within the human cytomegalovirus major immediate-early 86-kilodalton protein and the retinoblastoma protein required for physical and functional interaction with each other.

    PubMed Central

    Fortunato, E A; Sommer, M H; Yoder, K; Spector, D H

    1997-01-01

    The human cytomegalovirus major immediate-early 86-kDa protein (IE2 86) plays an important role in the trans activation and regulation of HCMV gene expression. Previously, we demonstrated that IE2 86 contains three regions (amino acids [aa] 86 to 135, 136 to 290, and 291 to 364) that can independently bind to in vitro-translated Rb when IE2 86 is produced as a glutathione S-transferase fusion protein (M. H. Sommer, A. L. Scully, and D. H. Spector, J. Virol. 68:6223-6231, 1994). In this report, we have elucidated the regions of Rb involved in binding to IE2 86 and have further analyzed the functional nature of the interaction between these two proteins. We find that two domains on Rb, the A/B pocket and the carboxy terminus, can each independently form a complex with IE2 86. In functional assays, we demonstrate that IE2 86 and another IE protein, IE1 72, can counter the enlarged flat cell phenotype, but not the G1/S block, which results from expression of wild-type Rb in the human osteosarcoma cell line Saos-2. Mutational analysis reveals that there are two domains on IE2 86 that can independently affect Rb function. One region (aa 241 to 369) includes the major Rb-binding domain, while the second maps to the amino-terminal region (aa 1 to 85) common to both IE2 86 and IE1 72. These data show that Rb and IE2 86 physically and functionally interact with each other via at least two separate domains and provide further support for the hypothesis that IE2 86 may exert its pleiotropic effects through the formation of multimeric protein complexes. PMID:9343168

  10. Broken-Line Functions with Unbroken Domains.

    ERIC Educational Resources Information Center

    Satianov, Pavel; Fried, Michael; Amit, Miriam

    1999-01-01

    Presents a method for introducing students to broken-line functions with unbroken domains. Concludes that a unit on broken-line functions should enhance students' understanding of the function concept. (ASK)

  11. Structure and Function of KH Domains

    SciTech Connect

    Valverde, R.; Regan, E

    2008-01-01

    The hnRNP K homology (KH) domain was first identified in the protein human heterogeneous nuclear ribonucleoprotein K (hnRNP K) 14 years ago. Since then, KH domains have been identified as nucleic acid recognition motifs in proteins that perform a wide range of cellular functions. KH domains bind RNA or ssDNA, and are found in proteins associated with transcriptional and translational regulation, along with other cellular processes. Several diseases, e.g. fragile X mental retardation syndrome and paraneoplastic disease, are associated with the loss of function of a particular KH domain. Here we discuss the progress made towards understanding both general and specific features of the molecular recognition of nucleic acids by KH domains. The typical binding surface of KH domains is a cleft that is versatile but that can typically accommodate only four unpaired bases. Van der Waals forces and hydrophobic interactions and, to a lesser extent, electrostatic interactions, contribute to the nucleic acid binding affinity. 'Augmented' KH domains or multiple copies of KH domains within a protein are two strategies that are used to achieve greater affinity and specificity of nucleic acid binding. Isolated KH domains have been seen to crystallize as monomers, dimers and tetramers, but no published data support the formation of noncovalent higher-order oligomers by KH domains in solution. Much attention has been given in the literature to a conserved hydrophobic residue (typically Ile or Leu) that is present in most KH domains. The interest derives from the observation that an individual with this Ile mutated to Asn, in the KH2 domain of fragile X mental retardation protein, exhibits a particularly severe form of the syndrome. The structural effects of this mutation in the fragile X mental retardation protein KH2 domain have recently been reported. We discuss the use of analogous point mutations at this position in other KH domains to dissect both structure and function.

  12. 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. PMID:27309309

  13. A Conserved START Domain Coenzyme Q-binding Polypeptide is Required for Efficient Q Biosynthesis, Respiratory Electron Transport, and Antioxidant Function in Saccharomyces cerevisiae

    PubMed Central

    Morvaridi, Susan; Saiki, Ryoichi; Johnson, Jarrett S.; Liau, Wei-Siang; Hirano, Kathleen; Kawashima, Tadashi; Ji, Ziming; Loo, Joseph A.; Shepherd, Jennifer N.; Clarke, Catherine F.

    2014-01-01

    Coenzyme Qn (ubiquinone or Qn) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail of n isoprene units. Saccharomyces cerevisiae coq1-coq9 mutants have defects in Q biosynthesis, lack Q6, are respiratory defective, and sensitive to stress imposed by polyunsaturated fatty acids. The hallmark phenotype of the Q-less yeast coq mutants is that respiration in isolated mitochondria can be rescued by the addition of Q2, a soluble Q analog. Yeast coq10 mutants share each of these phenotypes, with the surprising exception that they continue to produce Q6. Structure determination of the Caulobacter crescentus Coq10 homolog (CC1736) revealed a steroidogenic acute regulatory protein-related lipid transfer (START) domain, a hydrophobic tunnel known to bind specific lipids in other START domain family members. Here we show that purified CC1736 binds Q2, Q3, Q10, or demethoxy-Q3 in an equimolar ratio, but fails to bind 3-farnesyl-4-hydroxybenzoic acid, a farnesylated analog of an early Q-intermediate. Over-expression of C. crescentus CC1736 or COQ8 restores respiratory electron transport and antioxidant function of Q6 in the yeast coq10 null mutant. Studies with stable isotope ring precursors of Q reveal that early Q-biosynthetic intermediates accumulate in the coq10 mutant and de novo Q-biosynthesis is less efficient than in the wild-type yeast or rescued coq10 mutant. The results suggest that the Coq10 polypeptide:Q (protein:ligand) complex may serve essential functions in facilitating de novo Q biosynthesis and in delivering newly synthesized Q to one or more complexes of the respiratory electron transport chain. PMID:23270816

  14. The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex

    PubMed Central

    Ge, Chunmin; Che, Lixiao; Du, Chunying

    2015-01-01

    BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response. PMID:26683461

  15. The PAX3 paired domain and homeodomain function as a single binding module in vivo to regulate subnuclear localization and mobility by a mechanism that requires base-specific recognition.

    PubMed

    Corry, Gareth N; Raghuram, Nikhil; Missiaen, Kristal K; Hu, Ninghe; Hendzel, Michael J; Underhill, D Alan

    2010-09-10

    The transcription factor PAX3 is essential for myogenesis and neural crest development, and is one of several genes mutated in human Waardenburg syndrome. Analysis of disease-causing missense mutations in PAX3 has established the interdependence of its two DNA-binding domains, the paired domain (PD) and the homeodomain (HD), as well as defects in localization and mobility. Paradoxically, mutants that retained DNA binding activity exhibited the greatest defects in localization and mobility, regardless of the domain in which they reside. In the present study, structure-function analyses were used to determine the mechanistic basis of this effect. In the context of the isolated DNA-binding domains, HD mutants adopted an increase in mobility proportional to their loss in DNA binding, while PD mutants continued to display the inverse relationship observed in the full-length protein. At the structural level, this reflected an unexpected dependence on base-specific contacts in the PD, whereas HD mobility was more severely affected by loss of backbone contacts, as has been observed with other DNA-binding proteins. This requires that the HD switch to a base-specific mode in the full-length protein. Moreover, both domains underwent substantial reduction in mobility and altered localization when in a contiguous polypeptide with the endogenous linker segment. Notably, although the HD conferred localization to heterochromatin, this activity was masked when linked to the PD, despite the absence of determinants for subnuclear compartmentalization in the PD or linker. Last, the propensity for PAX3 heterochromatin localization was modulated by sequences at the amino and carboxy termini, supporting a model in which alternate conformations lead to unmasking of the HD. These data indicate that the PD and the HD functionally interact in vivo and behave as a single binding module whose mobility and localization are dependent on sequence-specific contacts. PMID:20643146

  16. The PR-Set7 binding domain of Riz1 is required for the H4K20me1-H3K9me1 trans-tail ‘histone code’ and Riz1 tumor suppressor function

    PubMed Central

    Congdon, Lauren M.; Sims, Jennifer K.; Tuzon, Creighton T.; Rice, Judd C.

    2014-01-01

    PR-Set7/Set8/KMT5a is the sole histone H4 lysine 20 monomethyltransferase (H4K20me1) in metazoans and is essential for proper cell division and genomic stability. We unexpectedly discovered that normal cellular levels of monomethylated histone H3 lysine 9 (H3K9me1) were also dependent on PR-Set7, but independent of its catalytic activity. This observation suggested that PR-Set7 interacts with an H3K9 monomethyltransferase to establish the previously reported H4K20me1-H3K9me1 trans-tail ‘histone code’. Here we show that PR-Set7 specifically and directly binds the C-terminus of the Riz1/PRDM2/KMT8 tumor suppressor and demonstrate that the N-terminal PR/SET domain of Riz1 preferentially monomethylates H3K9. The PR-Set7 binding domain was required for Riz1 nuclear localization and maintenance of the H4K20me1-H3K9me1 trans-tail ‘histone code’. Although Riz1 can function as a repressor, Riz1/H3K9me1 was dispensable for the repression of genes regulated by PR-Set7/H4K20me1. Frameshift mutations resulting in a truncated Riz1 incapable of binding PR-Set7 occur frequently in various aggressive cancers. In these cancer cells, expression of wild-type Riz1 restored tumor suppression by decreasing proliferation and increasing apoptosis. These phenotypes were not observed in cells expressing either the Riz1 PR/SET domain or PR-Set7 binding domain indicating that Riz1 methyltransferase activity and PR-Set7 binding domain are both essential for Riz1 tumor suppressor function. PMID:24423864

  17. DPP6 Domains Responsible for Its Localization and Function*

    PubMed Central

    Lin, Lin; Long, Laura K.; Hatch, Michael M.; Hoffman, Dax A.

    2014-01-01

    Dipeptidyl peptidase-like protein 6 (DPP6) is an auxiliary subunit of the Kv4 family of voltage-gated K+ channels known to enhance channel surface expression and potently accelerate their kinetics. DPP6 is a single transmembrane protein, which is structurally remarkable for its large extracellular domain. Included in this domain is a cysteine-rich motif, the function of which is unknown. Here we show that this cysteine-rich domain of DPP6 is required for its export from the ER and expression on the cell surface. Disulfide bridges formed at C349/C356 and C465/C468 of the cysteine-rich domain are necessary for the enhancement of Kv4.2 channel surface expression but not its interaction with Kv4.2 subunits. The short intracellular N-terminal and transmembrane domains of DPP6 associates with and accelerates the recovery from inactivation of Kv4.2, but the entire extracellular domain is necessary to enhance Kv4.2 surface expression and stabilization. Our findings show that the cysteine-rich domain of DPP6 plays an important role in protein folding of DPP6 that is required for transport of DPP6/Kv4.2 complexes out of the ER. PMID:25190807

  18. DPP6 domains responsible for its localization and function.

    PubMed

    Lin, Lin; Long, Laura K; Hatch, Michael M; Hoffman, Dax A

    2014-11-14

    Dipeptidyl peptidase-like protein 6 (DPP6) is an auxiliary subunit of the Kv4 family of voltage-gated K(+) channels known to enhance channel surface expression and potently accelerate their kinetics. DPP6 is a single transmembrane protein, which is structurally remarkable for its large extracellular domain. Included in this domain is a cysteine-rich motif, the function of which is unknown. Here we show that this cysteine-rich domain of DPP6 is required for its export from the ER and expression on the cell surface. Disulfide bridges formed at C349/C356 and C465/C468 of the cysteine-rich domain are necessary for the enhancement of Kv4.2 channel surface expression but not its interaction with Kv4.2 subunits. The short intracellular N-terminal and transmembrane domains of DPP6 associates with and accelerates the recovery from inactivation of Kv4.2, but the entire extracellular domain is necessary to enhance Kv4.2 surface expression and stabilization. Our findings show that the cysteine-rich domain of DPP6 plays an important role in protein folding of DPP6 that is required for transport of DPP6/Kv4.2 complexes out of the ER. PMID:25190807

  19. Architecture and function of metallopeptidase catalytic domains

    PubMed Central

    Cerdà-Costa, Núria; Gomis-Rüth, Francesc Xavier

    2014-01-01

    The cleavage of peptide bonds by metallopeptidases (MPs) is essential for life. These ubiquitous enzymes participate in all major physiological processes, and so their deregulation leads to diseases ranging from cancer and metastasis, inflammation, and microbial infection to neurological insults and cardiovascular disorders. MPs cleave their substrates without a covalent intermediate in a single-step reaction involving a solvent molecule, a general base/acid, and a mono-or dinuclear catalytic metal site. Most monometallic MPs comprise a short metal-binding motif (HEXXH), which includes two metal-binding histidines and a general base/acid glutamate, and they are grouped into the zincin tribe of MPs. The latter divides mainly into the gluzincin and metzincin clans. Metzincins consist of globular ∼130–270-residue catalytic domains, which are usually preceded by N-terminal pro-segments, typically required for folding and latency maintenance. The catalytic domains are often followed by C-terminal domains for substrate recognition and other protein–protein interactions, anchoring to membranes, oligomerization, and compartmentalization. Metzincin catalytic domains consist of a structurally conserved N-terminal subdomain spanning a five-stranded β-sheet, a backing helix, and an active-site helix. The latter contains most of the metal-binding motif, which is here characteristically extended to HEXXHXXGXX(H,D). Downstream C-terminal subdomains are generally shorter, differ more among metzincins, and mainly share a conserved loop—the Met-turn—and a C-terminal helix. The accumulated structural data from more than 300 deposited structures of the 12 currently characterized metzincin families reviewed here provide detailed knowledge of the molecular features of their catalytic domains, help in our understanding of their working mechanisms, and form the basis for the design of novel drugs. PMID:24596965

  20. Functional domain analysis of the Saccharomyces MAL-activator.

    PubMed

    Hu, Z; Gibson, A W; Kim, J H; Wojciechowicz, L A; Zhang, B; Michels, C A

    1999-08-01

    MAL63 of the MAL6 locus and its homologues at the other MAL loci encode transcription activators required for the maltose-inducible expression of the MAL structural genes. We carried out a deletion analysis of LexA-MAL63 gene fusions to localize the functional domains of the Mal63 MAL-activator protein. Our results indicate that the sequence-specific DNA-binding domain of Mal63p is contained in residues 1-100; that residues 60-283 constitute a functional core region including the transactivation domain; that residues 251-299 are required to inhibit the activation function of Mal63p; and that the rest of the C-terminal region of the protein contains a maltose-responsive domain that acts to relieve the inhibitory effect on the activation function. Abundant overproduction of Mal63p does not overcome the negative regulation of MAL gene expression in the absence of maltose, suggesting that a titratable MAL-specific repressor similar to Gal80p is not involved in the negative regulation of the MAL-activator. A model for maltose-inducible autoregulation of the MAL-activator is presented. PMID:10447589

  1. Project X functional requirements specification

    SciTech Connect

    Holmes, S.D.; Henderson, S.D.; Kephart, R.; Kerby, J.; Mishra, S.; Nagaitsev, S.; Tschirhart, R.; /Fermilab

    2011-03-01

    Project X is a multi-megawatt proton facility being developed to support intensity frontier research in elementary particle physics, with possible applications to nuclear physics and nuclear energy research, at Fermilab. A Functional Requirements Specification has been developed in order to establish performance criteria for the Project X complex in support of these multiple missions. This paper will describe the Functional Requirements for the Project X facility and the rationale for these requirements.

  2. Domain-Independent Scientific Function Finding

    NASA Astrophysics Data System (ADS)

    Schaffer, Cullen R.

    1990-01-01

    Programs such as Bacon, Abacus, Coper, Kepler and others are designed to find functional relationships of scientific significance in quantitative data without relying on the deep domain knowledge scientists normally bring to bear in analytic work. Whether these systems actually perform as intended is an open question, however. To date, they have been supported only by anecdotal evidence --reports that a desirable answer has been found in one or more selected and often artificial cases. In this dissertation, I thus attempt to develop, not only new approaches to domain -independent scientific function finding, but, equally, a rigorous methodology under which research into such methods can be conducted. A fundamental problem with previous work is that it has investigated scientific data analysis in the abstract --without referring to actual scientific data. By contrast, the work reported here is founded on a collection of 352 real scientific data sets. This empirical base supports a number of strong conclusions. First, while researchers working with artificial data have targeted complex multivariate relations, real data provides powerful evidence that even the simplest bivariate relationships are difficult to identify reliably. Second, despite its ubiquitous presence in previous work, the notion of heuristic search of a potentially explosive space of formulas appears to help very little with the problem of reliably identifying basic bivariate relationships. Instead, third, substantial performance improvement results from viewing function finding as a decision problem, the problem of classifying data sets reliably within a fixed--and quite limited--system of functional categories. This dissertation presents what I believe to be the strongest domain-independent scientific function-finding algorithm currently in existence and, certainly, the only one which has been rigorously demonstrated. At the same time, it suggests fundamental limitations in the power of such

  3. The Jak2V617F oncogene associated with myeloproliferative diseases requires a functional FERM domain for transformation and for expression of the Myc and Pim proto-oncogenes

    PubMed Central

    Wernig, Gerlinde; Gonneville, Jeffrey R.; Crowley, Brian J.; Rodrigues, Margret S.; Reddy, Mamatha M.; Hudon, Heidi E.; Walz, Christoph; Reiter, Andreas; Podar, Klaus; Royer, Yohan; Constantinescu, Stefan N.; Tomasson, Michael H.; Griffin, James D.; Gilliland, D. Gary

    2008-01-01

    The V617F activating point mutation in Jak2 is associated with a proportion of myeloproliferative disorders. In normal hematopoietic cells, Jak2 signals only when associated with a growth factor receptor, such as the erythropoietin receptor (EpoR). We sought to identify the molecular requirements for activation of Jak2V617F by introducing a point mutation in the FERM domain (Y114A), required for receptor binding. Whereas BaF3.EpoR cells are readily transformed by Jak2V617F to Epo independence, we found that the addition of the FERM domain mutation blocked transformation and the induction of reactive oxygen species. Further, while cells expressing Jak2V617F had constitutive activation of STAT5, cells expressing Jak2V617F/Y114A did not, suggesting that signaling is defective at a very proximal level. In addition, expression of the Myc and Pim proto-oncogenes by Jak2V617F was found to be FERM domain dependent. An inducible constitutively active STAT5 mutant expressed in BaF3 cells was sufficient to induce Myc and Pim. Finally, the FERM domain in Jak2V617F was also required for abnormal hematopoiesis in transduced primary murine fetal liver cells. Overall, our results suggest that constitutive activation of Jak2 requires an intact FERM domain for a transforming phenotype, and is necessary for activation of the major target of Jak2, STAT5. PMID:18216297

  4. Tool-Guided Domain-Specific, Systematic Requirements Management

    NASA Astrophysics Data System (ADS)

    Gerlich, Rainer; Gerlich, Ralf

    2010-08-01

    The importance of the quality of requirements for successful execution and completion of a project from a technical and contractual point of view is being recognized more and more. Many methods are targeted to improve the support for collecting requirements while still focusing on natural language. However, the ambiguities in the semantics of natural language are the biggest obstacles towards success. The approach presented in this paper focuses on the elements of a domain while keeping the expressiveness of natural names and terms and introducing clear semantics. This brings the advantage that immediate verification of the human-provided inputs is possible, immediate contributions to validation are available and inconsistencies can be detected by a tool immediately. This leads to guidance of an engineer by a tool towards consistent, complete and correct requirements - requirements of high quality - and eases maintenance for the same reasons. As most of the complexity is handled by the tool due to its good knowledge on the domain, the approach is scalable towards large specifications. Several examples of application domains are described which illustrate the universality and feasibility of the approach across domain boundaries.

  5. The WD40 Domain Is Required for LRRK2 Neurotoxicity

    PubMed Central

    Jorgensen, Nathan D.; Peng, Yong; Ho, Cherry C.-Y.; Rideout, Hardy J.; Petrey, Donald; Liu, Peng; Dauer, William T.

    2009-01-01

    Background Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson disease (PD). LRRK2 contains an “enzymatic core” composed of GTPase and kinase domains that is flanked by leucine-rich repeat (LRR) and WD40 protein-protein interaction domains. While kinase activity and GTP-binding have both been implicated in LRRK2 neurotoxicity, the potential role of other LRRK2 domains has not been as extensively explored. Principal Findings We demonstrate that LRRK2 normally exists in a dimeric complex, and that removing the WD40 domain prevents complex formation and autophosphorylation. Moreover, loss of the WD40 domain completely blocks the neurotoxicity of multiple LRRK2 PD mutations. Conclusion These findings suggest that LRRK2 dimerization and autophosphorylation may be required for the neurotoxicity of LRRK2 PD mutations and highlight a potential role for the WD40 domain in the mechanism of LRRK2-mediated cell death. PMID:20041156

  6. Project X functional requirements specification

    SciTech Connect

    Holmes, S.D.; Henderson, S.D.; Kephart, R.; Kerby, J.; Kourbanis, I.; Lebedev, V.; Mishra, S.; Nagaitsev, S.; Solyak, N.; Tschirhart, R.; /Fermilab

    2012-05-01

    Project X is a multi-megawatt proton facility being developed to support a world-leading program in Intensity Frontier physics at Fermilab. The facility is designed to support programs in elementary particle and nuclear physics, with possible applications to nuclear energy research. A Functional Requirements Specification has been developed in order to establish performance criteria for the Project X complex in support of these multiple missions, and to assure that the facility is designed with sufficient upgrade capability to provide U.S. leadership for many decades to come. This paper will briefly review the previously described Functional Requirements, and then discuss their recent evolution.

  7. Fosmid-Based Structure-Function Analysis Reveals Functionally Distinct Domains in the Cytoplasmic Domain of Drosophila Crumbs

    PubMed Central

    Klose, Sven; Flores-Benitez, David; Riedel, Falko; Knust, Elisabeth

    2013-01-01

    The evolutionarily conserved transmembrane protein Crumbs is required for epithelial polarity and morphogenesis in the embryo, control of tissue size in imaginal discs and morphogenesis of photoreceptor cells, and prevents light-dependent retinal degeneration. The small cytoplasmic domain contains two highly conserved regions, a FERM (i.e., protein 4.1/ezrin/radixin/moesin)-binding and a PDZ (i.e., postsynaptic density/discs large/ZO-1)-binding domain. Using a fosmid-based transgenomic approach, we analyzed the role of the two domains during invagination of the tracheae and the salivary glands in the Drosophila embryo. We provide data to show that the PDZ-binding domain is essential for the maintenance of cell polarity in both tissues. In contrast, in embryos expressing a Crumbs protein with an exchange of a conserved Tyrosine residue in the FERM-binding domain to an Alanine, both tissues are internalized, despite some initial defects in apical constriction, phospho-Moesin recruitment, and coordinated invagination movements. However, at later stages these embryos fail to undergo dorsal closure, germ band retraction, and head involution. In addition, frequent defects in tracheal fusion were observed. These results suggest stage and/or tissue specific binding partners. We discuss the power of this fosmid-based system for detailed structure-function analyses in comparison to the UAS/Gal4 system. PMID:23390593

  8. Differential function of Themis CABIT domains during T cell development.

    PubMed

    Okada, Toshiyuki; Nitta, Takeshi; Kaji, Kentaro; Takashima, Akiko; Oda, Hiroyo; Tamehiro, Norimasa; Goto, Motohito; Okamura, Tadashi; Patrick, Michael S; Suzuki, Harumi

    2014-01-01

    Themis (also named Gasp) is a newly identified Grb2-binding protein that is essential for thymocyte positive selection. Despite the possible involvement of Themis in TCR-mediated signal transduction, its function remains unresolved and controversial. Themis contains two functionally uncharacterized regions called CABIT (cysteine-containing, all-β in Themis) domains, a nuclear localization signal (NLS), and a proline-rich sequence (PRS). To elucidate the role of these motifs in Themis's function in vivo, we established a series of mutant Themis transgenic mice on a Themis(-/-) background. Deletion of the highly conserved Core motif of CABIT1 or CABIT2 (Core1 or Core2, respectively), the NLS, or the PRS abolished Grb2-association, as well as TCR-dependent tyrosine-phosphorylation and the ability to induce positive selection in the thymus. The NLS and Core1 motifs were required for the nuclear localization of Themis, whereas Core2 and PRS were not. Furthermore, expression of ΔCore1- but not ΔCore2-Themis conferred dominant negative-type inhibition on T cell development. Collectively, our current results indicate that PRS, NLS, CABIT1, and CABIT2 are all required for positive selection, and that each of the CABIT domains exerts distinct functions during positive selection. PMID:24586531

  9. Neurofascins are required to establish axonal domains for saltatory conduction.

    PubMed

    Sherman, Diane L; Tait, Steven; Melrose, Shona; Johnson, Richard; Zonta, Barbara; Court, Felipe A; Macklin, Wendy B; Meek, Stephen; Smith, Andrew J H; Cottrell, David F; Brophy, Peter J

    2005-12-01

    Voltage-gated sodium channels are concentrated in myelinated nerves at the nodes of Ranvier flanked by paranodal axoglial junctions. Establishment of these essential nodal and paranodal domains is determined by myelin-forming glia, but the mechanisms are not clear. Here, we show that two isoforms of Neurofascin, Nfasc155 in glia and Nfasc186 in neurons, are required for the assembly of these specialized domains. In Neurofascin-null mice, neither paranodal adhesion junctions nor nodal complexes are formed. Transgenic expression of Nfasc155 in the myelinating glia of Nfasc-/- nerves rescues the axoglial adhesion complex by recruiting the axonal proteins Caspr and Contactin to the paranodes. However, in the absence of Nfasc186, sodium channels remain diffusely distributed along the axon. Our study shows that the two major Neurofascins play essential roles in assembling the nodal and paranodal domains of myelinated axons; therefore, they are essential for the transition to saltatory conduction in developing vertebrate nerves. PMID:16337912

  10. Microdissection of Shoot Meristem Functional Domains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection–microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes th...

  11. The significance of requirements engineering for the medical domain.

    PubMed

    Kossmann, Mario

    2014-07-01

    This paper aims to raise awareness of the importance of Requirements Engineering (RE) for the successful and efficient development of high-quality systems and products for the medical domain. It does so by providing an introduction to RE from the viewpoints of project and programme management and systems engineering in general and by illustrating the usefulness of a sound RE approach to the development of a local healthcare system in a deprived region in central Africa. The paper concludes that RE is just as crucial for the development of systems and products in the medical domain, as it is for the development of systems in the aerospace industry or software systems in the consumer electronics industry; while the degree of detail and formality of how RE is used has to be tailored to fit the context in question. PMID:24953681

  12. Using Domain Requirements to Achieve Science-Oriented Provenance

    SciTech Connect

    Stephan, Eric G; Halter, Todd D; Critchlow, Terence J; Pinheiro Da Silva, Paulo; Salayandia, Leonardo

    2010-06-18

    Using Domain Requirements to Achieve Science-Oriented Provenance Eric Stephan1 Todd Halter1 Terence Critchlow1 Paulo Pinheiro da Silva2 Leonardo Salayandia2 1 Pacific Northwest National Laboratory, Richland WA, USA 2 University of Texas at El Paso, El Paso TX, USA Abstract. The US Department of Energy (DOE) Atmospheric Radi- ation Measurement Program (ARM) is adopting the use of formalized provenance to support observational data products produced by ARM operations and relied upon by researchers. Because of the diversity of needs in the climate community provenance will need to be conveyed in a domain-oriented context. This paper explores a use case where semantic abstract workflows (SAW) are employed as a means to filter, aggregate, and contextually describe the historical events responsible for the ARM data product the scientist is relying upon.

  13. Modelling protein functional domains in signal transduction using Maude

    NASA Technical Reports Server (NTRS)

    Sriram, M. G.

    2003-01-01

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

  14. Intellectual Growth in Children as a Function of Domain Specific and Domain General Working Memory Subgroups

    ERIC Educational Resources Information Center

    Swanson, H. Lee

    2011-01-01

    This study examined whether children's growth on measures of fluid (Raven Colored Progressive Matrices) and crystallized (reading and math achievement) intelligence was attributable to domain-specific or domain-general functions of working memory (WM). A sample of 290 elementary school children was tested on measures of intelligence across three…

  15. Functional domains in Fok I restriction endonuclease.

    PubMed Central

    Li, L; Wu, L P; Chandrasegaran, S

    1992-01-01

    The PCR was used to alter transcriptional and translational signals surrounding the Flavobacterium okeanokoites restriction endonuclease (fokIR) gene, so as to achieve high expression in Escherichia coli. By changing the ribosome-binding site sequence preceding the fokIR gene to match the consensus E. coli signal and by placing a positive retroregulator stem-loop sequence downstream of the gene, Fok I yield was increased to 5-8% of total cellular protein. Fok I was purified to homogeneity with phosphocellulose, DEAE-Sephadex, and gel chromatography, yielding 50 mg of pure Fok I endonuclease per liter of culture medium. The recognition and cleavage domains of Fok I were analyzed by trypsin digestion. Fok I in the absence of a DNA substrate cleaves into a 58-kDa carboxyl-terminal and 8-kDa amino-terminal fragment. The 58-kDa fragment does not bind the DNA substrate. Fok I in the presence of a DNA substrate cleaves into a 41-kDa amino-terminal fragment and a 25-kDa carboxyl-terminal fragment. On further digestion, the 41-kDa fragment degrades into 30-kDa amino-terminal and 11-kDa carboxyl-terminal fragments. The cleaved fragments both bind DNA substrates, as does the 41-kDa fragment. Gel-mobility-shift assays indicate that all the protein contacts necessary for the sequence-specific recognition of DNA substrates are encoded within the 41-kDa fragment. Thus, the 41-kDa amino-terminal fragment constitutes the Fok I recognition domain. The 25-kDa fragment, purified by using a DEAE-Sephadex column, cleaves nonspecifically both methylated (pACYCfokIM) and nonmethylated (pTZ19R) DNA substrates in the presence of MgCl2. Thus, the 25-kDa carboxyl-terminal fragment constitutes the Fok I cleavage domain. Images PMID:1584761

  16. Functional domains of the human epididymal protease inhibitor, eppin.

    PubMed

    McCrudden, Maelíosa T C; Dafforn, Tim R; Houston, David F; Turkington, Philip T; Timson, David J

    2008-04-01

    Eppin has two potential protease inhibitory domains: a whey acid protein or four disulfide core domain and a Kunitz domain. The protein is also reported to have antibacterial activity against Gram-negative bacteria. Eppin and its whey acid protein and Kunitz domains were expressed in Escherichia coli and their ability to inhibit proteases and kill bacteria compared. The Kunitz domain inhibits elastase (EC 3.4.21.37) to a similar extent as intact eppin, whereas the whey acid protein domain has no such activity. None of these fragments inhibits trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1) at the concentrations tested. In a colony forming unit assay, both domains have some antibacterial activity against E. coli, but this was not to the same degree as intact eppin or the two domains together. When bacterial respiratory electron transport was measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, eppin and its domains caused an increase in the rate of respiration. This suggests that the mechanism of cell killing may be partly through the permeablization of the bacterial inner membrane, resulting in uncoupling of respiratory electron transport and consequent collapse of the proton motive force. Thus, we conclude that although both of eppin's domains are involved in the protein's antibacterial activity, only the Kunitz domain is required for selective protease inhibition. PMID:18331357

  17. Identification of the topology and functional domains of PAQR10.

    PubMed

    Jin, Ting; Xu, Daqian; Ding, Qiurong; Zhang, Yixuan; Mao, Chenqian; Pan, Yi; Wang, Zhenzhen; Chen, Yan

    2012-05-01

    PAQR10 (progestin and adipoQ receptor 10) is a Golgi-localized protein that is able to enhance the retention and activation of Ras proteins in the Golgi apparatus, subsequently leading to a sustained ERK (extracellular-signal-regulated kinase) signalling. However, little is known about the topology and functional domains of PAQR10. In the present study, we extensively dissected and analysed the structure of PAQR10. The topology analysis reveals that PAQR10 is an integral membrane protein with its N-terminus facing the cytosol. Multiple domains, including the membrane-proximal region at the N-terminus, the membrane-proximal region at the C-terminus and the three loops facing the cytosol, were found to be required for PAQR10 to reside in the Golgi apparatus, to stimulate ERK phosphorylation and to tether Ras to the Golgi apparatus. Furthermore, when PAQR10 was artificially forced to be expressed in the endoplasmic reticulum, it could neither mobilize Ras to the Golgi apparatus nor increase ERK phosphorylation. Finally, the PAQR10 mutants that lost Golgi localization failed to promote differentiation of PC12 cells. Collectively, the results of the present study indicate that Golgi localization is indispensable for PAQR10 to implement its regulatory functions in the Ras signalling cascade. PMID:22339580

  18. Structural-Functional Analysis Reveals a Specific Domain Organization in Family GH20 Hexosaminidases

    PubMed Central

    Val-Cid, Cristina; Biarnés, Xevi; Faijes, Magda; Planas, Antoni

    2015-01-01

    Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20) always accompanied with an extra α-helix (GH20b-GH20-α), and Model B with only the catalytic GH20 domain. The large Bifidobacterium bifidum lacto-N-biosidase was used as a model protein to evaluate the minimal functional unit due to its interest and structural complexity. By expressing different truncated forms of this enzyme, we show that Model A architectures cannot be reduced to Model B. In particular, there are two structural requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always involves a remote element provided by a long loop from the catalytic domain itself or, when this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner. Particularly, the lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. We demonstrate restoration of activity of the inactive GH20b-GH20-α construct (model A architecture) by a complementation assay with the lectin-like domain. The engineering of minimal functional units of multidomain GH20 enzymes must consider these structural requirements. PMID:26024355

  19. The Pitx2c N-terminal domain is a critical interaction domain required for asymmetric morphogenesis

    PubMed Central

    Simard, Annie; Di Giorgio, Luciano; Amen, Melanie; Westwood, Ashley; Amendt, Brad A.; Ryan, Aimee K.

    2010-01-01

    The paired-like homeodomain transcription factor Pitx2c has an essential role in patterning the left-right axis. However, neither its transcriptional targets nor the molecular mechanisms through which it exerts its patterning function are known. Here we provide evidence that the N-terminal domain of Pitx2c is important for this activity. Overexpression of the Pitx2c N-terminus in ovo randomizes the direction of heart looping, the first morphological asymmetry conserved in vertebrate embryos. In addition, the Pitx2c N-terminal domain blocks the ability of Pitx2c to synergize with Nkx2.5 to transactivate the procollagen lysyl hydroxylase (Plod-1) promoter in transient transfection assays. A five amino acid region containing leucine-41 is required for both of these effects. Our data suggest that the Pitx2c N-terminal domain competes with endogenous Pitx2c for binding to a protein interaction partner that is required for the activation of genes that direct asymmetric morphogenesis along the left-right axis. PMID:19681163

  20. Functional domains of an ATP-dependent DNA ligase.

    PubMed

    Doherty, A J; Wigley, D B

    1999-01-01

    The crystal structure of an ATP-dependent DNA ligase from bacteriophage T7 revealed that the protein comprised two structural domains. In order to investigate the biochemical activities of these domains, we have overexpressed them separately and purified them to homogeneity. The larger N-terminal domain retains adenylation and ligase activities, though both at a reduced level. The adenylation activity of the large domain is stimulated by the presence of the smaller domain, suggesting that a conformational change is required for adenylation in the full length protein. The DNA binding properties of the two fragments have also been studied. The larger domain is able to band shift both single and double-stranded DNA, while the smaller fragment is only able to bind to double-stranded DNA. These data suggest that the specificity of DNA ligases for nick sites in DNA is produced by a combination of these different DNA binding activities in the intact enzyme. PMID:9878388

  1. Further insight into BRUTUS domain composition and functionality.

    PubMed

    Matthiadis, Anna; Long, Terri A

    2016-08-01

    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

  2. Identification of two functional domains within the arenavirus nucleoprotein.

    PubMed

    Levingston Macleod, Jesica M; D'Antuono, Alejandra; Loureiro, Maria Eugenia; Casabona, Juan Cruz; Gomez, Guillermo A; Lopez, Nora

    2011-03-01

    Tacaribe virus (TCRV) belongs to the Arenaviridae family. Its bisegmented negative-stranded RNA genome encodes the nucleoprotein (N), the precursor of the envelope glycoproteins, the polymerase (L), and a RING finger matrix (Z) protein. The 570-amino-acid N protein binds to viral RNA, forming nucleocapsids, which are the template for transcription and replication by the viral polymerase. We have previously shown that the interaction between N and Z is required for assembly of infectious virus-like particles (VLPs) (J. C. Casabona et al., J. Virol. 83:7029-7039, 2009). Here, we examine the functional organization of TCRV N protein. A series of deletions and point mutations were introduced into the N-coding sequence, and the ability of the mutants to sustain heterotypic (N-Z) or homotypic (N-N) interactions was analyzed. We found that N protein displays two functional domains. By using coimmunoprecipitation studies, VLP incorporation assays, and double immunofluorescence staining, the carboxy-terminal region of N was found to be required for N-Z interaction and also necessary for incorporation of N protein into VLPs. Moreover, further analysis of this region showed that the integrity of a putative zinc-finger motif, as well as its amino-flanking sequence (residues 461 to 489), are critical for Z binding and N incorporation into VLPs. In addition, we provide evidence of an essential role of the amino-terminal region of N protein for N-N interaction. In this regard, using reciprocal coimmunoprecipitation analysis, we identified a 28-residue region predicted to form a coiled-coil domain (residues 92 to 119) as a newly recognized molecular determinant of N homotypic interactions. PMID:21159858

  3. Identification of Two Functional Domains within the Arenavirus Nucleoprotein▿

    PubMed Central

    Levingston Macleod, Jesica M.; D'Antuono, Alejandra; Loureiro, Maria Eugenia; Casabona, Juan Cruz; Gomez, Guillermo A.; Lopez, Nora

    2011-01-01

    Tacaribe virus (TCRV) belongs to the Arenaviridae family. Its bisegmented negative-stranded RNA genome encodes the nucleoprotein (N), the precursor of the envelope glycoproteins, the polymerase (L), and a RING finger matrix (Z) protein. The 570-amino-acid N protein binds to viral RNA, forming nucleocapsids, which are the template for transcription and replication by the viral polymerase. We have previously shown that the interaction between N and Z is required for assembly of infectious virus-like particles (VLPs) (J. C. Casabona et al., J. Virol. 83:7029-7039, 2009). Here, we examine the functional organization of TCRV N protein. A series of deletions and point mutations were introduced into the N-coding sequence, and the ability of the mutants to sustain heterotypic (N-Z) or homotypic (N-N) interactions was analyzed. We found that N protein displays two functional domains. By using coimmunoprecipitation studies, VLP incorporation assays, and double immunofluorescence staining, the carboxy-terminal region of N was found to be required for N-Z interaction and also necessary for incorporation of N protein into VLPs. Moreover, further analysis of this region showed that the integrity of a putative zinc-finger motif, as well as its amino-flanking sequence (residues 461 to 489), are critical for Z binding and N incorporation into VLPs. In addition, we provide evidence of an essential role of the amino-terminal region of N protein for N-N interaction. In this regard, using reciprocal coimmunoprecipitation analysis, we identified a 28-residue region predicted to form a coiled-coil domain (residues 92 to 119) as a newly recognized molecular determinant of N homotypic interactions. PMID:21159858

  4. Functional diversity and interactions between the repeat domains of rat intestinal lactase.

    PubMed Central

    Jost, B; Duluc, I; Richardson, M; Lathe, R; Freund, J N

    1997-01-01

    Lactase-phlorizin hydrolase (LPH), a major digestive enzyme in the small intestine of newborns, is synthesized as a high-molecular-mass precursor comprising four tandemly repeated domains. Proteolytic cleavage of the precursor liberates the pro segment (LPHalpha) corresponding to domains I and II and devoid of known enzymic function. The mature enzyme (LPHbeta) comprises domains III and IV and is anchored in the brush border membrane via a C-terminal hydrophobic segment. To analyse the roles of the different domains of LPHalpha and LPHbeta, and the interactions between them, we have engineered a series of modified derivatives of the rat LPH precursor. These were expressed in cultured cells under the control of a cytomegalovirus promoter. The results show that recombinant LPHbeta harbouring both domains III and IV produces lactase activity. Neither domain III nor IV is alone sufficient to generate active enzyme, although the corresponding proteins are transport-competent. Tandem duplication of domains III or IV did not restore lactase activity, demonstrating the separate roles of both domains within LPHbeta. Further, the development of lactase activity did not require LPHalpha; however, LPHalpha potentiated the production of active LPHbeta but the individual LPHalpha subdomains I and II were unable to do so. Lactase activity and targeting required the C-terminal transmembrane anchor of LPH; this requirement was terminal transmembrane anchor or LPH; this requirement was not satisfied by the signal/anchor region of another digestive enzyme: sucrase-isomaltase. On the basis of this study we suggest that multiple levels of intramolecular interactions occur within the LPH precursor to produce the mature enzyme, and that the repeat domains of the precursor have distinct and specific functions in protein processing, substrate recognition and catalysis. We propose a functional model of LPHbeta in which substrate is channelled from an entry point located within domain II to

  5. Domain coloring of complex functions: an implementation-oriented introduction.

    PubMed

    Poelke, Konstantin; Polthier, Konrad

    2012-01-01

    This article gives a short overview of domain coloring for complex functions that have four-dimensional function graphs and therefore can't be visualized traditionally. The authors discuss several color schemes, focus on various aspects of complex functions, and provide Java-like pseudocode examples explaining the crucial ideas of the coloring algorithms to allow for easy reproduction. PMID:24806991

  6. Normal Myeloid Development Requires Both the Glutamine-Rich Transactivation Domain and the PEST Region of Transcription Factor PU.1 but Not the Potent Acidic Transactivation Domain

    PubMed Central

    Fisher, Robert C.; Olson, Marilyn C.; Pongubala, Jagan M. R.; Perkel, Jeffrey M.; Atchison, Michael L.; Scott, Edward W.; Simon, M. Celeste

    1998-01-01

    Gene targeting of transcription factor PU.1 results in an early block to fetal hematopoiesis, with no detectable lymphoid or myeloid cells produced in mouse embryos. Furthermore, PU.1−/− embryonic stem (ES) cells fail to differentiate into Mac-1+ and F4/80+ macrophages in vitro. We have previously shown that a PU.1 transgene under the control of its own promoter restores the ability of PU.1−/− ES cells to differentiate into macrophages. In this study, we take advantage of our PU.1−/− ES cell rescue system to genetically test which previously identified PU.1 functional domains are necessary for the development of mature macrophages. PU.1 functional domains include multiple N-terminal acidic and glutamine-rich transactivation domains, a PEST domain, several serine phosphorylation sites, and a C-terminal Ets DNA binding domain, all delineated and characterized by using standard biochemical and transactivational assays. By using the production of mature macrophages as a functional readout in our assay system, we have established that the glutamine-rich transactivation domain, a portion of the PEST domain, and the DNA binding domain are required for myelopoiesis. Deletion of three acidic domains, which exhibit potent transactivation potential in vitro, had no effect on the ability of PU.1 to promote macrophage development. Furthermore, mutagenesis of four independent sites of serine phosphorylation also had no effect on myelopoiesis. Collectively, our results indicate that PU.1 interacts with important regulatory proteins during macrophage development via the glutamine-rich and PEST domains. The PU.1−/− ES cell rescue system represents a powerful, in vitro strategy to functionally map domains of PU.1 essential for normal hematopoiesis and the generation of mature macrophages. PMID:9632818

  7. Identification of Ind transcription activation and repression domains required for dorsoventral patterning of the CNS.

    PubMed

    Von Ohlen, Tonia L; Moses, Cade

    2009-07-01

    Specification of cell fates across the dorsoventral axis of the central nervous system in Drosophila involves the subdivision of the neuroectoderm into three domains that give rise to three columns of neural precursor cells called neuroblasts. Ventral nervous system defective (Vnd), intermediate neuroblasts defective (Ind) and muscle segment homeobox (Msh) are expressed in the three columns from ventral to dorsal, respectively. The products of these genes play multiple important roles in formation and specification of the embryonic nervous system. Ind, for example, is known to play roles in two important processes. First, Ind is essential for formation of neuroblasts conjunction with SoxB class transcription factors. Sox class transcription factors are known to specify neural stem cells in vertebrates. Second, Ind plays an important role in patterning the CNS in conjunction with, vnd and msh, which is also similar to how vertebrates pattern their neural tube. This work focuses two important aspects of Ind function. First, we used multiple approaches to identify and characterize specific domains within the protein that confer repressor or activator ability. Currently, little is known about the presence of activation or repression domains within Ind. Here, we show that transcriptional repression by Ind requires multiple conserved domains within the protein, and that Ind has a transcriptional activation domain. Specifically, we have identified a novel domain, the Pst domain, that has transcriptional repression ability and appears to act independent of interaction with the co-repressor Groucho. This domain is highly conserved among insect species, but is not found in vertebrate Gsh class homeodomain proteins. Second, we show that Ind can and does repress vnd expression, but does so in a stage specific manner. We conclude from this that the function of Ind in regulating vnd expression is one of refinement and maintenance of the dorsal border. PMID:19348939

  8. Evolution of distinct EGF domains with specific functions

    PubMed Central

    Wouters, Merridee A.; Rigoutsos, Isidore; Chu, Carmen K.; Feng, Lina L.; Sparrow, Duncan B.; Dunwoodie, Sally L.

    2005-01-01

    EGF domains are extracellular protein modules cross-linked by three intradomain disulfides. Past studies suggest the existence of two types of EGF domain with three-disulfides, human EGF-like (hEGF) domains and complement C1r-like (cEGF) domains, but to date no functional information has been related to the two different types, and they are not differentiated in sequence or structure databases. We have developed new sequence patterns based on the different C-termini to search specifically for the two types of EGF domains in sequence databases. The exhibited sensitivity and specificity of the new pattern-based method represents a significant advancement over the currently available sequence detection techniques. We re-annotated EGF sequences in the latest release of Swiss-Prot looking for functional relationships that might correlate with EGF type. We show that important post-translational modifications of three-disulfide EGFs, including unusual forms of glycosylation and post-translational proteolytic processing, are dependent on EGF subtype. For example, EGF domains that are shed from the cell surface and mediate intercellular signaling are all hEGFs, as are all human EGF receptor family ligands. Additional experimental data suggest that functional specialization has accompanied subtype divergence. Based on our structural analysis of EGF domains with three-disulfide bonds and comparison to laminin and integrin-like EGF domains with an additional inter-domain disulfide, we propose that these hEGF and cEGF domains may have arisen from a four-disulfide ancestor by selective loss of different cysteine residues. PMID:15772310

  9. Functions and Requirements for the Transition Project

    SciTech Connect

    YANOCHKO, R.M.

    2000-04-24

    This document describes the functional requirement baseline for the Transition of 100 K Area Facilities Project (Transition Project). This baseline information consists of top-level functions, requirements, concept description, interface description, issues, and enabling assumptions.

  10. RING domain dimerization is essential for RNF4 function.

    PubMed

    Liew, Chu Wai; Sun, Huaiyu; Hunter, Tony; Day, Catherine L

    2010-10-01

    RNF4 [RING (really interesting new gene) finger protein 4] family ubiquitin ligases are RING E3 ligases that regulate the homoeostasis of SUMOylated proteins by promoting their ubiquitylation. In the present paper we report that the RING domain of RNF4 forms a stable dimer, and that dimerization is required for ubiquitin transfer. Our results suggest that the stability of the E2~ubiquitin thioester bond is regulated by RING domain dimerization. PMID:20681948

  11. CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation

    PubMed Central

    Narendra, Varun; Rocha, Pedro P.; An, Disi; Raviram, Ramya; Skok, Jane A.; Mazzoni, Esteban O.; Reinberg, Danny

    2015-01-01

    Polycomb and trithorax group proteins encode the epigenetic memory of cellular positional identity by establishing inheritable domains of repressive and active chromatin within the Hox clusters. Here, we demonstrate that the CCCTC-binding factor (CTCF) functions to insulate these adjacent yet antagonistic chromatin domains during embryonic stem cell differentiation into cervical motor neurons. Deletion of CTCF binding sites within the Hox clusters results in the expansion of active chromatin into the repressive domain. CTCF functions as an insulator by organizing Hox clusters into spatially disjoint domains. Ablation of CTCF binding disrupts topological boundaries such that caudal Hox genes leave the repressed domain and become subject to transcriptional activation. Hence, CTCF is required to insulate facultative heterochromatin from impinging euchromatin to produce discrete positional identities. PMID:25722416

  12. Intein Clustering Suggests Functional Importance in Different Domains of Life

    PubMed Central

    Novikova, Olga; Jayachandran, Pradeepa; Kelley, Danielle S.; Morton, Zachary; Merwin, Samantha; Topilina, Natalya I.; Belfort, Marlene

    2016-01-01

    Inteins, also called protein introns, are self-splicing mobile elements found in all domains of life. A bioinformatic survey of genomic data highlights a biased distribution of inteins among functional categories of proteins in both bacteria and archaea, with a strong preference for a single network of functions containing replisome proteins. Many nonorthologous, functionally equivalent replicative proteins in bacteria and archaea carry inteins, suggesting a selective retention of inteins in proteins of particular functions across domains of life. Inteins cluster not only in proteins with related roles but also in specific functional units of those proteins, like ATPase domains. This peculiar bias does not fully fit the models describing inteins exclusively as parasitic elements. In such models, evolutionary dynamics of inteins is viewed primarily through their mobility with the intein homing endonuclease (HEN) as the major factor of intein acquisition and loss. Although the HEN is essential for intein invasion and spread in populations, HEN dynamics does not explain the observed biased distribution of inteins among proteins in specific functional categories. We propose that the protein splicing domain of the intein can act as an environmental sensor that adapts to a particular niche and could increase the chance of the intein becoming fixed in a population. We argue that selective retention of some inteins might be beneficial under certain environmental stresses, to act as panic buttons that reversibly inhibit specific networks, consistent with the observed intein distribution. PMID:26609079

  13. Intein Clustering Suggests Functional Importance in Different Domains of Life.

    PubMed

    Novikova, Olga; Jayachandran, Pradeepa; Kelley, Danielle S; Morton, Zachary; Merwin, Samantha; Topilina, Natalya I; Belfort, Marlene

    2016-03-01

    Inteins, also called protein introns, are self-splicing mobile elements found in all domains of life. A bioinformatic survey of genomic data highlights a biased distribution of inteins among functional categories of proteins in both bacteria and archaea, with a strong preference for a single network of functions containing replisome proteins. Many nonorthologous, functionally equivalent replicative proteins in bacteria and archaea carry inteins, suggesting a selective retention of inteins in proteins of particular functions across domains of life. Inteins cluster not only in proteins with related roles but also in specific functional units of those proteins, like ATPase domains. This peculiar bias does not fully fit the models describing inteins exclusively as parasitic elements. In such models, evolutionary dynamics of inteins is viewed primarily through their mobility with the intein homing endonuclease (HEN) as the major factor of intein acquisition and loss. Although the HEN is essential for intein invasion and spread in populations, HEN dynamics does not explain the observed biased distribution of inteins among proteins in specific functional categories. We propose that the protein splicing domain of the intein can act as an environmental sensor that adapts to a particular niche and could increase the chance of the intein becoming fixed in a population. We argue that selective retention of some inteins might be beneficial under certain environmental stresses, to act as panic buttons that reversibly inhibit specific networks, consistent with the observed intein distribution. PMID:26609079

  14. Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins.

    PubMed Central

    Gillooly, D J; Simonsen, A; Stenmark, H

    2001-01-01

    PtdIns3P is a phosphoinositide 3-kinase product that has been strongly implicated in regulating membrane trafficking in both mammalian and yeast cells. PtdIns3P has been shown to be specifically located on membranes associated with the endocytic pathway. Proteins that contain FYVE zinc-finger domains are recruited to PtdIns3P-containing membranes. Structural information is now available concerning the interaction between FYVE domains and PtdIns3P. A number of proteins have been identified which contain a FYVE domain, and in this review we discuss the functions of PtdIns3P and its FYVE-domain-containing effector proteins in membrane trafficking, cytoskeletal regulation and receptor signalling. PMID:11284710

  15. Immunoglobulin-domain proteins required for maintenance of ventral nerve cord organization.

    PubMed

    Aurelio, Oscar; Hall, David H; Hobert, Oliver

    2002-01-25

    During development, neurons extend axons along defined routes to specific target cells. We show that additional mechanisms ensure that axons maintain their correct positioning in defined axonal tracts. After termination of axonal outgrowth and target recognition, axons in the ventral nerve cord (VNC) of Caenorhabditis elegans require the presence of a specific VNC neuron, PVT, to maintain their correct positioning in the left and right fascicles of the VNC. PVT may exert its stabilizing function by the temporally tightly controlled secretion of 2-immunoglobulin (Ig)-domain proteins encoded by the zig genes. Dedicated axon maintenance mechanisms may be widely used to ensure the preservation of functional neuronal circuitries. PMID:11809975

  16. Domain requirements for DNA unwinding by mycobacterial UvrD2, an essential DNA helicase.

    PubMed

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

    2008-09-01

    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

  17. An updated view on the structure and function of PYRIN domains

    PubMed Central

    Chu, Lan Hoang; Gangopadhyay, Anu; Dorfleutner, Andrea; Stehlik, Christian

    2014-01-01

    The PYRIN domain (PYD) is a protein-protein interaction domain, which belongs to the death domain fold (DDF) superfamily. It is best known for its signaling function in innate immune responses and particularly in the assembly of inflammasomes, which are large protein complexes that allow the induced proximity-mediated activation of caspase-1 and subsequently the release of pro-inflammatory cytokines. The molecular mechanism of inflammasome assembly was only recently elucidated and specifically requires PYD oligomerization. Here we discuss the recent advances in our understanding of PYD signaling and its regulation by PYD-only proteins. PMID:25451010

  18. Integral estimates for differentiable functions on irregular domains

    SciTech Connect

    Besov, Oleg V

    2011-02-11

    Integral representations for functions and their partial derivatives in terms of some fixed system of partial derivatives are constructed on irregular domains in a Euclidean space. Embedding theorems for Sobolev-type spaces into a Lebesgue space are established and the norms of the derivatives are estimated. Bibliography: 17 titles.

  19. Interdependence of the Rad50 hook and globular domain functions

    PubMed Central

    Hohl, Marcel; Kochańczyk, Tomasz; Tous, Cristina; Aguilera, Andrés; Krężel, Artur; Petrini, John H J

    2015-01-01

    SUMMARY Rad50 contains a conserved Zn2+ coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here we focused on rad50 mutations flanking the Zn2+-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end-joining, and DNA double strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled coil and globular ATPase domain, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions. PMID:25601756

  20. PROSITE, a protein domain database for functional characterization and annotation.

    PubMed

    Sigrist, Christian J A; Cerutti, Lorenzo; de Castro, Edouard; Langendijk-Genevaux, Petra S; Bulliard, Virginie; Bairoch, Amos; Hulo, Nicolas

    2010-01-01

    PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of these profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. PROSITE is largely used for the annotation of domain features of UniProtKB/Swiss-Prot entries. Among the 983 (DNA-binding) domains, repeats and zinc fingers present in Swiss-Prot (release 57.8 of 22 September 2009), 696 ( approximately 70%) are annotated with PROSITE descriptors using information from ProRule. In order to allow better functional characterization of domains, PROSITE developments focus on subfamily specific profiles and a new profile building method giving more weight to functionally important residues. Here, we describe AMSA, an annotated multiple sequence alignment format used to build a new generation of generalized profiles, the migration of ScanProsite to Vital-IT, a cluster of 633 CPUs, and the adoption of the Distributed Annotation System (DAS) to facilitate PROSITE data integration and interchange with other sources. The latest version of PROSITE (release 20.54, of 22 September 2009) contains 1308 patterns, 863 profiles and 869 ProRules. PROSITE is accessible at: http://www.expasy.org/prosite/. PMID:19858104

  1. PROSITE, a protein domain database for functional characterization and annotation

    PubMed Central

    Sigrist, Christian J. A.; Cerutti, Lorenzo; de Castro, Edouard; Langendijk-Genevaux, Petra S.; Bulliard, Virginie; Bairoch, Amos; Hulo, Nicolas

    2010-01-01

    PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of these profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. PROSITE is largely used for the annotation of domain features of UniProtKB/Swiss-Prot entries. Among the 983 (DNA-binding) domains, repeats and zinc fingers present in Swiss-Prot (release 57.8 of 22 September 2009), 696 (∼70%) are annotated with PROSITE descriptors using information from ProRule. In order to allow better functional characterization of domains, PROSITE developments focus on subfamily specific profiles and a new profile building method giving more weight to functionally important residues. Here, we describe AMSA, an annotated multiple sequence alignment format used to build a new generation of generalized profiles, the migration of ScanProsite to Vital-IT, a cluster of 633 CPUs, and the adoption of the Distributed Annotation System (DAS) to facilitate PROSITE data integration and interchange with other sources. The latest version of PROSITE (release 20.54, of 22 September 2009) contains 1308 patterns, 863 profiles and 869 ProRules. PROSITE is accessible at: http://www.expasy.org/prosite/. PMID:19858104

  2. Function of a Conserved Checkpoint Recruitment Domain in ATRIP Proteins▿

    PubMed Central

    Ball, Heather L.; Ehrhardt, Mark R.; Mordes, Daniel A.; Glick, Gloria G.; Chazin, Walter J.; Cortez, David

    2007-01-01

    The ATR (ATM and Rad3-related) kinase is essential to maintain genomic integrity. ATR is recruited to DNA lesions in part through its association with ATR-interacting protein (ATRIP), which in turn interacts with the single-stranded DNA binding protein RPA (replication protein A). In this study, a conserved checkpoint protein recruitment domain (CRD) in ATRIP orthologs was identified by biochemical mapping of the RPA binding site in combination with nuclear magnetic resonance, mutagenesis, and computational modeling. Mutations in the CRD of the Saccharomyces cerevisiae ATRIP ortholog Ddc2 disrupt the Ddc2-RPA interaction, prevent proper localization of Ddc2 to DNA breaks, sensitize yeast to DNA-damaging agents, and partially compromise checkpoint signaling. These data demonstrate that the CRD is critical for localization and optimal DNA damage responses. However, the stimulation of ATR kinase activity by binding of topoisomerase binding protein 1 (TopBP1) to ATRIP-ATR can occur independently of the interaction of ATRIP with RPA. Our results support the idea of a multistep model for ATR activation that requires separable localization and activation functions of ATRIP. PMID:17339343

  3. Mapping a kingdom-specific functional domain of squalene synthase.

    PubMed

    Linscott, Kristin B; Niehaus, Thomas D; Zhuang, Xun; Bell, Stephen A; Chappell, Joe

    2016-09-01

    Squalene synthase catalyzes the first committed step in sterol biosynthesis and consists of both an amino-terminal catalytic domain and a carboxy-terminal domain tethering the enzyme to the ER membrane. While the overall architecture of this enzyme is identical in eukaryotes, it was previously shown that plant and animal genes cannot complement a squalene synthase knockout mutation in yeast unless the carboxy-terminal domain is swapped for one of fungal origin. This implied a unique component of the fungal carboxy-terminal domain was responsible for the complementation phenotype. To identify this motif, we used Saccharomyces cerevisiae with a squalene synthase knockout mutation, and expressed intact and chimeric squalene synthases originating from fungi, plants, and animals. In contrast to previous observations, all enzymes tested could partially complement the knockout mutation when the genes were weakly expressed. However, when highly expressed, non-fungal squalene synthases could not complement the yeast mutation and instead led to the accumulation of a toxic intermediate(s) as defined by mutations of genes downstream in the ergosterol pathway. Restoration of the complete complementation phenotype was mapped to a 26-amino acid hinge region linking the catalytic and membrane-spanning domains specific to fungal squalene synthases. Over-expression of the C-terminal domain containing a hinge domain from fungi, not from animals or plants, led to growth inhibition of wild-type yeast. Because this hinge region is unique to and highly conserved within each kingdom of life, the data suggests that the hinge domain plays an essential functional role, such as assembly of ergosterol multi-enzyme complexes in fungi. PMID:27320012

  4. Recognition of additional roles for immunoglobulin domains in immune function

    PubMed Central

    Cannon, John P.; Dishaw, Larry J.; Haire, Robert N.; Litman, Ronda T.; Ostrov, David A.; Litman, Gary W.

    2010-01-01

    Characterization of immune receptors found in phylogenetically disparate species at the genetic, structural and functional levels has provided unique insight into the evolutionary acquisition of immune function. The roles of variable- and intermediate-type immunoglobulin (Ig) domains in direct recognition of ligands and other functions are far wider than previously anticipated. Common mechanisms of multigene family diversification and expansion as well as unique adaptations that relate to function continue to provide unique insight into the numerous patterns, processes and complex interactions that regulate the host response to infectious challenge. PMID:20004115

  5. Protein function annotation using protein domain family resources.

    PubMed

    Das, Sayoni; Orengo, Christine A

    2016-01-15

    As a result of the genome sequencing and structural genomics initiatives, we have a wealth of protein sequence and structural data. However, only about 1% of these proteins have experimental functional annotations. As a result, computational approaches that can predict protein functions are essential in bridging this widening annotation gap. This article reviews the current approaches of protein function prediction using structure and sequence based classification of protein domain family resources with a special focus on functional families in the CATH-Gene3D resource. PMID:26434392

  6. Independent Biological and Biochemical Functions for Individual Structural Domains of Drosophila Linker Histone H1.

    PubMed

    Kavi, Harsh; Emelyanov, Alexander V; Fyodorov, Dmitry V; Skoultchi, Arthur I

    2016-07-15

    Linker histone H1 is among the most abundant components of chromatin. H1 has profound effects on chromosome architecture. H1 also helps to tether DNA- and histone-modifying enzymes to chromatin. Metazoan linker histones have a conserved tripartite structure comprising N-terminal, globular, and long, unstructured C-terminal domains. Here we utilize truncated Drosophila H1 polypeptides in vitro and H1 mutant transgenes in vivo to interrogate the roles of these domains in multiple biochemical and biological activities of H1. We demonstrate that the globular domain and the proximal part of the C-terminal domain are essential for H1 deposition into chromosomes and for the stability of H1-chromatin binding. The two domains are also essential for fly viability and the establishment of a normal polytene chromosome structure. Additionally, through interaction with the heterochromatin-specific histone H3 Lys-9 methyltransferase Su(var)3-9, the H1 C-terminal domain makes important contributions to formation and H3K9 methylation of heterochromatin as well as silencing of transposons in heterochromatin. Surprisingly, the N-terminal domain does not appear to be required for any of these functions. However, it is involved in the formation of a single chromocenter in polytene chromosomes. In summary, we have discovered that linker histone H1, similar to core histones, exerts its multiple biological functions through independent, biochemically separable activities of its individual structural domains. PMID:27226620

  7. Two distinct domains of protein 4.1 critical for assembly of functional nuclei in vitro.

    PubMed

    Krauss, Sharon Wald; Heald, Rebecca; Lee, Gloria; Nunomura, Wataru; Gimm, J Aura; Mohandas, Narla; Chasis, Joel Anne

    2002-11-15

    Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4.1 is essential for nuclear assembly and identify two distinct 4.1 domains, initially characterized in cytoskeletal interactions, that have crucial and versatile functions in nuclear assembly. PMID:12171917

  8. The structure-function relationships in Drosophila neurotactin show that cholinesterasic domains may have adhesive properties.

    PubMed Central

    Darboux, I; Barthalay, Y; Piovant, M; Hipeau-Jacquotte, R

    1996-01-01

    Neurotactin (Nrt), a Drosophila transmembrane glycoprotein which is expressed in neuronal and epithelial tissues during embryonic and larval stages, exhibits heterophilic adhesive properties. The extracellular domain is composed of a catalytically inactive cholinesterase-like domain. A three-dimensional model deduced from the crystal structure of Torpedo acetylcholinesterase (AChE) has been constructed for Nrt and suggests that its extracellular domain is composed of two sub-domains organized around a gorge: an N-terminal region, whose three-dimensional structure is almost identical to that of Torpedo AChE, and a less conserved C-terminal region. By using truncated Nrt molecules and a homotypic cell aggregation assay which involves a soluble ligand activity, it has been possible to show that the adhesive function is localized in the N-terminal region of the extracellular domain comprised between His347 and His482. The C-terminal region of the protein can be removed without impairing Nrt adhesive properties, suggesting that the two sub-domains are structurally independent. Chimeric molecules in which the Nrt cholinesterase-like domain has been replaced by homologous domains from Drosophila AChE, Torpedo AChE or Drosophila glutactin (Glt), share similar adhesive properties. These properties may require the presence of Nrt cytoplasmic and transmembrane domains since authentic Drosophila AChE does not behave as an adhesive molecule when transfected in S2 cells. Images PMID:8890157

  9. The Pilus Usher Controls Protein Interactions via Domain Masking and is Functional as an Oligomer

    PubMed Central

    Werneburg, Glenn T.; Henderson, Nadine S.; Portnoy, Erica B.; Sarowar, Samema; Hultgren, Scott J.; Li, Huilin; Thanassi, David G.

    2015-01-01

    The chaperone-usher (CU) pathway assembles organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Biogenesis of pili by the CU pathway requires a periplasmic chaperone and an outer membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate binding site, but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which serves as a switch controlling usher activation. We demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions, and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria. PMID:26052892

  10. Identification of novel targets of MYC whose transcription requires the essential MbII domain.

    PubMed

    Zhang, Xiao-yong; DeSalle, Lauren M; McMahon, Steven B

    2006-02-01

    The MYC oncoprotein is among the most potent regulators of cell cycle progression and malignant transformation in human cells. Current models suggest that much of MYC's role in these processes is related to its ability to regulate the transcription of downstream target genes that encode the ultimate effector proteins. In addition to its carboxy-terminal DNA binding and dimerization domains, an enigmatic motif in the amino terminus termed MbII is required for all of MYC's biological activities. In spite of historical observations demonstrating the absolute requirement for MbII in these biological functions, clues implicating this domain in target gene transcription have only recently appeared. Based on this emerging link between MbII and transcriptional activation, we hypothesized that the identification of individual MYC targets whose transactivation requires MbII would help define the essential downstream effectors of MYC in transformation and cell cycle progression. In hopes of directly identifying new MbII-dependent MYC target genes, an expression profiling screen was conducted. This screen resulted in our identification of ten novel downstream targets of MYC. As a proof of principle, we recently demonstrated using RNAi-mediated depletion that one of these targets, the metastasis regulator MTA1, is absolutely required for MYC mediated transformation. Here we report the identity of these previously uncharacterized MYC targets and discuss their potential roles in MYC function. In addition, we attempt to reconcile the historical and contemporary evidence linking MbII to transcriptional activation. PMID:16434883

  11. Individual Carboxypeptidase D domains have both redundant and unique functions in Drosophila development and behavior

    PubMed Central

    Sidyelyeva, Galyna; Wegener, Christian; Schoenfeld, Brian P.; Bell, Aaron J.; Baker, Nicholas E.; McBride, Sean M. J.; Fricker, Lloyd D.

    2010-01-01

    Metallocarboxypeptidase D (CPD) functions in protein and peptide processing. The Drosophila CPD svr gene undergoes alternative splicing, producing forms containing 1–3 active or inactive CP domains. To investigate the function of the various CP domains, we created transgenic flies expressing specific forms of CPD in the embryonic-lethal svrPG33 mutant. All constructs containing an active CP domain rescued the lethality with varying degrees, and full viability required inactive CP domain-3. Transgenic flies overexpressing active CP domain-1 or -2 were similar to each other and to the viable svr mutants, with pointed wing shape, enhanced ethanol sensitivity, and decreased cold sensitivity. The transgenes fully compensated for a long-term memory deficit observed in the viable svr mutants. Overexpression of CP domain-1 or -2 reduced the levels of Lys/Arg-extended adipokinetic hormone intermediates. These findings suggest that CPD domains-1 and -2 have largely redundant functions in the processing of growth factors, hormones, and neuropeptides. PMID:20386952

  12. Functional and topological diversity of LOV domain photoreceptors

    PubMed Central

    Glantz, Spencer T.; Carpenter, Eric J.; Melkonian, Michael; Boyden, Edward S.; Wong, Gane Ka-Shu; Chow, Brian Y.

    2016-01-01

    Light–oxygen–voltage sensitive (LOV) flavoproteins are ubiquitous photoreceptors that mediate responses to environmental cues. Photosensory inputs are transduced into signaling outputs via structural rearrangements in sensor domains that consequently modulate the activity of an effector domain or multidomain clusters. Establishing the diversity in effector function and sensor–effector topology will inform what signaling mechanisms govern light-responsive behaviors across multiple kingdoms of life and how these signals are transduced. Here, we report the bioinformatics identification of over 6,700 candidate LOV domains (including over 4,000 previously unidentified sequences from plants and protists), and insights from their annotations for ontological function and structural arrangements. Motif analysis identified the sensors from ∼42 million ORFs, with strong statistical separation from other flavoproteins and non-LOV members of the structurally related Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family. Conserved-domain analysis determined putative light-regulated function and multidomain topologies. We found that for certain effectors, sensor–effector linker length is discretized based on both phylogeny and the preservation of α-helical heptad repeats within an extended coiled-coil linker structure. This finding suggests that preserving sensor–effector orientation is a key determinant of linker length, in addition to ancestry, in LOV signaling structure–function. We found a surprisingly high prevalence of effectors with functions previously thought to be rare among LOV proteins, such as regulators of G protein signaling, and discovered several previously unidentified effectors, such as lipases. This work highlights the value of applying genomic and transcriptomic technologies to diverse organisms to capture the structural and functional variation in photosensory proteins that are vastly important in adaptation, photobiology, and

  13. Functional and topological diversity of LOV domain photoreceptors.

    PubMed

    Glantz, Spencer T; Carpenter, Eric J; Melkonian, Michael; Gardner, Kevin H; Boyden, Edward S; Wong, Gane Ka-Shu; Chow, Brian Y

    2016-03-15

    Light-oxygen-voltage sensitive (LOV) flavoproteins are ubiquitous photoreceptors that mediate responses to environmental cues. Photosensory inputs are transduced into signaling outputs via structural rearrangements in sensor domains that consequently modulate the activity of an effector domain or multidomain clusters. Establishing the diversity in effector function and sensor-effector topology will inform what signaling mechanisms govern light-responsive behaviors across multiple kingdoms of life and how these signals are transduced. Here, we report the bioinformatics identification of over 6,700 candidate LOV domains (including over 4,000 previously unidentified sequences from plants and protists), and insights from their annotations for ontological function and structural arrangements. Motif analysis identified the sensors from ∼42 million ORFs, with strong statistical separation from other flavoproteins and non-LOV members of the structurally related Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family. Conserved-domain analysis determined putative light-regulated function and multidomain topologies. We found that for certain effectors, sensor-effector linker length is discretized based on both phylogeny and the preservation of α-helical heptad repeats within an extended coiled-coil linker structure. This finding suggests that preserving sensor-effector orientation is a key determinant of linker length, in addition to ancestry, in LOV signaling structure-function. We found a surprisingly high prevalence of effectors with functions previously thought to be rare among LOV proteins, such as regulators of G protein signaling, and discovered several previously unidentified effectors, such as lipases. This work highlights the value of applying genomic and transcriptomic technologies to diverse organisms to capture the structural and functional variation in photosensory proteins that are vastly important in adaptation, photobiology, and optogenetics

  14. LIM Homeobox Domain 2 Is Required for Corneal Epithelial Homeostasis

    PubMed Central

    Sartaj, Rachel; Chee, Ru‐ik; Yang, Jing; Wan, Pengxia; Liu, Aihong; Guaiquil, Victor; Fuchs, Elaine

    2016-01-01

    Abstract The cornea requires constant epithelial renewal to maintain clarity for appropriate vision. A subset of stem cells residing at the limbus is primarily responsible for maintaining corneal epithelium homeostasis. Trauma and disease may lead to stem cell deficiency and therapeutic targeting to replenish the stemness capacity has been stalled by the lack of reliable corneal epithelial stem cell markers. Here we identified the location of Lhx2 in mice (mLhx2) cornea and conjunctival tissue using an Lhx2eGFP reporter model and in human tissues (hLHX2). Lhx2 localized to the basal cells of central cornea, the conjunctiva and the entire limbal epithelium in humans and mice. To ascribe a functional role we generated Lhx2 conditional knockout (cKO) mice and the phenotypic effects in corneas were analyzed by slit lamp microscopy, in cell‐based assays and in a model of corneal epithelium debridement. Immunodetection on corneal sections were used to visualize conjunctivalization, a sign of limbal barrier failure. Lhx2cKO mice produced reduced body hair and spontaneous epithelial defects in the cornea that included neovascularization, perforation with formation of scar tissue and opacification. Cell based assays showed that Lhx2cKO derived corneal epithelial cells have a significantly lower capacity to form colonies over time and delayed wound‐healing recovery when compared to wildtype cells. Repeated corneal epithelial wounding resulted in decreased re‐epithelialization and multiple cornea lesions in Lhx2cKO mice compared to normal recovery seen in wildtype mice. We conclude that Lhx2 is required for maintenance of the corneal epithelial cell compartment and the limbal barrier. Stem Cells 2016;34:493–503 PMID:26661907

  15. LIM Homeobox Domain 2 Is Required for Corneal Epithelial Homeostasis.

    PubMed

    Sartaj, Rachel; Chee, Ru-ik; Yang, Jing; Wan, Pengxia; Liu, Aihong; Guaiquil, Victor; Fuchs, Elaine; Rosenblatt, Mark I

    2016-02-01

    The cornea requires constant epithelial renewal to maintain clarity for appropriate vision. A subset of stem cells residing at the limbus is primarily responsible for maintaining corneal epithelium homeostasis. Trauma and disease may lead to stem cell deficiency and therapeutic targeting to replenish the stemness capacity has been stalled by the lack of reliable corneal epithelial stem cell markers. Here we identified the location of Lhx2 in mice (mLhx2) cornea and conjunctival tissue using an Lhx2eGFP reporter model and in human tissues (hLHX2). Lhx2 localized to the basal cells of central cornea, the conjunctiva and the entire limbal epithelium in humans and mice. To ascribe a functional role we generated Lhx2 conditional knockout (cKO) mice and the phenotypic effects in corneas were analyzed by slit lamp microscopy, in cell-based assays and in a model of corneal epithelium debridement. Immunodetection on corneal sections were used to visualize conjunctivalization, a sign of limbal barrier failure. Lhx2cKO mice produced reduced body hair and spontaneous epithelial defects in the cornea that included neovascularization, perforation with formation of scar tissue and opacification. Cell based assays showed that Lhx2cKO derived corneal epithelial cells have a significantly lower capacity to form colonies over time and delayed wound-healing recovery when compared to wildtype cells. Repeated corneal epithelial wounding resulted in decreased re-epithelialization and multiple cornea lesions in Lhx2cKO mice compared to normal recovery seen in wildtype mice. We conclude that Lhx2 is required for maintenance of the corneal epithelial cell compartment and the limbal barrier. PMID:26661907

  16. 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.

  17. The IRBIT domain adds new functions to the AHCY family.

    PubMed

    Devogelaere, Benoit; Sammels, Eva; De Smedt, Humbert

    2008-07-01

    During the past few years, the IRBIT domain has emerged as an important add-on of S-adenosyl-L-homocystein hydrolase (AHCY), thereby creating the new family of AHCY-like proteins. In this review, we discuss the currently available data on this new family of proteins. We describe the IRBIT domain as a unique part of these proteins and give an overview of its regulation via (de)phosphorylation and proteolysis. The second part of this review is focused on the potential functions of the AHCY-like proteins. We propose that the IRBIT domain serves as an anchor for targeting AHCY-like proteins towards cytoplasmic targets. This leads to regulation of (i) intracellular Ca2+ via the inositol 1,4,5-trisphosphate receptor (IP3R), (ii) intracellular pH via the Na+/HCO3 - cotransporters (NBCs); whereas inactivation of the IRBIT domain induces (iii) nuclear translocation and regulation of AHCY activity. Dysfunction of AHCY-like proteins will disturb these three important functions, with various biological implications. PMID:18536033

  18. Evolutionary relationships among Rel domains indicate functional diversification by recombination.

    PubMed

    Graef, I A; Gastier, J M; Francke, U; Crabtree, G R

    2001-05-01

    The recent sequencing of several complete genomes has made it possible to track the evolution of large gene families by their genomic structure. Following the large-scale association of exons encoding domains with well defined functions in invertebrates could be useful in predicting the function of complex multidomain proteins in mammals produced by accretion of domains. With this objective, we have determined the genomic structure of the 14 genes in invertebrates and vertebrates that contain rel domains. The sequence encoding the rel domain is defined by intronic boundaries and has been recombined with at least three structurally and functionally distinct genomic sequences to generate coding sequences for: (i) the rel/Dorsal/NFkappaB proteins that are retained in the cytoplasm by IkB-like proteins; (ii) the NFATc proteins that sense calcium signals and undergo cytoplasmic-to-nuclear translocation in response to dephosphorylation by calcineurin; and (iii) the TonEBP tonicity-responsive proteins. Remarkably, a single exon in each NFATc family member encodes the entire Ca(2+)/calcineurin sensing region, including nuclear import/export, calcineurin-binding, and substrate regions. The Rel/Dorsal proteins and the TonEBP proteins are present in Drosophila but not Caenorhabditis elegans. On the other hand, the calcium-responsive NFATc proteins are present only in vertebrates, suggesting that the NFATc family is dedicated to functions specific to vertebrates such as a recombinational immune response, cardiovascular development, and vertebrate-specific aspects of the development and function of the nervous system. PMID:11344309

  19. A Naturally-Occurring Transcript Variant of MARCO Reveals the SRCR Domain is Critical for Function

    PubMed Central

    Novakowski, Kyle E.; Huynh, Angela; Han, SeongJun; Dorrington, Michael G.; Yin, Charles; Tu, Zhongyuan; Pelka, Peter; Whyte, Peter; Guarné, Alba; Sakamoto, Kaori; Bowdish, Dawn M.E.

    2016-01-01

    Macrophage receptor with collagenous structure (MARCO) is a Class A Scavenger Receptor (cA-SR) that recognizes and phagocytoses of a wide variety of pathogens. Most cA-SRs that contain a C-terminal Scavenger Receptor Cysteine Rich (SRCR) domain use the proximal collagenous domain to bind ligands. In contrast, for the role of the SRCR domain of MARCO in phagocytosis, adhesion and pro-inflammatory signalling is less clear. The discovery of a naturally-occurring transcript variant lacking the SRCR domain, MARCOII, provided the opportunity to study the role of the SRCR domain of MARCO. We tested whether the SRCR domain is required for ligand binding, promoting downstream signalling, and enhancing cellular adhesion. Unlike cells expressing full-length MARCO, ligand binding was abolished in MARCOII-expressing cells. Furthermore, co-expression of MARCO and MARCOII impaired phagocytic function, indicating that MARCOII acts as a dominant negative variant. Unlike MARCO, expression of MARCOII did not enhance Toll-Like Receptor 2 (TLR2)-mediated pro-inflammatory signalling in response to bacterial stimulation. MARCO-expressing cells were more adherent and exhibited a dendritic-like phenotype, while MARCOII-expressing cells were less adherent and did not exhibit changes in morphology. These data suggest the SRCR domain of MARCO is the key domain in modulating ligand binding, enhancing downstream pro-inflammatory signalling, and MARCO-mediated cellular adhesion. PMID:26888252

  20. The Drosophila fork head domain protein crocodile is required for the establishment of head structures.

    PubMed Central

    Häcker, U; Kaufmann, E; Hartmann, C; Jürgens, G; Knöchel, W; Jäckle, H

    1995-01-01

    The fork head (fkh) domain defines the DNA-binding region of a family of transcription factors which has been implicated in regulating cell fate decisions across species lines. We have cloned and molecularly characterized the crocodile (croc) gene which encodes a new family member from Drosophila. croc is expressed in the head anlagen of the blastoderm embryo under the control of the anterior, the dorsoventral and the terminal maternal organizer systems. The croc mutant phenotype indicates that the croc wild-type gene is required to function as an early patterning gene in the anterior-most blastoderm head segment anlage and for the establishment of a specific head skeletal structure that derives from the non-adjacent intercalary segment at a later stage of embryogenesis. As an early patterning gene, croc exerts unusual properties which do not allow it to be grouped among the established segmentation genes. A single-site mutation within the croc fkh domain, which causes a replacement of the first out of four conserved amino acid residues thought to be involved in the coordinate binding of Mg2+, abolishes the DNA binding of the protein in vitro. In view of the resulting lack-of-function mutant phenotype, it appears likely that metal binding by the affected region of the fkh domain is crucial for proper folding of the DNA-binding structure. Images PMID:7489720

  1. Photonic-crystal time-domain simulations using Wannier functions.

    PubMed

    Blum, Christian; Wolff, Christian; Busch, Kurt

    2011-01-15

    We present a Wannier-function-based time-domain method for photonic-crystal integrated optical circuits. In contrast to other approaches, this method allows one to trade CPU time against memory consumption and therefore is particularly well suited for the treatment of large-scale systems. As an illustration, we apply the method to the design of a photonic-crystal-based sensor, which utilizes a dual Mach-Zehnder-Fano interferometer. PMID:21263535

  2. Factorized domain wall partition functions in trigonometric vertex models

    NASA Astrophysics Data System (ADS)

    Foda, O.; Wheeler, M.; Zuparic, M.

    2007-10-01

    We obtain factorized domain wall partition functions for two sets of trigonometric vertex models: (1) the N-state Deguchi Akutsu models, for N \\in \\{2, 3, 4\\} (and conjecture the result for all N>=5), and (2) the sl(r+1|s+1) Perk Schultz models, for \\{r, s \\in \\mathbb {N}\\} , where (given the symmetries of these models) the result is independent of {r,s}.

  3. Crystal Structure of CRN-4: Implications for Domain Function in Apoptotic DNA Degradation▿

    PubMed Central

    Hsiao, Yu-Yuan; Nakagawa, Akihisa; Shi, Zhonghao; Mitani, Shohei; Xue, Ding; Yuan, Hanna S.

    2009-01-01

    Cell death related nuclease 4 (CRN-4) is one of the apoptotic nucleases involved in DNA degradation in Caenorhabditis elegans. To understand how CRN-4 is involved in apoptotic DNA fragmentation, we analyzed CRN-4's biochemical properties, in vivo cell functions, and the crystal structures of CRN-4 in apo-form, Mn2+-bound active form, and Er3+-bound inactive form. CRN-4 is a dimeric nuclease with the optimal enzyme activity in cleaving double-stranded DNA in apoptotic salt conditions. Both mutational studies and the structures of the Mn2+-bound CRN-4 revealed the geometry of the functional nuclease active site in the N-terminal DEDDh domain. The C-terminal domain, termed the Zn-domain, contains basic surface residues ideal for nucleic acid recognition and is involved in DNA binding, as confirmed by deletion assays. Cell death analysis in C. elegans further demonstrated that both the nuclease active site and the Zn-domain are required for crn-4's function in apoptosis. Combining all of the data, we suggest a structural model where chromosomal DNA is bound at the Zn-domain and cleaved at the DEDDh nuclease domain in CRN-4 when the cell is undergoing apoptosis. PMID:18981218

  4. Rules and Norms: Requirements for Rule Interchange Languages in the Legal Domain

    NASA Astrophysics Data System (ADS)

    Gordon, Thomas F.; Governatori, Guido; Rotolo, Antonino

    In this survey paper we summarize the requirements for rule interchange languages for applications in the legal domain and use these requirements to evaluate RuleML, SBVR, SWRL and RIF. We also present the Legal Knowledge Interchange Format (LKIF), a new rule interchange format developed specifically for applications in the legal domain.

  5. Dual amyloid domains promote differential functioning of the chaplin proteins during Streptomyces aerial morphogenesis

    PubMed Central

    Capstick, David S.; Jomaa, Ahmad; Hanke, Chistopher; Ortega, Joaquin; Elliot, Marie A.

    2011-01-01

    The chaplin proteins are functional amyloids found in the filamentous Streptomyces bacteria. These secreted proteins are required for the aerial development of Streptomyces coelicolor, and contribute to an intricate rodlet ultrastructure that decorates the surfaces of aerial hyphae and spores. S. coelicolor encodes eight chaplin proteins. Previous studies have revealed that only three of these proteins (ChpC, ChpE, and ChpH) are necessary for promoting aerial development, and of these three, ChpH is the primary developmental determinant. Here, we show that the model chaplin, ChpH, contains two amyloidogenic domains: one in the N terminus and one in the C terminus of the mature protein. These domains have different polymerization properties as determined using fluorescence spectroscopy, secondary structure analyses, and electron microscopy. We coupled these in vitro assays with in vivo genetic studies to probe the connection between ChpH amyloidogenesis and its biological function. Using mutational analyses, we demonstrated that both N- and C-terminal amyloid domains of ChpH were required for promoting aerial hypha formation, while the N-terminal domain was dispensable for assembly of the rodlet ultrastructure. These results suggest that there is a functional differentiation of the dual amyloid domains in the chaplin proteins. PMID:21628577

  6. The CA domain of the respiratory complex I is required for normal embryogenesis in Arabidopsis thaliana.

    PubMed

    Córdoba, Juan Pablo; Marchetti, Fernanda; Soto, Débora; Martin, María Victoria; Pagnussat, Gabriela Carolina; Zabaleta, Eduardo

    2016-03-01

    The NADH-ubiquinone oxidoreductase [complex I (CI), EC 1.6.5.3] of the mitochondrial respiratory chain is the principal entry point of electrons, and vital in maintaining metabolism and the redox balance. In a variety of eukaryotic organisms, except animal and fungi (Opisthokonta), it contains an extra domain composed of putative gamma carbonic anhydrases subunits, named the CA domain, which was proposed to be essential for complex I assembly. There are two kinds of carbonic anhydrase subunits: CAs (of which there are three) and carbonic anhydrase-like proteins (CALs) (of which there are two). In plants, the CA domain has been linked to photorespiration. In this work, we report that Arabidopsis mutant plants affected in two specific CA subunits show a lethal phenotype. Double homozygous knockouts ca1ca2 embryos show a significant developmental delay compared to the non-homozygous embryos, which show a wild-type (WT) phenotype in the same silique. Mutant embryos show impaired mitochondrial membrane potential and mitochondrial reactive oxygen species (ROS) accumulation. The characteristic embryo greening does not take place and fewer but larger oil bodies are present. Although seeds look dark brown and wrinkled, they are able to germinate 12 d later than WT seeds. However, they die immediately, most likely due to oxidative stress.Since the CA domain is required for complex I biogenesis, it is predicted that in ca1ca2 mutants no complex I could be formed, triggering the lethal phenotype. The in vivo composition of a functional CA domain is proposed. PMID:26721503

  7. The transactivation domain AF-2 but not the DNA-binding domain of the estrogen receptor is required to inhibit differentiation of avian erythroid progenitors.

    PubMed

    von Lindern, M; Boer, L; Wessely, O; Parker, M; Beug, H

    1998-02-01

    Earlier work demonstrated that an activated estrogen receptor (ER) is required for long-term self-renewal of c-ErbB-expressing avian erythroid progenitors. Here, we demonstrate that activation of the ER does not only arrest or retard differentiation of early progenitors but that it affects erythroid differentiation at all stages of erythroid maturation. A search for genes whose expression is affected by the ER showed that the 17beta-estradiol-activated receptor suppressed the differentiation-associated up-regulation of Gata-1, SCL-1, and globin genes in partially mature cells. In the same cells, the expression of carbonic anhydrase II (CAII) and histone H5 was enhanced. This led to premature expression of CAII, a possible explanation for the toxic effects of overexpressed ER. Repression specifically required the transactivation domain AF-2, but neither an intact DNA-binding domain (DBD) nor the AF-1 domain. The transcriptional activation of CAII, however, required both an intact AF-2 and a functional DBD. The requirement for the AF-2, but not the DBD, suggested that the ER may compete with other nuclear hormone receptors for transcriptional coactivators that bind AF-2, a domain well conserved within this family of transcription factors. We show, however, that this model does not apply for the most likely candidate, the avian thyroid hormone receptor. PMID:9482667

  8. Phenotypic lentivirus screens to identify functional single domain antibodies.

    PubMed

    Schmidt, Florian I; Hanke, Leo; Morin, Benjamin; Brewer, Rebeccah; Brusic, Vesna; Whelan, Sean P J; Ploegh, Hidde L

    2016-01-01

    Manipulation of proteins is key in assessing their in vivo function. Although genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we have developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway. PMID:27573105

  9. The RNA recognition motif domains of RBM5 are required for RNA binding and cancer cell proliferation inhibition

    SciTech Connect

    Zhang, Lei; Zhang, Qing; Yang, Yu; Wu, Chuanfang

    2014-02-14

    Highlights: • RNA recognition motif domains of RBM5 are essential for cell proliferation inhibition. • RNA recognition motif domains of RBM5 are essential for apoptosis induction. • RNA recognition motif domains of RBM5 are essential for RNA binding. • RNA recognition motif domains of RBM5 are essential for caspase-2 alternative splicing. - Abstract: RBM5 is a known putative tumor suppressor gene that has been shown to function in cell growth inhibition by modulating apoptosis. RBM5 also plays a critical role in alternative splicing as an RNA binding protein. However, it is still unclear which domains of RBM5 are required for RNA binding and related functional activities. We hypothesized the two putative RNA recognition motif (RRM) domains of RBM5 spanning from amino acids 98–178 and 231–315 are essential for RBM5-mediated cell growth inhibition, apoptosis regulation, and RNA binding. To investigate this hypothesis, we evaluated the activities of the wide-type and mutant RBM5 gene transfer in low-RBM5 expressing A549 cells. We found that, unlike wild-type RBM5 (RBM5-wt), a RBM5 mutant lacking the two RRM domains (RBM5-ΔRRM), is unable to bind RNA, has compromised caspase-2 alternative splicing activity, lacks cell proliferation inhibition and apoptosis induction function in A549 cells. These data provide direct evidence that the two RRM domains of RBM5 are required for RNA binding and the RNA binding activity of RBM5 contributes to its function on apoptosis induction and cell growth inhibition.

  10. Functional Significance of SRJ Domain Mutations in CITED2

    PubMed Central

    Cosgrove, Catherine; Braganca, Jose; Cuenda, Ana; Bamforth, Simon D.; Schneider, Jürgen E.; Watkins, Hugh; Keavney, Bernard; Davies, Benjamin; Bhattacharya, Shoumo

    2012-01-01

    CITED2 is a transcriptional co-activator with 3 conserved domains shared with other CITED family members and a unique Serine-Glycine Rich Junction (SRJ) that is highly conserved in placental mammals. Loss of Cited2 in mice results in cardiac and aortic arch malformations, adrenal agenesis, neural tube and placental defects, and partially penetrant defects in left-right patterning. By screening 1126 sporadic congenital heart disease (CHD) cases and 1227 controls, we identified 19 variants, including 5 unique non-synonymous sequence variations (N62S, R92G, T166N, G180-A187del and A187T) in patients. Many of the CHD-specific variants identified in this and previous studies cluster in the SRJ domain. Transient transfection experiments show that T166N mutation impairs TFAP2 co-activation function and ES cell proliferation. We find that CITED2 is phosphorylated by MAPK1 in vitro at T166, and that MAPK1 activation enhances the coactivation function of CITED2 but not of CITED2-T166N. In order to investigate the functional significance in vivo, we generated a T166N mutation of mouse Cited2. We also used PhiC31 integrase-mediated cassette exchange to generate a Cited2 knock-in allele replacing the mouse Cited2 coding sequence with human CITED2 and with a mutant form deleting the entire SRJ domain. Mouse embryos expressing only CITED2-T166N or CITED2-SRJ-deleted alleles surprisingly show no morphological abnormalities, and mice are viable and fertile. These results indicate that the SRJ domain is dispensable for these functions of CITED2 in mice and that mutations clustering in the SRJ region are unlikely to be the sole cause of the malformations observed in patients with sporadic CHD. Our results also suggest that coding sequence mutations observed in case-control studies need validation using in vivo models and that predictions based on structural conservation and in vitro functional assays, or even in vivo global loss of function models, may be insufficient. PMID:23082118

  11. Domain function dissection and catalytic properties of Listeria monocytogenes p60 protein with bacteriolytic activity.

    PubMed

    Yu, Minfeng; Zuo, Jinrong; Gu, Hao; Guo, Minliang; Yin, Yuelan

    2015-12-01

    The major extracellular protein p60 of Listeria monocytogenes (Lm-p60) is an autolysin that can hydrolyze the peptidoglycan of bacterial cell wall and has been shown to be required for L. monocytogenes virulence. The predicted three-dimensional structure of Lm-p60 showed that Lm-p60 could be split into two independent structural domains at the amino acid residue 270. Conserved motif analysis showed that V30, D207, S395, and H444 are the key amino acid residues of the corresponding motifs. However, not only the actual functions of these two domains but also the catalytic properties of Lm-p60 are unclear. We try to express recombinant Lm-p60 and identify the functions of two domains by residue substitution (V30A, D207A, S395A, and H444A) and peptide truncation. The C-terminal domain was identified as catalytic element and N-terminal domain as substrate recognition and binding element. Either N-terminal domain truncation or C-terminal domain truncation presents corresponding biological activity. The catalytic activity of Lm-p60 with a malfunctioned substrate-binding domain was decreased, while the substrate binding was not affected by a mulfunctioned catalytic domain. With turbidimetric method, we determined the optimal conditions for the bacteriolytic activity of Lm-p60 against Micrococcus lysodeikficus. The assay for the effect of Lm-p60 on the bacteriolytic activity of lysozyme revealed that the combined use of Lm-p60 protein with lysozyme showed a strong synergistic effect on the bacteriolytic activity. PMID:26363556

  12. Phylogenomic and functional domain analysis of polyketide synthases in Fusarium

    SciTech Connect

    Brown, Daren W.; Butchko, Robert A.; Baker, Scott E.; Proctor, Robert H.

    2012-02-01

    Fusarium species are ubiquitous in nature, cause a range of plant diseases, and produce a variety of chemicals often referred to as secondary metabolites. Although some fungal secondary metabolites affect plant growth or protect plants from other fungi and bacteria, their presence in grain based food and feed is more often associated with a variety of diseases in plants and in animals. Many of these structurally diverse metabolites are derived from a family of related enzymes called polyketide synthases (PKSs). A search of genomic sequence of Fusarium verticillioides, F. graminearum, F. oxysporum and Nectria haematococca (anamorph F. solani) identified a total of 58 PKS genes. To gain insight into how this gene family evolved and to guide future studies, we conducted a phylogenomic and functional domain analysis. The resulting genealogy suggested that Fusarium PKSs represent 34 different groups responsible for synthesis of different core metabolites. The analyses indicate that variation in the Fusarium PKS gene family is due to gene duplication and loss events as well as enzyme gain-of-function due to the acquisition of new domains or of loss-of-function due to nucleotide mutations. Transcriptional analysis indicate that the 16 F. verticillioides PKS genes are expressed under a range of conditions, further evidence that they are functional genes that confer the ability to produce secondary metabolites.

  13. Human Systems Integration: Requirements and Functional Decomposition

    NASA Technical Reports Server (NTRS)

    Berson, Barry; Gershzohn, Gary; Boltz, Laura; Wolf, Russ; Schultz, Mike

    2005-01-01

    This deliverable was intended as an input to the Access 5 Policy and Simulation Integrated Product Teams. This document contains high-level pilot functionality for operations in the National Airspace System above FL430. Based on the derived pilot functions the associated pilot information and control requirements are given.

  14. Functional classification of CATH superfamilies: a domain-based approach for protein function annotation

    PubMed Central

    Das, Sayoni; Lee, David; Sillitoe, Ian; Dawson, Natalie L.; Lees, Jonathan G.; Orengo, Christine A.

    2015-01-01

    Motivation: Computational approaches that can predict protein functions are essential to bridge the widening function annotation gap especially since <1.0% of all proteins in UniProtKB have been experimentally characterized. We present a domain-based method for protein function classification and prediction of functional sites that exploits functional sub-classification of CATH superfamilies. The superfamilies are sub-classified into functional families (FunFams) using a hierarchical clustering algorithm supervised by a new classification method, FunFHMMer. Results: FunFHMMer generates more functionally coherent groupings of protein sequences than other domain-based protein classifications. This has been validated using known functional information. The conserved positions predicted by the FunFams are also found to be enriched in known functional residues. Moreover, the functional annotations provided by the FunFams are found to be more precise than other domain-based resources. FunFHMMer currently identifies 110 439 FunFams in 2735 superfamilies which can be used to functionally annotate > 16 million domain sequences. Availability and implementation: All FunFam annotation data are made available through the CATH webpages (http://www.cathdb.info). The FunFHMMer webserver (http://www.cathdb.info/search/by_funfhmmer) allows users to submit query sequences for assignment to a CATH FunFam. Contact: sayoni.das.12@ucl.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26139634

  15. Infected cell protein 0 functional domains and their coordination in herpes simplex virus replication.

    PubMed

    Gu, Haidong

    2016-02-12

    Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen that establishes latent infection in ganglia neurons. Its unique life cycle requires a balanced "conquer and compromise" strategy to deal with the host anti-viral defenses. One of HSV-1 α (immediate early) gene products, infected cell protein 0 (ICP0), is a multifunctional protein that interacts with and modulates a wide range of cellular defensive pathways. These pathways may locate in different cell compartments, which then migrate or exchange factors upon stimulation, for the purpose of a concerted and effective defense. ICP0 is able to simultaneously attack multiple host pathways by either degrading key restrictive factors or modifying repressive complexes. This is a viral protein that contains an E3 ubiquitin ligase, translocates among different cell compartments and interacts with major defensive complexes. The multiple functional domains of ICP0 can work independently and at the same time coordinate with each other. Dissecting the functional domains of ICP0 and delineating the coordination of these domains will help us understand HSV-1 pathogenicity as well as host defense mechanisms. This article focuses on describing individual ICP0 domains, their biochemical properties and their implication in HSV-1 infection. By putting individual domain functions back into the picture of host anti-viral defense network, this review seeks to elaborate the complex interactions between HSV-1 and its host. PMID:26870669

  16. Infected cell protein 0 functional domains and their coordination in herpes simplex virus replication

    PubMed Central

    Gu, Haidong

    2016-01-01

    Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen that establishes latent infection in ganglia neurons. Its unique life cycle requires a balanced “conquer and compromise” strategy to deal with the host anti-viral defenses. One of HSV-1 α (immediate early) gene products, infected cell protein 0 (ICP0), is a multifunctional protein that interacts with and modulates a wide range of cellular defensive pathways. These pathways may locate in different cell compartments, which then migrate or exchange factors upon stimulation, for the purpose of a concerted and effective defense. ICP0 is able to simultaneously attack multiple host pathways by either degrading key restrictive factors or modifying repressive complexes. This is a viral protein that contains an E3 ubiquitin ligase, translocates among different cell compartments and interacts with major defensive complexes. The multiple functional domains of ICP0 can work independently and at the same time coordinate with each other. Dissecting the functional domains of ICP0 and delineating the coordination of these domains will help us understand HSV-1 pathogenicity as well as host defense mechanisms. This article focuses on describing individual ICP0 domains, their biochemical properties and their implication in HSV-1 infection. By putting individual domain functions back into the picture of host anti-viral defense network, this review seeks to elaborate the complex interactions between HSV-1 and its host. PMID:26870669

  17. Both tails and the centromere targeting domain of CENP-A are required for centromere establishment

    PubMed Central

    Logsdon, Glennis A.; Barrey, Evelyne J.; Bassett, Emily A.; DeNizio, Jamie E.; Guo, Lucie Y.; Panchenko, Tanya; Dawicki-McKenna, Jennine M.; Heun, Patrick

    2015-01-01

    The centromere—defined by the presence of nucleosomes containing the histone H3 variant, CENP-A—is the chromosomal locus required for the accurate segregation of chromosomes during cell division. Although the sequence determinants of human CENP-A required to maintain a centromere were reported, those that are required for early steps in establishing a new centromere are unknown. In this paper, we used gain-of-function histone H3 chimeras containing various regions unique to CENP-A to investigate early events in centromere establishment. We targeted histone H3 chimeras to chromosomally integrated Lac operator sequences by fusing each of the chimeras to the Lac repressor. Using this approach, we found surprising contributions from a small portion of the N-terminal tail and the CENP-A targeting domain in the initial recruitment of two essential constitutive centromere proteins, CENP-C and CENP-T. Our results indicate that the regions of CENP-A required for early events in centromere establishment differ from those that are required for maintaining centromere identity. PMID:25713413

  18. Structure-Based Functional Analyses of Domains II and III of Pseudorabies Virus Glycoprotein H

    PubMed Central

    Böhm, Sebastian W.; Eckroth, Elisa; Backovic, Marija; Klupp, Barbara G.; Rey, Felix A.; Fuchs, Walter

    2014-01-01

    ABSTRACT Enveloped viruses utilize membrane fusion for entry into, and release from, host cells. For entry, members of the Herpesviridae require at least three envelope glycoproteins: the homotrimeric gB and a heterodimer of gH and gL. The crystal structures of three gH homologues, including pseudorabies virus (PrV) gH, revealed four conserved domains. Domain II contains a planar β-sheet (“fence”) and a syntaxin-like bundle of three α-helices (SLB), similar to those found in eukaryotic fusion proteins, potentially executing an important role in gH function. To test this hypothesis, we introduced targeted mutations into the PrV gH gene, which either disrupt the helices of the SLB by introduction of proline residues or covalently join them by artificial intramolecular disulfide bonds between themselves, to the adjacent fence region, or to domain III. Disruption of either of the three α-helices of the SLB (A250P, V275P, V298P) severely affected gH function in in vitro fusion assays and replication of corresponding PrV mutants. Considerable defects in fusion activity of gH, as well as in penetration kinetics and cell-to-cell spread of PrV mutants, were also observed after disulfide linkage of two α-helices within the SLB (A284C-S291C) or between SLB and domain III (H251C-L432C), as well as by insertions of additional cysteine pairs linking fence, SLB, and domain III. In vitro fusion activity of mutated gH could be partly restored by reduction of the artificial disulfide bonds. Our results indicate that the structure and flexibility of the SLB are relevant for the function of PrV gH in membrane fusion. IMPORTANCE Mutational analysis based on crystal structures of proteins is a powerful tool to understand protein function. Here, we continued our study of pseudorabies virus gH, a part of the core fusion machinery of herpesviruses. We previously showed that the “flap” region in domain IV of PrV gH is important for its function. We now demonstrate that mutations

  19. Structure and function of the interacting domains of Spire and Fmn-family formins

    SciTech Connect

    Vizcarra, Christina L.; Kreutz, Barry; Rodal, Avital A.; Toms, Angela V.; Lu, Jun; Zheng, Wei; Quinlan, Margot E.; Eck, Michael J.

    2012-07-11

    Evidence for cooperation between actin nucleators is growing. The WH2-containing nucleator Spire and the formin Cappuccino interact directly, and both are essential for assembly of an actin mesh during Drosophila oogenesis. Their interaction requires the kinase noncatalytic C-lobe domain (KIND) domain of Spire and the C-terminal tail of the formin. Here we describe the crystal structure of the KIND domain of human Spir1 alone and in complex with the tail of Fmn2, a mammalian ortholog of Cappuccino. The KIND domain is structurally similar to the C-lobe of protein kinases. The Fmn2 tail is coordinated in an acidic cleft at the base of the domain that appears to have evolved via deletion of a helix from the canonical kinase fold. Our functional analysis of Cappuccino reveals an unexpected requirement for its tail in actin assembly. In addition, we find that the KIND/tail interaction blocks nucleation by Cappuccino and promotes its displacement from filament barbed ends providing insight into possible modes of cooperation between Spire and Cappuccino.

  20. Molecular dissection of radixin: distinct and interdependent functions of the amino- and carboxy-terminal domains

    PubMed Central

    1995-01-01

    The ERM proteins--ezrin, radixin, and moesin--occur in particular cortical cytoskeletal structures. Several lines of evidence suggest that they interact with both cytoskeletal elements and plasma membrane components. Here we described the properties of full-length and truncated radixin polypeptides expressed in transfected cells. In stable transfectants, exogenous full-length radixin behaves much like endogenous ERM proteins, localizing to the same cortical structures. However, the presence of full-length radixin or its carboxy-terminal domain in cortical structures correlates with greatly diminished staining of endogenous moesin in those structures, suggesting that radixin and moesin compete for a limiting factor required for normal associations in the cell. The results also reveal distinct roles for the amino- and carboxy-terminal domains. At low levels relative to endogenous radixin, the carboxy-terminal polypeptide is associated with most of the correct cortical targets except cleavage furrows. In contrast, the amino-terminal polypeptide is diffusely localized throughout the cell. Low level expression of full-length radixin or either of the truncated polypeptides has no detectable effect on cell physiology. However, high level expression of the carboxy-terminal domain dramatically disrupts normal cytoskeletal structures and functions. At these high levels, the amino-terminal polypeptide does localize to cortical structures, but does not affect the cells. We conclude that the behavior of radixin in cells depends upon activities contributed by separate domains of the protein, but also requires modulating interactions between those domains. PMID:7744951

  1. Functional synergy between the Munc13 C-terminal C1 and C2 domains

    PubMed Central

    Liu, Xiaoxia; Seven, Alpay Burak; Camacho, Marcial; Esser, Victoria; Xu, Junjie; Trimbuch, Thorsten; Quade, Bradley; Su, Lijing; Ma, Cong; Rosenmund, Christian; Rizo, Josep

    2016-01-01

    Neurotransmitter release requires SNARE complexes to bring membranes together, NSF-SNAPs to recycle the SNAREs, Munc18-1 and Munc13s to orchestrate SNARE complex assembly, and Synaptotagmin-1 to trigger fast Ca2+-dependent membrane fusion. However, it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly, and how the actions of their multiple domains are integrated. Reconstitution, liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1, C2B and C2C domains of Munc13-1, indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain. Our reconstitution data also suggest that Munc18-1, Munc13-1, NSF, αSNAP and the SNAREs are critical to form a ‘primed’ state that does not fuse but is ready for fast fusion upon Ca2+ influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion. DOI: http://dx.doi.org/10.7554/eLife.13696.001 PMID:27213521

  2. Functional synergy between the Munc13 C-terminal C1 and C2 domains.

    PubMed

    Liu, Xiaoxia; Seven, Alpay Burak; Camacho, Marcial; Esser, Victoria; Xu, Junjie; Trimbuch, Thorsten; Quade, Bradley; Su, Lijing; Ma, Cong; Rosenmund, Christian; Rizo, Josep

    2016-01-01

    Neurotransmitter release requires SNARE complexes to bring membranes together, NSF-SNAPs to recycle the SNAREs, Munc18-1 and Munc13s to orchestrate SNARE complex assembly, and Synaptotagmin-1 to trigger fast Ca(2+)-dependent membrane fusion. However, it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly, and how the actions of their multiple domains are integrated. Reconstitution, liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1, C2B and C2C domains of Munc13-1, indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain. Our reconstitution data also suggest that Munc18-1, Munc13-1, NSF, αSNAP and the SNAREs are critical to form a 'primed' state that does not fuse but is ready for fast fusion upon Ca(2+) influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion. PMID:27213521

  3. Functional Behavioral Assessments: Legal Requirements and Challenges.

    ERIC Educational Resources Information Center

    Drasgow, Erik; Yell, Mitchell L.

    2001-01-01

    This article presents the legal requirements of the Individuals with Disabilities Education Act Amendments regarding Individualized Education Programs (IEP), functional behavioral assessments, and behavior intervention plans for special education students with problem behavior; describes the initial policy letters and due process hearings that…

  4. 47 CFR 80.1081 - Functional requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 5 2010-10-01 2010-10-01 false Functional requirements. 80.1081 Section 80.1081 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment...

  5. 47 CFR 80.1081 - Functional requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 5 2013-10-01 2013-10-01 false Functional requirements. 80.1081 Section 80.1081 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment...

  6. 47 CFR 80.1081 - Functional requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 5 2011-10-01 2011-10-01 false Functional requirements. 80.1081 Section 80.1081 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment...

  7. Functional Foods Baseline and Requirements Analysis

    NASA Technical Reports Server (NTRS)

    Cooper, M. R.; Bermudez-Aguirre, L. D.; Douglas, G.

    2015-01-01

    Current spaceflight foods were evaluated to determine if their nutrient profile supports positioning as a functional food and if the stability of the bioactive compound within the food matrix over an extended shelf-life correlated with the expected storage duration during the mission. Specifically, the research aims were: Aim A. To determine the amount of each nutrient in representative spaceflight foods immediately after processing and at predetermined storage time to establish the current nutritional state. Aim B. To identify the requirements to develop foods that stabilize these nutrients such that required concentrations are maintained in the space food system throughout long duration missions (up to five years). Aim C. To coordinate collaborations with health and performance groups that may require functional foods as a countermeasure.

  8. Functional overlap between conserved and diverged KH domains in Saccharomyces cerevisiae SCP160

    PubMed Central

    Brykailo, Melissa A.; Corbett, Anita H.; Fridovich-Keil, Judith L.

    2007-01-01

    The K homology (KH) domain is a remarkably versatile and highly conserved RNA-binding motif. Classical KH domains include a characteristic pattern of hydrophobic residues, a Gly-X-X-Gly (GXXG) segment, and a variable loop. KH domains typically occur in clusters, with some retaining their GXXG sequence (conserved), while others do not (diverged). As a first step towards addressing whether GXXG is essential for KH-domain function, we explored the roles of conserved and diverged KH domains in Scp160p, a multiple-KH-domain-containing protein in Saccharomyces cerevisiae. We specifically wanted to know (1) whether diverged KH domains were essential for Scp160p function, and (2) whether diverged KH domains could functionally replace conserved KH domains. To address these questions, we deleted and/or interchanged conserved and diverged KH domains of Scp160p and expressed the mutated alleles in yeast. Our results demonstrated that the answer to each question was yes. Both conserved and diverged KH domains are essential for Scp160p function, and diverged KH domains can function in place of conserved KH domains. These findings challenge the prevailing notions about the requisite features of a KH domain and raise the possibility that there may be more functional KH domains in the proteome than previously appreciated. PMID:17264125

  9. Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase.

    PubMed Central

    Leberer, E; Wu, C; Leeuw, T; Fourest-Lieuvin, A; Segall, J E; Thomas, D Y

    1997-01-01

    Ste20p from Saccharomyces cerevisiae belongs to the Ste20p/p65PAK family of protein kinases which are highly conserved from yeast to man and regulate conserved mitogen-activated protein kinase pathways. Ste20p fulfills multiple roles in pheromone signaling, morphological switching and vegetative growth and binds Cdc42p, a Rho-like small GTP binding protein required for polarized morphogenesis. We have analyzed the functional consequences of mutations that prevent binding of Cdc42p to Ste20p. The complete amino-terminal, non-catalytic half of Ste20p, including the conserved Cdc42p binding domain, was dispensable for heterotrimeric G-protein-mediated pheromone signaling. However, the Cdc42p binding domain was necessary for filamentous growth in response to nitrogen starvation and for an essential function that Ste20p shares with its isoform Cla4p during vegetative growth. Moreover, the Cdc42p binding domain was required for cell-cell adhesion during conjugation. Subcellular localization of wild-type and mutant Ste20p fused to green fluorescent protein showed that the Cdc42p binding domain is needed to direct localization of Ste20p to regions of polarized growth. These results suggest that Ste20p is regulated in different developmental pathways by different mechanisms which involve heterotrimeric and small GTP binding proteins. PMID:9009270

  10. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

    PubMed Central

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M.

    2016-01-01

    ABSTRACT The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed

  11. Gas Test Loop Functional and Technical Requirements

    SciTech Connect

    Glen R. Longhurst; Soli T. Khericha; James L. Jones

    2004-09-01

    This document defines the technical and functional requirements for a gas test loop (GTL) to be constructed for the purpose of providing a high intensity fast-flux irradiation environment for developers of advanced concept nuclear reactors. This capability is needed to meet fuels and materials testing requirements of the designers of Generation IV (GEN IV) reactors and other programs within the purview of the Advanced Fuel Cycle Initiative (AFCI). Space nuclear power development programs may also benefit by the services the GTL will offer. The overall GTL technical objective is to provide developers with the means for investigating and qualifying fuels and materials needed for advanced reactor concepts. The testing environment includes a fast-flux neutron spectrum of sufficient intensity to perform accelerated irradiation testing. Appropriate irradiation temperature, gaseous environment, test volume, diagnostics, and access and handling features are also needed. This document serves to identify those requirements as well as generic requirements applicable to any system of this kind.

  12. Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors.

    PubMed

    Zhao, Li-Hua; Yin, Yanting; Yang, Dehua; Liu, Bo; Hou, Li; Wang, Xiaoxi; Pal, Kuntal; Jiang, Yi; Feng, Yang; Cai, Xiaoqing; Dai, Antao; Liu, Mingyao; Wang, Ming-Wei; Melcher, Karsten; Xu, H Eric

    2016-07-15

    G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation. PMID:27226600

  13. ERAD of proteins containing aberrant transmembrane domains requires ubiquitylation of cytoplasmic lysine residues

    PubMed Central

    Briant, Kit; Koay, Yee-Hui; Otsuka, Yuka; Swanton, Eileithyia

    2015-01-01

    ABSTRACT Clearance of misfolded proteins from the endoplasmic reticulum (ER) is mediated by the ubiquitin-proteasome system in a process known as ER-associated degradation (ERAD). The mechanisms through which proteins containing aberrant transmembrane domains are degraded by ERAD are poorly understood. To address this question, we generated model ERAD substrates based on CD8 with either a non-native transmembrane domain but a folded ER luminal domain (CD8TMD*), or the native transmembrane domain but a misfolded luminal domain (CD8LUM*). Although both chimeras were degraded by ERAD, we found that the location of the folding defect determined the initial site of ubiquitylation. Ubiquitylation of cytoplasmic lysine residues was required for the extraction of CD8TMD* from the ER membrane during ERAD, whereas CD8LUM* continued to be degraded in the absence of cytoplasmic lysine residues. Cytoplasmic lysine residues were also required for degradation of an additional ERAD substrate containing an unassembled transmembrane domain and when a non-native transmembrane domain was introduced into CD8LUM*. Our results suggest that proteins with defective transmembrane domains are removed from the ER through a specific ERAD mechanism that depends upon ubiquitylation of cytoplasmic lysine residues. PMID:26446255

  14. The Impact of Acute Phase Domain-Specific Cognitive Function on Post-stroke Functional Recovery

    PubMed Central

    Park, Jihong; Lee, Gangpyo; Lee, Shi-Uk

    2016-01-01

    Objective To assess whether the cognitive function in the acute stage evaluated by domain-specific neuropsychological assessments would be an independent predictor of functional outcome after stroke. Methods Forty patients underwent 4 domain-specific neuropsychological examinations about 3 weeks after the onset of stroke. The tests included the Boston Naming Test (BNT), the construction recall test (CRT), the construction praxis test (CPT), and the verbal fluency test (VFT). The Korean version of Modified Barthel Index (K-MBI) at 3 months and the modified Rankin Scale (mRS) at 6 months were investigated as functional outcome after stroke. Functional improvement was assessed using the change in K-MBI during the first 3 months and subjects were dichotomized into 'good status' and 'poor status' according to mRS at 6 months. The domain-specific cognitive function along with other possible predictors for functional outcome was examined using regression analysis. Results The z-score of CPT (p=0.044) and CRT (p<0.001) were independent predictors for functional improvement measured by the change in K-MBI during the first 3 months after stroke. The z-score of CPT (p=0.049) and CRT (p=0.048) were also independent predictors of functional status at post-stroke 6 months assessed by mRS. Conclusion Impairment in visuospatial construction and memory within one month after stroke can be an independent prognostic factor of functional outcome. Domain-specific neuropsychological assessments could be considered in patients with stroke in the acute phase to predict long-term functional outcome. PMID:27152270

  15. Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain.

    PubMed

    Pike, Brietta L; Tenis, Nora; Heierhorst, Jörg

    2004-09-17

    Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways. PMID:15271990

  16. Structure and function of the epidermal growth factor domain of P-selectin.

    PubMed

    Freedman, S J; Sanford, D G; Bachovchin, W W; Furie, B C; Baleja, J D; Furie, B

    1996-10-29

    P-selectin is a multidomain adhesion protein on the surface of activated platelets and endothelial cells that functions in the recruitment of leukocytes to the site of inflammation. The amino-terminal lectin and EGF domains constitute the ligand recognition unit. We have produced a synthetic 40-residue P-selectin EGF domain (P-sel:EGF) to examine the structure and function of this domain independent of P-selectin. The peptide was folded in vitro and exhibited the same disulfide bonding pattern as other EGF-like domains. P-sel:EGF did not inhibit P-selectin-mediated cellular adhesion assays, indicating that the lectin domain is also required. We undertook the study of the P-selectin EGF by 1H NMR to determine its structure independent of the lectin domain and to compare its structure to that of E-selectin determined crystallographically [Graves et al. (1994) Nature 367, 532]. Although the binding of P-selectin to its carbohydrate ligand is calcium dependent, and some EGF domains have calcium binding sites, addition of calcium had no effect on the NMR spectrum or on the pH-induced changes. Nearly complete resonance assignments were made from 2D 1H NMR spectra at pH 6.0. Two sections of antiparallel beta-sheet were identified on the basis of the pattern of long-range NOEs, 3JHN alpha coupling constants, and slowly exchanging amides. The solution structure of the peptide backbone was determined using distance geometry and simulated annealing calculations. The backbone RMSD to the geometric average for 19 final structures is 0.64 +/- 0.17 A. The resulting fold closely resembles that of other EGF-like peptides, including the E-selectin EGF domain (RMSD approximately 1.08 A). However, compared to the E-selectin EGF structure which also contains the lectin domain, some residues from 1-11 are less ordered, and novel contacts occur between the amino terminus and the core beta-sheet. Despite marked structural homology of the selectin polypeptide backbones, the selectin EGF

  17. Structural and Functional Dissection of the Abp1 ADFH Actin-binding Domain Reveals Versatile In Vivo Adapter Functions

    SciTech Connect

    Quintero-Monzon,O.; Rodal, A.; Strokopytov, B.; Almo, S.; Goode, B.

    2005-01-01

    Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1-actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.

  18. Measurement of retinal physiology using functional Fourier domain OCT concepts

    NASA Astrophysics Data System (ADS)

    Leitgeb, R. A.; Bachmann, A. H.; Villiger, M.; Michaely, R.; Blatter, C.; Lasser, T.; Pache, C.; Pircher, M.

    2007-02-01

    Fourier Domain OCT proved to be an outstanding tool for measuring 3D retinal structures with high sensitivity, resolution, and speed. We extended the FDOCT concept towards functional imaging by analyzing the spectroscopic tissue properties, polarization contrast and Doppler velocity imaging. Differential spectral contrast FDOCT allows optical staining of retinal tomograms and to contrast tissue of high pigmentation such as the retinal pigment epithelium (RPE). The latter shows strong correlation if compared to polarization sensitive OCT images. First implementations of Doppler FDOCT systems demonstrated the capability of measuring in-vivo retinal blood flow profiles and pulsatility. We developed a new concept of Doppler FDOCT that allows measuring also large flow velocities typically close to the optic nerve head. Studies of retinal perfusion based on Laser Doppler Flowmetry (LDF) demonstrated the high sensitivity of blood flow to external stimuli. We performed first experiments of studying retinal perfusion in response to flicker stimulation. An increase in vessel diameter by 11% and of flow velocity by 49% was measured. We believe that a multi-modal functional imaging concept is of high value for an accurate and early diagnosis and understanding of retinal pathologies and pathogenesis.

  19. Structural and functional analysis of the NLRP4 pyrin domain.

    PubMed

    Eibl, Clarissa; Grigoriu, Simina; Hessenberger, Manuel; Wenger, Julia; Puehringer, Sandra; Pinheiro, Anderson S; Wagner, Roland N; Proell, Martina; Reed, John C; Page, Rebecca; Diederichs, Kay; Peti, Wolfgang

    2012-09-18

    NLRP4 is a member of the nucleotide-binding and leucine-rich repeat receptor (NLR) family of cytosolic receptors and a member of an inflammation signaling cascade. Here, we present the crystal structure of the NLRP4 pyrin domain (PYD) at 2.3 Å resolution. The NLRP4 PYD is a member of the death domain (DD) superfamily and adopts a DD fold consisting of six α-helices tightly packed around a hydrophobic core, with a highly charged surface that is typical of PYDs. Importantly, however, we identified several differences between the NLRP4 PYD crystal structure and other PYD structures that are significant enough to affect NLRP4 function and its interactions with binding partners. Notably, the length of helix α3 and the α2-α3 connecting loop in the NLRP4 PYD are unique among PYDs. The apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein whose interactions with a number of distinct PYDs are believed to be critical for activation of the inflammatory response. Here, we use co-immunoprecipitation, yeast two-hybrid, and nuclear magnetic resonance chemical shift perturbation analysis to demonstrate that, despite being important for activation of the inflammatory response and sharing several similarities with other known ASC-interacting PYDs (i.e., ASC2), NLRP4 does not interact with the adaptor protein ASC. Thus, we propose that the factors governing homotypic PYD interactions are more complex than the currently accepted model, which states that complementary charged surfaces are the main determinants of PYD-PYD interaction specificity. PMID:22928810

  20. The pilus usher controls protein interactions via domain masking and is functional as an oligomer

    SciTech Connect

    Werneburg, Glenn T.; Li, Huilin; Henderson, Nadine S.; Portnoy, Erica B.; Sarowar, Samema; Hultgren, Scott J.; Thanassi, David G.

    2015-06-08

    The chaperone/usher (CU) pathway is responsible for biogenesis of organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Assembly and secretion of pili by the CU pathway requires a dedicated periplasmic chaperone and a multidomain outer membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate binding site, but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which served as a central switch controlling usher activation. In addition, we demonstrate that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions, and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria.

  1. The pilus usher controls protein interactions via domain masking and is functional as an oligomer

    DOE PAGESBeta

    Werneburg, Glenn T.; Li, Huilin; Henderson, Nadine S.; Portnoy, Erica B.; Sarowar, Samema; Hultgren, Scott J.; Thanassi, David G.

    2015-06-08

    The chaperone/usher (CU) pathway is responsible for biogenesis of organelles termed pili or fimbriae in Gram-negative bacteria. Type 1 pili expressed by uropathogenic Escherichia coli are prototypical structures assembled by the CU pathway. Assembly and secretion of pili by the CU pathway requires a dedicated periplasmic chaperone and a multidomain outer membrane protein termed the usher (FimD). We show that the FimD C-terminal domains provide the high-affinity substrate binding site, but that these domains are masked in the resting usher. Domain masking requires the FimD plug domain, which served as a central switch controlling usher activation. In addition, we demonstratemore » that usher molecules can act in trans for pilus biogenesis, providing conclusive evidence for a functional usher oligomer. These results reveal mechanisms by which molecular machines such as the usher regulate and harness protein-protein interactions, and suggest that ushers may interact in a cooperative manner during pilus assembly in bacteria.« less

  2. Drosophila Rolling Blackout Displays Lipase Domain-Dependent and Independent Endocytic Functions Downstream of Dynamin

    PubMed Central

    Vijayakrishnan, Niranjana; Phillips, Scott E.; Broadie, Kendal

    2010-01-01

    Drosophila temperature-sensitive rolling blackout (rbots) mutants display a total block of endocytosis in non-neuronal cells and a weaker, partial defect at neuronal synapses. RBO is an integral plasma membrane protein and is predicted to be a serine esterase. To determine if lipase activity is required for RBO function, we mutated the catalytic serine 358 to alanine in the G-X-S-X-G active site, and assayed genomic rescue of rbo mutant non-neuronal and neuronal phenotypes. The rboS358A mutant is unable to rescue rbo null 100% embryonic lethality, indicating that the lipase-domain is critical for RBO essential function. Likewise, the rboS358A mutant cannot provide any rescue of endocytic blockade in rbots Garland cells, demonstrating that the lipase-domain is indispensable for non-neuronal endocytosis. In contrast, rbots conditional paralysis, synaptic transmission block and synapse endocytic defects are all fully rescued by the rboS358A mutant, showing that the RBO lipase-domain is dispensable in neuronal contexts. We identified a synthetic lethal interaction between rbots and the well-characterized dynamin GTPase conditional shibire (shits1) mutant. In both non-neuronal cells and neuronal synapses, shits1;rbots phenocopies shits1 endocytic defects, indicating that dynamin and RBO act in the same pathway, with dynamin functioning upstream of RBO. We conclude that RBO possesses both lipase-domain dependent and scaffolding functions with differential requirements in non-neuronal versus neuronal endocytosis mechanisms downstream of dynamin GTPase activity. PMID:21029287

  3. The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis

    PubMed Central

    Beck, Kirsten; Vannini, Alessandro; Cramer, Patrick; Lipps, Georg

    2010-01-01

    The plasmid pRN1 encodes for a multifunctional replication protein with primase, DNA polymerase and helicase activity. The minimal region required for primase activity encompasses amino-acid residues 40–370. While the N-terminal part of that minimal region (residues 47–247) folds into the prim/pol domain and bears the active site, the structure and function of the C-terminal part (residues 248–370) is unknown. Here we show that the C-terminal part of the minimal region folds into a compact domain with six helices and is stabilized by a disulfide bond. Three helices superimpose well with the C-terminal domain of the primase of the bacterial broad host range plasmid RSF1010. Structure-based site-directed mutagenesis shows that the C-terminal helix of the helix bundle domain is required for primase activity although it is distant to the active site in the crystallized conformation. Furthermore, we identified mutants of the C-terminal domain, which are defective in template binding, dinucleotide formation and conformation change prior to DNA extension. PMID:20511586

  4. An exploration of function analysis and function allocation in the commercial flight domain

    NASA Technical Reports Server (NTRS)

    Mcguire, James C.; Zich, John A.; Goins, Richard T.; Erickson, Jeffery B.; Dwyer, John P.; Cody, William J.; Rouse, William B.

    1991-01-01

    The applicability is explored of functional analysis methods to support cockpit design. Specifically, alternative techniques are studied for ensuring an effective division of responsibility between the flight crew and automation. A functional decomposition is performed of the commercial flight domain to provide the information necessary to support allocation decisions and demonstrate methodology for allocating functions to flight crew or to automation. The function analysis employed 'bottom up' and 'top down' analyses and demonstrated the comparability of identified functions, using the 'lift off' segment of the 'take off' phase as a test case. The normal flight mission and selected contingencies were addressed. Two alternative methods for using the functional description in the allocation of functions between man and machine were investigated. The two methods were compared in order to ascertain their relative strengths and weaknesses. Finally, conclusions were drawn regarding the practical utility of function analysis methods.

  5. The crystal structure of Mtr4 reveals a novel arch domain required for rRNA processing

    SciTech Connect

    Jackson, R.N.; Robinson, H.; Klauer, A. A.; Hintze, B. J.; van Hoof, A.; Johnson, S. J.

    2010-07-01

    The essential RNA helicase, Mtr4, performs a critical role in RNA processing and degradation as an activator of the nuclear exosome. The molecular basis for this vital function is not understood and detailed analysis is significantly limited by the lack of structural data. In this study, we present the crystal structure of Mtr4. The structure reveals a new arch-like domain that is specific to Mtr4 and Ski2 (the cytosolic homologue of Mtr4). In vivo and in vitro analyses demonstrate that the Mtr4 arch domain is required for proper 5.8S rRNA processing, and suggest that the arch functions independently of canonical helicase activity. In addition, extensive conservation along the face of the putative RNA exit site highlights a potential interface with the exosome. These studies provide a molecular framework for understanding fundamental aspects of helicase function in exosome activation, and more broadly define the molecular architecture of Ski2-like helicases.

  6. The auto-inhibitory domain and ATP-independent microtubule-binding region of Kinesin heavy chain are major functional domains for transport in the Drosophila germline

    PubMed Central

    Williams, Lucy S.; Ganguly, Sujoy; Loiseau, Philippe; Ng, Bing Fu; Palacios, Isabel M.

    2014-01-01

    The major motor Kinesin-1 provides a key pathway for cell polarization through intracellular transport. Little is known about how Kinesin works in complex cellular surroundings. Several cargos associate with Kinesin via Kinesin light chain (KLC). However, KLC is not required for all Kinesin transport. A putative cargo-binding domain was identified in the C-terminal tail of fungal Kinesin heavy chain (KHC). The tail is conserved in animal KHCs and might therefore represent an alternative KLC-independent cargo-interacting region. By comprehensive functional analysis of the tail during Drosophila oogenesis we have gained an understanding of how KHC achieves specificity in its transport and how it is regulated. This is, to our knowledge, the first in vivo structural/functional analysis of the tail in animal Kinesins. We show that the tail is essential for all functions of KHC except Dynein transport, which is KLC dependent. These tail-dependent KHC activities can be functionally separated from one another by further characterizing domains within the tail. In particular, our data show the following. First, KHC is temporally regulated during oogenesis. Second, the IAK domain has an essential role distinct from its auto-inhibitory function. Third, lack of auto-inhibition in itself is not necessarily detrimental to KHC function. Finally, the ATP-independent microtubule-binding motif is required for cargo localization. These results stress that two unexpected highly conserved domains, namely the auto-inhibitory IAK and the auxiliary microtubule-binding motifs, are crucial for transport by Kinesin-1 and that, although not all cargos are conserved, their transport involves the most conserved domains of animal KHCs. PMID:24257625

  7. Intrinsic HER4/4ICD transcriptional activation domains are required for STAT5A activated gene expression.

    PubMed

    Han, Wen; Sfondouris, Mary E; Semmes, Eleanor C; Meyer, Alicia M; Jones, Frank E

    2016-10-30

    The epidermal growth factor receptor family member HER4 undergoes proteolytic processing at the cell surface to release the HER4 intracellular domain (4ICD) nuclear protein. Interestingly, 4ICD directly interacts with STAT5 and functions as an obligate STAT5 nuclear chaperone. Once in the nucleus 4ICD binds with STAT5 at STAT5 target genes, dramatically potentiating STAT5 transcriptional activation. These observations raise the possibility that 4ICD directly coactivates STAT5 gene expression. Using both yeast and mammalian transactivation reporter assays, we performed truncations of 4ICD fused to a GAL4 DNA binding domain and identified two independent 4ICD transactivation domains located between residues 1022 and 1090 (TAD1) and 1192 and 1225 (TAD2). The ability of the 4ICD DNA binding domain fusions to transactivate reporter gene expression required deletion of the intrinsic tyrosine kinase domain. In addition, we identified the 4ICD carboxyl terminal TVV residues, a PDZ domain binding motif (PDZ-DBM), as a potent transcriptional repressor. The transactivation activity of the HER4 carboxyl terminal domain lacking the tyrosine kinase (CTD) was significantly lower than similar EGFR or HER2 CTD. However, deletion of the HER4 CTD PDZ-DBM enhanced HER4 CTD transactivation to levels equivalent to the EGFR and HER2 CTDs. To determine if 4ICD TAD1 and TAD2 have a physiologically relevant role in STAT5 transactivation, we coexpressed 4ICD or 4ICD lacking TAD2 or both TAD1 and TAD2 with STAT5 in a luciferase reporter assay. Our results demonstrate that each 4ICD TAD contributes additively to STAT5A transactivation and the ability of STAT5A to transactivate the β-casein promoter requires the 4ICD TADs. Taken together, published data and our current results demonstrate that both 4ICD nuclear chaperone and intrinsic coactivation activities are essential for STAT5 regulated gene expression. PMID:27502417

  8. Clustering of protein domains for functional and evolutionary studies

    PubMed Central

    Goldstein, Pavle; Zucko, Jurica; Vujaklija, Dušica; Kriško, Anita; Hranueli, Daslav; Long, Paul F; Etchebest, Catherine; Basrak, Bojan; Cullum, John

    2009-01-01

    Background The number of protein family members defined by DNA sequencing is usually much larger than those characterised experimentally. This paper describes a method to divide protein families into subtypes purely on sequence criteria. Comparison with experimental data allows an independent test of the quality of the clustering. Results An evolutionary split statistic is calculated for each column in a protein multiple sequence alignment; the statistic has a larger value when a column is better described by an evolutionary model that assumes clustering around two or more amino acids rather than a single amino acid. The user selects columns (typically the top ranked columns) to construct a motif. The motif is used to divide the family into subtypes using a stochastic optimization procedure related to the deterministic annealing EM algorithm (DAEM), which yields a specificity score showing how well each family member is assigned to a subtype. The clustering obtained is not strongly dependent on the number of amino acids chosen for the motif. The robustness of this method was demonstrated using six well characterized protein families: nucleotidyl cyclase, protein kinase, dehydrogenase, two polyketide synthase domains and small heat shock proteins. Phylogenetic trees did not allow accurate clustering for three of the six families. Conclusion The method clustered the families into functional subtypes with an accuracy of 90 to 100%. False assignments usually had a low specificity score. PMID:19832975

  9. Time domain functional NIRS imaging for human brain mapping.

    PubMed

    Torricelli, Alessandro; Contini, Davide; Pifferi, Antonio; Caffini, Matteo; Re, Rebecca; Zucchelli, Lucia; Spinelli, Lorenzo

    2014-01-15

    This review is aimed at presenting the state-of-the-art of time domain (TD) functional near-infrared spectroscopy (fNIRS). We first introduce the physical principles, the basics of modeling and data analysis. Basic instrumentation components (light sources, detection techniques, and delivery and collection systems) of a TD fNIRS system are described. A survey of past, existing and next generation TD fNIRS systems used for research and clinical studies is presented. Performance assessment of TD fNIRS systems and standardization issues are also discussed. Main strengths and weakness of TD fNIRS are highlighted, also in comparison with continuous wave (CW) fNIRS. Issues like quantification of the hemodynamic response, penetration depth, depth selectivity, spatial resolution and contrast-to-noise ratio are critically examined, with the help of experimental results performed on phantoms or in vivo. Finally we give an account on the technological developments that would pave the way for a broader use of TD fNIRS in the neuroimaging community. PMID:23747285

  10. Weighted mutual information analysis substantially improves domain-based functional network models

    PubMed Central

    Shim, Jung Eun; Lee, Insuk

    2016-01-01

    Motivation: Functional protein–protein interaction (PPI) networks elucidate molecular pathways underlying complex phenotypes, including those of human diseases. Extrapolation of domain–domain interactions (DDIs) from known PPIs is a major domain-based method for inferring functional PPI networks. However, the protein domain is a functional unit of the protein. Therefore, we should be able to effectively infer functional interactions between proteins based on the co-occurrence of domains. Results: Here, we present a method for inferring accurate functional PPIs based on the similarity of domain composition between proteins by weighted mutual information (MI) that assigned different weights to the domains based on their genome-wide frequencies. Weighted MI outperforms other domain-based network inference methods and is highly predictive for pathways as well as phenotypes. A genome-scale human functional network determined by our method reveals numerous communities that are significantly associated with known pathways and diseases. Domain-based functional networks may, therefore, have potential applications in mapping domain-to-pathway or domain-to-phenotype associations. Availability and Implementation: Source code for calculating weighted mutual information based on the domain profile matrix is available from www.netbiolab.org/w/WMI. Contact: Insuklee@yonsei.ac.kr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27207946

  11. LRIG1 extracellular domain: structure and function analysis.

    PubMed

    Xu, Yibin; Soo, Priscilla; Walker, Francesca; Zhang, Hui Hua; Redpath, Nicholas; Tan, Chin Wee; Nicola, Nicos A; Adams, Timothy E; Garrett, Thomas P; Zhang, Jian-Guo; Burgess, Antony W

    2015-05-22

    We have expressed and purified three soluble fragments of the human LRIG1-ECD (extracellular domain): the LRIG1-LRR (leucine-rich repeat) domain, the LRIG1-3Ig (immunoglobulin-like) domain, and the LRIG1-LRR-1Ig fragment using baculovirus vectors in insect cells. The two LRIG1 domains crystallised so that we have been able to determine the three-dimensional structures at 2.3Å resolution. We developed a three-dimensional structure for the LRIG1-ECD using homology modelling based on the LINGO-1 structure. The LRIG1-LRR domain and the LRIG1-LRR-1Ig fragment are monomers in solution, whereas the LRIG1-3Ig domain appears to be dimeric. We could not detect any binding of the LRIG1 domains or the LRIG1-LRR-1Ig fragment to the EGF receptor (EGFR), either in solution using biosensor analysis or when the EGFR was expressed on the cell surface. The FLAG-tagged LRIG1-LRR-1Ig fragment binds weakly to colon cancer cells regardless of the presence of EGFRs. Similarly, neither the soluble LRIG1-LRR nor the LRIG1-3Ig domains nor the full-length LRIG1 co-expressed in HEK293 cells inhibited ligand-stimulated activation of cell-surface EGFR. PMID:25765764

  12. The Dysferlin Domain-Only Protein, Spo73, Is Required for Prospore Membrane Extension in Saccharomyces cerevisiae.

    PubMed

    Okumura, Yuuya; Nakamura, Tsuyoshi S; Tanaka, Takayuki; Inoue, Ichiro; Suda, Yasuyuki; Takahashi, Tetsuo; Nakanishi, Hideki; Nakamura, Shugo; Gao, Xiao-Dong; Tachikawa, Hiroyuki

    2016-01-01

    Sporulation of Saccharomyces cerevisiae is a developmental process in which an ascus containing four haploid spores forms from a diploid cell. During this process, newly formed membrane structures called prospore membranes extend along the nuclear envelope and engulf and package daughter nuclei along with cytosol and organelles to form precursors of spores. Proteins involved in prospore membrane extension, Vps13 and Spo71, have recently been reported; however, the overall mechanism of membrane extension remains unclear. Here, we identified Spo73 as an additional factor involved in prospore membrane extension. Analysis of a spo73∆ mutant revealed that it shows defects similar to those of a spo71∆ mutant during prospore membrane formation. Spo73 localizes to the prospore membrane, and this localization is independent of Spo71 and Vps13. In contrast, a Spo73 protein carrying mutations in a surface basic patch mislocalizes to the cytoplasm and overexpression of Spo71 can partially rescue localization to the prospore membrane. Similar to spo71∆ mutants, spo73∆ mutants display genetic interactions with the mutations in the SMA2 and SPO1 genes involved in prospore membrane bending. Further, our bioinformatic analysis revealed that Spo73 is a dysferlin domain-only protein. Thus, these results suggest that a dysferlin domain-only protein, Spo73, functions with a dual pleckstrin homology domain protein, Spo71, in prospore membrane extension. Analysis of Spo73 will provide insights into the conserved function of dysferlin domains, which is related to dysferlinopathy. IMPORTANCE Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall formation

  13. The Dysferlin Domain-Only Protein, Spo73, Is Required for Prospore Membrane Extension in Saccharomyces cerevisiae

    PubMed Central

    Okumura, Yuuya; Nakamura, Tsuyoshi S.; Tanaka, Takayuki; Inoue, Ichiro; Suda, Yasuyuki; Takahashi, Tetsuo; Nakanishi, Hideki; Nakamura, Shugo; Gao, Xiao-Dong

    2015-01-01

    ABSTRACT Sporulation of Saccharomyces cerevisiae is a developmental process in which an ascus containing four haploid spores forms from a diploid cell. During this process, newly formed membrane structures called prospore membranes extend along the nuclear envelope and engulf and package daughter nuclei along with cytosol and organelles to form precursors of spores. Proteins involved in prospore membrane extension, Vps13 and Spo71, have recently been reported; however, the overall mechanism of membrane extension remains unclear. Here, we identified Spo73 as an additional factor involved in prospore membrane extension. Analysis of a spo73∆ mutant revealed that it shows defects similar to those of a spo71∆ mutant during prospore membrane formation. Spo73 localizes to the prospore membrane, and this localization is independent of Spo71 and Vps13. In contrast, a Spo73 protein carrying mutations in a surface basic patch mislocalizes to the cytoplasm and overexpression of Spo71 can partially rescue localization to the prospore membrane. Similar to spo71∆ mutants, spo73∆ mutants display genetic interactions with the mutations in the SMA2 and SPO1 genes involved in prospore membrane bending. Further, our bioinformatic analysis revealed that Spo73 is a dysferlin domain-only protein. Thus, these results suggest that a dysferlin domain-only protein, Spo73, functions with a dual pleckstrin homology domain protein, Spo71, in prospore membrane extension. Analysis of Spo73 will provide insights into the conserved function of dysferlin domains, which is related to dysferlinopathy. IMPORTANCE Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall

  14. Functional and topological characteristics of mammalian regulatory domains

    PubMed Central

    Symmons, Orsolya; Uslu, Veli Vural; Tsujimura, Taro; Ruf, Sandra; Nassari, Sonya; Schwarzer, Wibke; Ettwiller, Laurence; Spitz, François

    2014-01-01

    Long-range regulatory interactions play an important role in shaping gene-expression programs. However, the genomic features that organize these activities are still poorly characterized. We conducted a large operational analysis to chart the distribution of gene regulatory activities along the mouse genome, using hundreds of insertions of a regulatory sensor. We found that enhancers distribute their activities along broad regions and not in a gene-centric manner, defining large regulatory domains. Remarkably, these domains correlate strongly with the recently described TADs, which partition the genome into distinct self-interacting blocks. Different features, including specific repeats and CTCF-binding sites, correlate with the transition zones separating regulatory domains, and may help to further organize promiscuously distributed regulatory influences within large domains. These findings support a model of genomic organization where TADs confine regulatory activities to specific but large regulatory domains, contributing to the establishment of specific gene expression profiles. PMID:24398455

  15. Functional Service Domain Architecture Management: Building the Foundation for Situational Method Engineering

    NASA Astrophysics Data System (ADS)

    Stock, Daniel; Winter, Robert; Mayer, Jörg H.

    Functional service domains are logical design artifacts that are intended to achieve better business/IT alignment. Their widespread utilization clearly indicates their perceived usefulness in managing the complexity of aligning business structures with IT structures. However, a common understanding of functional service domains and the associated principles that govern their design and evolution is still missing. So far, the literature provides only little guidance in closing this gap. This article contributes to the foundations that allow for the design of a situational method for functional service domain architecture management. Reviewing current literature, a framework is pro posed that supports the identification of functional service domain architecture management patterns. Based on a better understanding of functional domain architecture management approaches, situational method engineering for functional domains can be applied by identifying context types and goal vectors, designing fragments, and associating successfully adopted method fragments with specific situations. The validity of the proposed framework is tested by five case studies.

  16. Significance of the N-terminal domain for the function of chloroplast cpn20 chaperonin.

    PubMed

    Bonshtien, Anat L; Weiss, Celeste; Vitlin, Anna; Niv, Adina; Lorimer, George H; Azem, Abdussalam

    2007-02-16

    Chaperonins cpn60 and cpn10 are essential proteins involved in cellular protein folding. Plant chloroplasts contain a unique version of the cpn10 co-chaperonin, cpn20, which consists of two homologous cpn10-like domains (N-cpn20 and C-cpn20) that are connected by a short linker region. Although cpn20 seems to function like other single domain cpn10 oligomers, the structure and specific functions of the domains are not understood. We mutated amino acids in the "mobile loop" regions of N-cpn20, C-cpn20 or both: a highly conserved glycine, which was shown to be important for flexibility of the mobile loop, and a leucine residue shown to be involved in binding of co-chaperonin to chaperonin. The mutant proteins were purified and their oligomeric structure validated by gel filtration, native gel electrophoresis, and circular dichroism. Functional assays of protein refolding and inhibition of GroEL ATPase both showed (i) mutation of the conserved glycine reduced the activity of cpn20, whether in N-cpn20 (G32A) or C-cpn20 (G130A). The same mutation in the bacterial cpn10 (GroES G24A) had no effect on activity. (ii) Mutations in the highly conserved leucine of N-cpn20 (L35A) and in the corresponding L27A of GroES resulted in inactive protein. (iii) In contrast, mutant L133A, in which the conserved leucine of C-cpn20 was altered, retained 55% activity. We conclude that the structure of cpn20 is much more sensitive to alterations in the mobile loop than is the structure of GroES. Moreover, only N-cpn20 is necessary for activity of cpn20. However, full and efficient functioning requires both domains. PMID:17178727

  17. The tRNA-binding moiety in GCN2 contains a dimerization domain that interacts with the kinase domain and is required for tRNA binding and kinase activation

    PubMed Central

    Qiu, Hongfang; Dong, Jinsheng; Hu, Cuihua; Francklyn, Christopher S.; Hinnebusch, Alan G.

    2001-01-01

    GCN2 stimulates translation of GCN4 mRNA in amino acid-starved cells by phosphorylating translation initiation factor 2. GCN2 is activated by binding of uncharged tRNA to a domain related to histidyl-tRNA synthetase (HisRS). The HisRS-like region contains two dimerization domains (HisRS-N and HisRS-C) required for GCN2 function in vivo but dispensable for dimerization by full-length GCN2. Residues corresponding to amino acids at the dimer interface of Escherichia coli HisRS were required for dimerization of recombinant HisRS-N and for tRNA binding by full-length GCN2, suggesting that HisRS-N dimerization promotes tRNA binding and kinase activation. HisRS-N also interacted with the protein kinase (PK) domain, and a deletion impairing this interaction destroyed GCN2 function without reducing tRNA binding; thus, HisRS-N–PK interaction appears to stimulate PK function. The C-terminal domain of GCN2 (C-term) interacted with the PK domain in a manner disrupted by an activating PK mutation (E803V). These results suggest that the C-term is an autoinhibitory domain, counteracted by tRNA binding. We conclude that multiple domain interactions, positive and negative, mediate the activation of GCN2 by uncharged tRNA. PMID:11250908

  18. Functionally relevant motions of haloalkane dehalogenases occur in the specificity-modulating cap domains

    PubMed Central

    Otyepka, Michal; Damborský, Jiří

    2002-01-01

    One-nanosecond molecular dynamics trajectories of three haloalkane dehalogenases (DhlA, LinB, and DhaA) are compared. The main domain was rigid in all three dehalogenases, whereas the substrate specificity-modulating cap domains showed considerably higher mobility. The functionally relevant motions were spread over the entire cap domain in DhlA, whereas they were more localized in LinB and DhaA. The highest amplitude of essential motions of DhlA was noted in the α4′-helix-loop-α4-helix region, formerly proposed to participate in the large conformation change needed for product release. The highest amplitude of essential motions of LinB and DhaA was observed in the random coil before helix 4, linking two domains of these proteins. This flexibility is the consequence of the modular composition of haloalkane dehalogenases. Two members of the catalytic triad, that is, the nucleophile and the base, showed a very high level of rigidity in all three dehalogenases. This rigidity is essential for their function. One of the halide-stabilizing residues, important for the catalysis, shows significantly higher flexibility in DhlA compared with LinB and DhaA. Enhanced flexibility may be required for destabilization of the electrostatic interactions during the release of the halide ion from the deeply buried active site of DhlA. The exchange of water molecules between the enzyme active site and bulk solvent was very different among the three dehalogenases. The differences could be related to the flexibility of the cap domains and to the number of entrance tunnels. PMID:11967377

  19. Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression.

    PubMed Central

    Wek, R C; Ramirez, M; Jackson, B M; Hinnebusch, A G

    1990-01-01

    GCN4 is a transcriptional activator of amino acid-biosynthetic genes in the yeast Saccharomyces cerevisiae. GCN2, a translational activator of GCN4 expression, contains a domain homologous to the catalytic subunit of eucaryotic protein kinases. Substitution of a highly conserved lysine residue in the kinase domain abolished GCN2 regulatory function in vivo and its ability to autophosphorylate in vitro, indicating that GCN2 acts as a protein kinase in stimulating GCN4 expression. Elevated GCN2 gene dosage led to derepression of GCN4 under nonstarvation conditions; however, we found that GCN2 mRNA and protein levels did not increase in wild-type cells in response to amino acid starvation. Therefore, it appears that GCN2 protein kinase function is stimulated posttranslationally in amino acid-starved cells. Three dominant-constitutive GCN2 point mutations were isolated that led to derepressed GCN4 expression under nonstarvation conditions. Two of the GCN2(Con) mutations mapped in the kinase domain itself. The third mapped just downstream from a carboxyl-terminal segment homologous to histidyl-tRNA synthetase (HisRS), which we suggested might function to detect uncharged tRNA in amino acid-starved cells and activate the adjacent protein kinase moiety. Deletions and substitutions in the HisRS-related sequences and in the carboxyl-terminal segment in which one of the GCN2(Con) mutation mapped abolished GCN2 positive regulatory function in vivo without lowering autophosphorylation activity in vitro. These results suggest that sequences flanking the GCN2 protein kinase moiety are positive-acting domains required to increase recognition of physiological substrates or lower the requirement for uncharged tRNA to activate kinase activity under conditions of amino acid starvation. Images PMID:2188100

  20. Subcellular patterning: axonal domains with specialized structure and function

    PubMed Central

    Normand, Elizabeth A.; Rasband, Matthew N.

    2015-01-01

    Myelinated axons are patterned into discrete and often repeating domains responsible for the efficient and rapid transmission of electrical signals. These domains include nodes of Ranvier and axon initial segments. Disruption of axonal patterning leads to nervous system dysfunction. In this review we introduce the concept of subcellular patterning as applied to axons and discuss how these patterning events depend on both intrinsic, cytoskeletal mechanisms, and extrinsic, myelinating-glia dependent mechanisms. PMID:25710532

  1. Human FAD synthase is a bi-functional enzyme with a FAD hydrolase activity in the molybdopterin binding domain.

    PubMed

    Giancaspero, Teresa Anna; Galluccio, Michele; Miccolis, Angelica; Leone, Piero; Eberini, Ivano; Iametti, Stefania; Indiveri, Cesare; Barile, Maria

    2015-09-25

    FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is involved in the biochemical pathway for converting riboflavin into FAD. Human FADS exists in different isoforms. Two of these have been characterized and are localized in different subcellular compartments. hFADS2 containing 490 amino acids shows a two domain organization: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and a resembling molybdopterin-binding (MPTb) domain. By a multialignment of hFADS2 with other MPTb containing proteins of various organisms from bacteria to plants, the critical residues for hydrolytic function were identified. A homology model of the MPTb domain of hFADS2 was built, using as template the solved structure of a T. acidophilum enzyme. The capacity of hFADS2 to catalyse FAD hydrolysis was revealed. The recombinant hFADS2 was able to hydrolyse added FAD in a Co(2+) and mersalyl dependent reaction. The recombinant PAPS reductase domain is not able to perform the same function. The mutant C440A catalyses the same hydrolytic function of WT with no essential requirement for mersalyl, thus indicating the involvement of C440 in the control of hydrolysis switch. The enzyme C440A is also able to catalyse hydrolysis of FAD bound to the PAPS reductase domain, which is quantitatively converted into FMN. PMID:26277395

  2. Functional analysis of the chromo domain of HP1.

    PubMed Central

    Platero, J S; Hartnett, T; Eissenberg, J C

    1995-01-01

    Heterochromatin protein 1 (HP1) is a non-histone chromosomal protein in Drosophila with dosage-dependent effects on heterochromatin-mediated gene silencing. An evolutionarily conserved amino acid sequence in the N-terminal half of HP1 (the 'chromo domain') shares > 60% sequence identity with a motif found in the Polycomb protein, a silencer of homeotic genes. We report here that point mutations in the HP1 chromo domain abolish the ability of HP1 to promote gene silencing. We show that the HP1 chromo domain, like the Polycomb chromo domain, has chromosome binding activity, but to distinct chromosomal sites. We constructed a chimeric HP1-Polycomb protein, consisting of the chromo domain of Polycomb in the context of HP1, and show that it binds to both heterochromatin and Polycomb binding sites in polytene chromosomes. In flies expressing chimeric HP1-Polycomb protein, endogenous HP1 is mislocalized to Polycomb binding sites, and endogenous polycomb is misdirected to the heterochromatic chromocenter, suggesting that both proteins are recruited to their distinct chromosomal binding sites through protein-protein contacts. Chimeric HP1-Polycomb protein expression in transgenic flies promotes heterochromatin-mediated gene silencing, supporting the view that the chromo domain homology reflects a common mechanistic basis for homeotic and heterochromatic silencing. Images PMID:7664737

  3. Frequency domain transfer function identification using the computer program SYSFIT

    SciTech Connect

    Trudnowski, D.J.

    1992-12-01

    Because the primary application of SYSFIT for BPA involves studying power system dynamics, this investigation was geared toward simulating the effects that might be encountered in studying electromechanical oscillations in power systems. Although the intended focus of this work is power system oscillations, the studies are sufficiently genetic that the results can be applied to many types of oscillatory systems with closely-spaced modes. In general, there are two possible ways of solving the optimization problem. One is to use a least-squares optimization function and to write the system in such a form that the problem becomes one of linear least-squares. The solution can then be obtained using a standard least-squares technique. The other method involves using a search method to obtain the optimal model. This method allows considerably more freedom in forming the optimization function and model, but it requires an initial guess of the system parameters. SYSFIT employs this second approach. Detailed investigations were conducted into three main areas: (1) fitting to exact frequency response data of a linear system; (2) fitting to the discrete Fourier transformation of noisy data; and (3) fitting to multi-path systems. The first area consisted of investigating the effects of alternative optimization cost function options; using different optimization search methods; incorrect model order, missing response data; closely-spaced poles; and closely-spaced pole-zero pairs. Within the second area, different noise colorations and levels were studied. In the third area, methods were investigated for improving fitting results by incorporating more than one system path. The following is a list of guidelines and properties developed from the study for fitting a transfer function to the frequency response of a system using optimization search methods.

  4. Functional performance requirements for seismic network upgrade

    SciTech Connect

    Lee, R.C.

    1991-08-18

    The SRL seismic network, established in 1976, was developed to monitor site and regional seismic activity that may have any potential to impact the safety or reduce containment capability of existing and planned structures and systems at the SRS, report seismic activity that may be relevant to emergency preparedness, including rapid assessments of earthquake location and magnitude, and estimates of potential on-site and off-site damage to facilities and lifelines for mitigation measures. All of these tasks require SRL seismologists to provide rapid analysis of large amounts of seismic data. The current seismic network upgrade, the subject of this Functional Performance Requirements Document, is necessary to improve system reliability and resolution. The upgrade provides equipment for the analysis of the network seismic data and replacement of old out-dated equipment. The digital network upgrade is configured for field station and laboratory digital processing systems. The upgrade consists of the purchase and installation of seismic sensors,, data telemetry digital upgrades, a dedicated Seismic Data Processing (SDP) system (already in procurement stage), and a Seismic Signal Analysis (SSA) system. The field stations and telephone telemetry upgrades include equipment necessary for three remote station upgrades including seismic amplifiers, voltage controlled oscillators, pulse calibrators, weather protection (including lightning protection) systems, seismometers, seismic amplifiers, and miscellaneous other parts. The central receiving and recording station upgrades will include discriminators, helicopter amplifier, omega timing system, strong motion instruments, wide-band velocity sensors, and other miscellaneous equipment.

  5. CNS Myelination Requires Cytoplasmic Dynein Function

    PubMed Central

    Yang, Michele L.; Shin, Jimann; Kearns, Christina A.; Langworthy, Melissa M.; Snell, Heather; Walker, Macie B.; Appel, Bruce

    2014-01-01

    Background Cytoplasmic dynein provides the main motor force for minus-end-directed transport of cargo on microtubules. Within the vertebrate central nervous system (CNS), proliferation, neuronal migration and retrograde axon transport are among the cellular functions known to require dynein. Accordingly, mutations of DYNC1H1, which encodes the heavy chain subunit of cytoplasmic dynein, have been linked to developmental brain malformations and axonal pathologies. Oligodendrocytes, the myelinating glial cell type of the CNS, migrate from their origins to their target axons and subsequently extend multiple long processes that ensheath axons with specialized insulating membrane. These processes are filled with microtubules, which facilitate molecular transport of myelin components. However, whether oligodendrocytes require cytoplasmic dynein to ensheath axons with myelin is not known. Results We identified a mutation of zebrafish dync1h1 in a forward genetic screen that caused a deficit of oligodendrocytes. Using in vivo imaging and gene expression analyses, we additionally found evidence that dync1h1 promotes axon ensheathment and myelin gene expression. Conclusions In addition to its well known roles in axon transport and neuronal migration, cytoplasmic dynein contributes to neural development by promoting myelination. PMID:25488883

  6. N-terminal domain-mediated homodimerization is required for photoreceptor activity of Arabidopsis CRYPTOCHROME 1.

    PubMed

    Sang, Yi; Li, Qing-Hua; Rubio, Vicente; Zhang, Yan-Chun; Mao, Jian; Deng, Xing-Wang; Yang, Hong-Quan

    2005-05-01

    Cryptochromes (CRY) are blue light receptors that share sequence similarity with photolyases, flavoproteins that catalyze the repair of UV light-damaged DNA. Transgenic Arabidopsis thaliana seedlings expressing the C-terminal domains of the Arabidopsis CRY fused to beta-glucuronidase (GUS) display a constitutive photomorphogenic (COP) phenotype, indicating that the signaling mechanism of Arabidopsis CRY is mediated through the C-terminal domain. The role of the Arabidopsis CRY N-terminal photolyase-like domain in CRY action remains poorly understood. Here, we report the essential role of the Arabidopsis CRY1 N-terminal domain (CNT1) in the light activation of CRY1 photoreceptor activity. Yeast two-hybrid assay, in vitro binding, in vivo chemical cross-linking, gel filtration, and coimmunoprecipitation studies indicate that CRY1 homodimerizes in a light-independent manner. Mutagenesis and transgenic studies demonstrate that CNT1-mediated dimerization is required for light activation of the C-terminal domain of CRY1 (CCT1). Transgenic data and native gel electrophoresis studies suggest that multimerization of GUS is both responsible and required for mediating a COP phenotype on fusion to CCT1. These results indicate that the properties of the GUS multimer are analogous to those of the light-modified CNT1 dimer. Irradiation with blue light modifies the properties of the CNT1 dimer, resulting in a change in CCT1, activating CCT1, and eventually triggering the CRY1 signaling pathway. PMID:15805487

  7. Linking Functional Domains of the Human Insulin Receptor with the Bacterial Aspartate Receptor

    NASA Astrophysics Data System (ADS)

    Ellis, Leland; Morgan, David O.; Koshland, Daniel E.; Clauser, Eric; Moe, Gregory R.; Bollag, Gideon; Roth, Richard A.; Rutter, William J.

    1986-11-01

    A hybrid receptor has been constructed that is composed of the extracellular domain of the human insulin receptor fused to the transmembrane and cytoplasmic domains of the bacterial aspartate chemoreceptor. This hybrid protein can be expressed in rodent (CHO) cells and displays several functional features comparable to wild-type insulin receptor. It is localized to the cell surface, binds insulin with high affinity, forms oligomers, and is recognized by conformation-specific monoclonal antibodies. Although most of the expressed protein accumulates as a 180-kDa proreceptor, some processed 135-kDa receptor can be detected on the cell surface by covalent cross-linking. Expression of the hybrid receptor inhibits the insulin-activated uptake of 2-deoxyglucose by CHO cells. Thus, this hybrid is partially functional and can be processed; however, it is incapable of native transmembrane signaling. The results indicate that the intact domains of different types of receptors can retain some of the native features in a hybrid molecule but specific requirements will need to be satisfied for transmembrane signaling.

  8. Recombinant preparation and functional studies of EspI ATP binding domain from Mycobacterium tuberculosis.

    PubMed

    Chen, Hanyu; Wang, Huilin; Sun, Tao; Tian, Shuangliang; Lin, Donghai; Guo, Chenyun

    2016-07-01

    The ESX-1 secretion system of Mycobacterium tuberculosis is required for the virulence of tubercle bacillus. EspI, the ESX-1 secretion-associated protein in Mycobacterium tuberculosis (MtEspI), is involved in repressing the activity of ESX-1-mediated secretion when the cellular ATP level is low. The ATP binding domain of MtEspI plays a crucial role in this regulatory process. However, further structural and functional studies of MtEspI are hindered due to the bottleneck of obtaining stable and pure recombinant protein. In this study, we systematically analyzed the structure and function of MtEspI using bioinformatics tools and tried various expression constructs to recombinantly express full-length and truncated MtEspI ATP binding domain. Finally, we prepared pure and stable MtEspI ATP binding domain, MtEspI415-493, in Escherichia coli by fusion expression and purification with dual tag, Glutathione S-transferase (GST) tag and (His)6 tag. (31)P NMR titration assay indicated that MtEspI415-493 possessed a moderate affinity (∼μM) for ATP and the residue K425 was located at the binding site. The protocol described here may provide a train of thought for recombinant preparation of other ESX-1 secretion-associated proteins. PMID:27017992

  9. Structural and functional characterization of an atypical activation domain in erythroid Kruppel-like factor (EKLF).

    PubMed

    Mas, Caroline; Lussier-Price, Mathieu; Soni, Shefali; Morse, Thomas; Arseneault, Geneviève; Di Lello, Paola; Lafrance-Vanasse, Julien; Bieker, James J; Omichinski, James G

    2011-06-28

    Erythroid Krüppel-like factor (EKLF) plays an important role in erythroid development by stimulating β-globin gene expression. We have examined the details by which the minimal transactivation domain (TAD) of EKLF (EKLFTAD) interacts with several transcriptional regulatory factors. We report that EKLFTAD displays homology to the p53TAD and, like the p53TAD, can be divided into two functional subdomains (EKLFTAD1 and EKLFTAD2). Based on sequence analysis, we found that EKLFTAD2 is conserved in KLF2, KLF4, KLF5, and KLF15. In addition, we demonstrate that EKLFTAD2 binds the amino-terminal PH domain of the Tfb1/p62 subunit of TFIIH (Tfb1PH/p62PH) and four domains of CREB-binding protein/p300. The solution structure of the EKLFTAD2/Tfb1PH complex indicates that EKLFTAD2 binds Tfb1PH in an extended conformation, which is in contrast to the α-helical conformation seen for p53TAD2 in complex with Tfb1PH. These studies provide detailed mechanistic information into EKLFTAD functions as well as insights into potential interactions of the TADs of other KLF proteins. In addition, they suggest that not only have acidic TADs evolved so that they bind using different conformations on a common target, but that transitioning from a disordered to a more ordered state is not a requirement for their ability to bind multiple partners. PMID:21670263

  10. The RFTS Domain of Raf2 Is Required for Cul4 Interaction and Heterochromatin Integrity in Fission Yeast

    PubMed Central

    White, Sharon A.; Buscaino, Alessia; Sanchez-Pulido, Luis; Ponting, Chris P.; Nowicki, Matthew W.; Allshire, Robin C.

    2014-01-01

    Centromeric heterochromatin assembly in fission yeast is critical for faithful chromosome segregation at mitosis. Its assembly requires a concerted pathway of events whereby the RNA interference (RNAi) pathway guides H3K9 methylation to target sequences. H3K9 methylation, a hallmark of heterochromatin structure, is mediated by the single histone methyltransferase Clr4 (equivalent to metazoan Suv3-9), a component of the CLRC complex. Loss of or defects in CLRC components disrupts heterochromatin formation due to loss of H3K9 methylation, thus an intact, fully functional CLRC complex is required for heterochromatin integrity. Despite its importance, little is known about the contribution of the CLRC component Raf2 to H3K9 methylation and heterochromatin assembly. We demonstrate that Raf2 is concentrated at centromeres and contrary to other analyses, we find that loss of Raf2 does not affect CENP-ACnp1 localisation or recruitment to centromeres. Our sequence alignments show that Raf2 contains a Replication Foci Targeting Sequence (RFTS) domain homologous to the RFTS domain of the human DNA methyltransferase DNMT1. We show that the Raf2 RFTS domain is required for centromeric heterochromatin formation as its mutation disrupts H3K9 methylation but not the processing of centromeric transcripts into small interfering RNAs (siRNAs) by the RNAi pathway. Analysis of biochemical interactions demonstrates that the RFTS domain mediates an interaction between Raf2 and the CLRC component Cul4. We conclude that the RFTS domain of Raf2 is a protein interaction module that plays an important role in heterochromatin formation at centromeres. PMID:25090107

  11. Oncogenic transformation by vrel requires an amino-terminal activation domain

    SciTech Connect

    Kamens, J.; Brent, R. . Dept. of Molecular Biology); Richardson, P.; Gilmore, T. . Dept. of Biology); Mosialos, G. . Dept. of Chemistry)

    1990-06-01

    The mechanism by which the products of the v-{ital rel} oncogene, the corresponding c-{ital rel} proto-oncogene, and the related {ital dorsal} gene of {ital Drosophila melanogaster} exert their effects is not clear. The authors show that the v-{ital rel}, chicken c-{ital rel}, and {ital dorsal} proteins activated gene expression when fused to LexA sequences and bound to DNA upstream of target genes in {ital Saccharomyces cerevisiae}. They have defined two distinct activation regions in the c-{ital rel} protein. Region I, located in the amino-terminal half of {ital rel} and {ital dorsal} proteins, contains no stretches of glutamines, prolines, or acidic amino acids and therefore may be a novel activation domain. Lesions in the v-{ital rel} protein that diminished or abolished oncogenic transformation of avian spleen cells correspondingly affected transcription activation by region I. Region II, located in the carboxy terminus of the c-{ital rel} protein, is highly acidic. Region II is not present in the v-{ital rel} protein or in a transforming mutant derivative of the c-{ital rel} protein. The authors' results show that the oncogenicity of Rel proteins requires activation region I and suggest that the biological function of {ital rel} and {ital dorsal} proteins depends on transcription activation by this region.

  12. An essential single domain response regulator required for normal cell division and differentiation in Caulobacter crescentus.

    PubMed Central

    Hecht, G B; Lane, T; Ohta, N; Sommer, J M; Newton, A

    1995-01-01

    Signal transduction pathways mediated by sensor histidine kinases and cognate response regulators control a variety of physiological processes in response to environmental conditions. Here we show that in Caulobacter crescentus these systems also play essential roles in the regulation of polar morphogenesis and cell division. Previous studies have implicated histidine kinase genes pleC and divJ in the regulation of these developmental events. We now report that divK encodes an essential, cell cycle-regulated homolog of the CheY/Spo0F subfamily and present evidence that this protein is a cognate response regulator of the histidine kinase PleC. The purified kinase domain of PleC, like that of DivJ, can serve as an efficient phosphodonor to DivK and as a phospho-DivK phosphatase. Based on these and earlier genetic results we propose that PleC and DivK are members of a signal transduction pathway that couples motility and stalk formation to completion of a late cell division cycle event. Gene disruption experiments and the filamentous phenotype of the conditional divK341 mutant reveal that DivK also functions in an essential signal transduction pathway required for cell division, apparently in response to another histidine kinase. We suggest that phosphotransfer mediated by these two-component signal transduction systems may represent a general mechanism regulating cell differentiation and cell division in response to successive cell cycle checkpoints. Images PMID:7664732

  13. The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation

    PubMed Central

    Hennig, Janosch; Zou, Peijian; Nössner, Elfriede; Ling, Paul D.; Sattler, Michael; Kempkes, Bettina

    2015-01-01

    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. PMID:26024477

  14. The Evolution of Extracellular Fibrillins and Their Functional Domains

    PubMed Central

    Piha-Gossack, Adam; Sossin, Wayne; Reinhardt, Dieter P.

    2012-01-01

    Fibrillins constitute the major backbone of multifunctional microfibrils in elastic and non-elastic extracellular matrices, and are known to interact with several binding partners including tropoelastin and integrins. Here, we study the evolution of fibrillin proteins. Following sequence collection from 39 organisms representative of the major evolutionary groups, molecular evolutionary genetics and phylogeny inference software were used to generate a series of evolutionary trees using distance-based and maximum likelihood methods. The resulting trees support the concept of gene duplication as a means of generating the three vertebrate fibrillins. Beginning with a single fibrillin sequence found in invertebrates and jawless fish, a gene duplication event, which coincides with the appearance of elastin, led to the creation of two genes. One of the genes significantly evolved to become the gene for present-day fibrillin-1, while the other underwent evolutionary changes, including a second duplication, to produce present-day fibrillin-2 and fibrillin-3. Detailed analysis of several sequences and domains within the fibrillins reveals distinct similarities and differences across various species. The RGD integrin-binding site in TB4 of all fibrillins is conserved in cephalochordates and vertebrates, while the integrin-binding site within cbEGF18 of fibrillin-3 is a recent evolutionary change. The proline-rich domain in fibrillin-1, glycine-rich domain in fibrillin-2 and proline-/glycine-rich domain in fibrillin-3 are found in all analyzed tetrapod species, whereas it is completely replaced with an EGF-like domain in cnidarians, arthropods, molluscs and urochordates. All collected sequences contain the first 9-cysteine hybrid domain, and the second 8-cysteine hybrid domain with exception of arthropods containing an atypical 10-cysteine hybrid domain 2. Furin cleavage sites within the N- and C-terminal unique domains were found for all analyzed fibrillin sequences

  15. Conserved Features of the PB2 627 Domain Impact Influenza Virus Polymerase Function and Replication

    PubMed Central

    Kirui, James; Bucci, Michael D.; Poole, Daniel S.

    2014-01-01

    ABSTRACT Successful replication of influenza virus requires the coordinated expression of viral genes and replication of the genome by the viral polymerase, composed of the subunits PA, PB1, and PB2. Polymerase activity is regulated by both viral and host factors, yet the mechanisms of regulation and how they contribute to viral pathogenicity and tropism are poorly understood. To characterize these processes, we created a series of mutants in the 627 domain of the PB2 subunit. This domain contains a conserved “P[F/P]AAAPP” sequence motif and the well-described amino acid 627, whose identity regulates host range. A lysine present at position 627 in most mammalian viral isolates creates a basic face on the domain surface and confers high-level activity in humans compared to the glutamic acid found at this position in avian isolates. Mutation of the basic face or the P[F/P]AAAPP motif impaired polymerase activity, assembly of replication complexes, and viral replication. Most of these residues are required for general polymerase activity, whereas PB2 K586 and R589 were preferentially required for function in human versus avian cells. Thus, these data identify residues in the 627 domain and other viral proteins that regulate polymerase activity, highlighting the importance of the surface charge and structure of this domain for virus replication and host adaptation. IMPORTANCE Influenza virus faces barriers to transmission across species as it emerges from its natural reservoir in birds to infect mammals. The viral polymerase is an important regulator of this process and undergoes discrete changes to adapt to replication in mammals. Many of these changes occur in the polymerase subunit PB2. Here we describe the systematic analysis of a key region in PB2 that controls species-specific polymerase activity. We report the importance of conserved residues that contribute to the overall charge of the protein as well as those that likely affect protein structure. These

  16. Structure, function, and tethering of DNA-binding domains in σ⁵⁴ transcriptional activators.

    PubMed

    Vidangos, Natasha; Maris, Ann E; Young, Anisa; Hong, Eunmi; Pelton, Jeffrey G; Batchelor, Joseph D; Wemmer, David E

    2013-12-01

    We compare the structure, activity, and linkage of DNA-binding domains (DBDs) from σ(54) transcriptional activators and discuss how the properties of the DBDs and the linker to the neighboring domain are affected by the overall properties and requirements of the full proteins. These transcriptional activators bind upstream of specific promoters that utilize σ(54)-polymerase. Upon receiving a signal the activators assemble into hexamers, which then, through adenosine triphosphate (ATP) hydrolysis, drive a conformational change in polymerase that enables transcription initiation. We present structures of the DBDs of activators nitrogen regulatory protein C 1 (NtrC1) and Nif-like homolog 2 (Nlh2) from the thermophile Aquifex aeolicus. The structures of these domains and their relationship to other parts of the activators are discussed. These structures are compared with previously determined structures of the DBDs of NtrC4, NtrC, ZraR, and factor for inversion stimulation. The N-terminal linkers that connect the DBDs to the central domains in NtrC1 and Nlh2 were studied and found to be unstructured. Additionally, a crystal structure of full-length NtrC1 was solved, but density of the DBDs was extremely weak, further indicating that the linker between ATPase and DBDs functions as a flexible tether. Flexible linking of ATPase and DBDs is likely necessary to allow assembly of the active hexameric ATPase ring. The comparison of this set of activators also shows clearly that strong dimerization of the DBD only occurs when other domains do not dimerize strongly. PMID:23818155

  17. Functions of sorting nexin 17 domains and recognition motif for P-selectin trafficking.

    PubMed

    Knauth, Peter; Schlüter, Thomas; Czubayko, Martin; Kirsch, Cornelia; Florian, Volker; Schreckenberger, Susan; Hahn, Heidi; Bohnensack, Ralf

    2005-04-01

    SNX17 is a member of the sorting nexin family (SNX), a group of hydrophilic proteins whose common characteristic property is a phox homology (PX) domain. The PX domain directs SNXs to phosphatidylinositides containing membranes of the endosomal compartment, where the SNXs are involved in the sorting of transmembrane proteins. SNX17 is known to interact with P-selectin and the LDL receptor family. Here, we report that the PX domain of SNX17 specifically binds to phosphatidylinositol 3-phosphate-containing membranes. The functional part of SNX17 that binds P-selectin or Patched (PTCH) consists of a truncated FERM domain and a unique C terminus together (FC-unit). In a yeast two-hybrid analysis a putative recognition motif for the FC-unit was revealed within P-selectin as FxNaa(F/Y). When HepG2 cells overexpress P-selectin together with SNX17, SNX17 changes its distribution from early endosomes to lysobisphosphatidic acid-containing late endosomes. Furthermore, overexpressed SNX17 restrains P-selectin in the outer membrane of the late endosomal compartment, thus preventing the normal lysosomal accumulation of P-selectin. These results suggest that the PX domain is necessary for the intracellular localisation, while the FC-unit is required for cargo recognition. We hypothesise that the expression level of SNX17 may regulate the lysosomal degradation, at least for P-selectin, by suppressing its entry into the inner vesicles of the multi-vesicular bodies (MVBs). PMID:15769472

  18. Structural and functional conservation of key domains in InsP[subscript 3] and ryanodine receptors

    SciTech Connect

    Seo, Min-Duk; Velamakanni, Saroj; Ishiyama, Noboru; Stathopulos, Peter B.; Rossi, Ana M.; Khan, Samir A.; Dale, Philippa; Li, Congmin; Ames, James B.; Ikura, Mitsuhiko; Taylor, Colin W.

    2012-07-11

    Inositol-1,4,5-trisphosphate receptors (InsP{sub 3}Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca{sup 2+} channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP{sub 3}R gating is initiated by InsP{sub 3} binding to the InsP{sub 3}-binding core (IBC, residues 224-604 of InsP{sub 3}R1) and it requires the suppressor domain (SD, residues 1-223 of InsP{sub 3}R1). Here we present structures of the amino-terminal region (NT, residues 1-604) of rat InsP{sub 3}R1 with (3.6 {angstrom}) and without (3.0 {angstrom}) InsP{sub 3} bound. The arrangement of the three NT domains, SD, IBC-{beta} and IBC-{alpha}, identifies two discrete interfaces ({alpha} and {beta}) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR1 (ref. 9). The orientations of the three domains when docked into a tetrameric structure of InsP{sub 3}R and of the ABC domains docked into RyR are remarkably similar. The importance of the {alpha}-interface for activation of InsP{sub 3}R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations. Binding of InsP{sub 3} causes partial closure of the clam-like IBC, disrupting the {beta}-interface and pulling the SD towards the IBC. This reorients an exposed SD loop ('hotspot' (HS) loop) that is essential for InsP{sub 3}R activation. The loop is conserved in RyR and includes mutations that are associated with malignant hyperthermia and central core disease. The HS loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A domain of RyR functionally replaced the SD in full-length InsP{sub 3}R, and an InsP{sub 3}R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP{sub 3} and blocked by ryanodine. Activation mechanisms are conserved between InsP{sub 3}R and Ry

  19. Cellulase Linkers Are Optimized Based on Domain Type and Function: Insights from Sequence Analysis, Biophysical Measurements, and Molecular Simulation

    PubMed Central

    Sammond, Deanne W.; Payne, Christina M.; Brunecky, Roman; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

    2012-01-01

    Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs) to carbohydrate-binding modules (CBMs). Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests that cellulase

  20. Functional Analysis of a Bacterial Antifreeze Protein Indicates a Cooperative Effect between Its Two Ice-Binding Domains.

    PubMed

    Wang, Chen; Oliver, Erin E; Christner, Brent C; Luo, Bing-Hao

    2016-07-19

    Antifreeze proteins make up a class of ice-binding proteins (IBPs) that are possessed and expressed by certain cold-adapted organisms to enhance their freezing tolerance. Here we report the biophysical and functional characterization of an IBP discovered in a bacterium recovered from a deep glacial ice core drilled at Vostok Station, Antarctica (IBPv). Our study showed that the recombinant protein rIBPv exhibited a thermal hysteresis of 2 °C at concentrations of >50 μM, effectively inhibited ice recrystallization, and enhanced bacterial viability during freeze-thaw cycling. Circular dichroism scans indicated that rIBPv mainly consists of β strands, and its denaturing temperature was 53.5 °C. Multiple-sequence alignment of homologous IBPs predicted that IBPv contains two ice-binding domains, a feature unique among known IBPs. To examine functional differences between the IBPv domains, each domain was cloned, expressed, and purified. The second domain (domain B) expressed greater ice binding activity. Data from thermal hysteresis and gel filtration assays supported the idea that the two domains cooperate to achieve a higher ice binding effect by forming heterodimers. However, physical linkage of the domains was not required for this effect. PMID:27359086

  1. Functional analysis of the transmembrane domain in paramyxovirus F protein-mediated membrane fusion

    PubMed Central

    Bissonnette, Mei Lin Z.; Donald, Jason E.; DeGrado, William F.; Jardetzky, Theodore S.; Lamb, Robert A.

    2009-01-01

    To enter cells enveloped viruses use fusion-mediating glycoproteins to facilitate the merger of the viral and host cell membranes. These glycoproteins undergo large-scale irreversible refolding during membrane fusion. The paramyxovirus parainfluenza virus 5 (PIV5) mediates membrane merger through its fusion protein (F). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion. The TM domain of F is particularly interesting in that it is potentially unusually long; multiple calculations suggest a TM helix length between 25 and 48 residues. Oxidative cross-linking of single cysteine substitutions indicates the F TM trimer forms a helical bundle within the membrane. To assess the functional role of the PIV5 F protein TM domain, alanine scanning mutagenesis was performed. Two residues located in the outer leaflet of the bilayer are critical for fusion. Multiple amino acid substitutions at these positions indicate the physical properties of the side chain play a critical role in supporting or blocking fusion. Analysis of intermediate steps in F protein refolding indicated that the mutants were not trapped at the open stalk intermediate or the prehairpin intermediate. Incorporation of a known F protein destabilizing mutation that causes a hyperfusogenic phenotype restored fusion activity to the mutants. Further, altering the curvature of the lipid bilayer by addition of oleic acid promoted fusion of the F protein mutants. In aggregate, these data indicate that the TM domain plays a functional role in fusion beyond merely anchoring the protein in the viral envelope and that it can affect the structures and steady-state concentrations of the various conformational intermediates en route to the final postfusion state. We suggest that the unusual length of this TM helix might allow it to serve as a template for formation of or specifically stabilize the lipid stalk intermediate in fusion. PMID:19121325

  2. LRR Conservation Mapping to Predict Functional Sites within Protein Leucine-Rich Repeat Domains

    PubMed Central

    Helft, Laura; Reddy, Vignyan; Chen, Xiyang; Koller, Teresa; Federici, Luca; Fernández-Recio, Juan; Gupta, Rishabh; Bent, Andrew

    2011-01-01

    Computational prediction of protein functional sites can be a critical first step for analysis of large or complex proteins. Contemporary methods often require several homologous sequences and/or a known protein structure, but these resources are not available for many proteins. Leucine-rich repeats (LRRs) are ligand interaction domains found in numerous proteins across all taxonomic kingdoms, including immune system receptors in plants and animals. We devised Repeat Conservation Mapping (RCM), a computational method that predicts functional sites of LRR domains. RCM utilizes two or more homologous sequences and a generic representation of the LRR structure to identify conserved or diversified patches of amino acids on the predicted surface of the LRR. RCM was validated using solved LRR+ligand structures from multiple taxa, identifying ligand interaction sites. RCM was then used for de novo dissection of two plant microbe-associated molecular pattern (MAMP) receptors, EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2). In vivo testing of Arabidopsis thaliana EFR and FLS2 receptors mutagenized at sites identified by RCM demonstrated previously unknown functional sites. The RCM predictions for EFR, FLS2 and a third plant LRR protein, PGIP, compared favorably to predictions from ODA (optimal docking area), Consurf, and PAML (positive selection) analyses, but RCM also made valid functional site predictions not available from these other bioinformatic approaches. RCM analyses can be conducted with any LRR-containing proteins at www.plantpath.wisc.edu/RCM, and the approach should be modifiable for use with other types of repeat protein domains. PMID:21789174

  3. Agrobacterium VirB10 domain requirements for type IV secretion and T pilus biogenesis

    PubMed Central

    Jakubowski, Simon J.; Kerr, Jennifer E.; Garza, Isaac; Krishnamoorthy, Vidhya; Bayliss, Richard; Waksman, Gabriel; Christie, Peter J.

    2013-01-01

    Summary Agrobacterium tumefaciens VirB10 couples inner membrane (IM) ATP energy consumption to substrate transfer through the VirB/D4 type IV secretion (T4S) channel and also mediates biogenesis of the virB-encoded T pilus. Here, we determined the functional importance of VirB10 domains denoted as the: (i) N-terminal cytoplasmic region, (ii) transmembrane (TM) α-helix, (iii) proline-rich region (PRR) and (iv) C-terminal β-barrel domain. Mutations conferring a transfer- and pilus-minus (Tra−, Pil−) phenotype included PRR deletion and β-barrel substitution mutations that prevented VirB10 interaction with the outer membrane (OM) VirB7–VirB9 channel complex. Mutations permissive for substrate transfer but blocking pilus production (Tra+, Pil−) included a cytoplasmic domain deletion and TM domain insertion mutations. Another class of Tra+ mutations also selectively disrupted pilus biogenesis but caused release of pilin monomers to the milieu; these mutations included deletions of α-helical projections extending from the β-barrel domain. Our findings, together with results of Cys accessibility studies, indicate that VirB10 stably integrates into the IM, extends via its PRR across the periplasm, and interacts via its β-barrel domain with the VirB7–VirB9 channel complex. The data further support a model that distinct domains of VirB10 regulate formation of the secretion channel or the T pilus. PMID:19054325

  4. Functional dissection of the global repressor Tup1 in yeast: dominant role of the C-terminal repression domain.

    PubMed Central

    Zhang, Zhizhou; Varanasi, Ushasri; Trumbly, Robert J

    2002-01-01

    In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general repressor of transcription. Tup1 and Cyc8 are required for repression of diverse families of genes coordinately controlled by glucose repression, mating type, and other mechanisms. This repression is mediated by recruitment of the Cyc8-Tup1 complex to target promoters by sequence-specific DNA-binding proteins. We created a library of XhoI linker insertions and internal in-frame deletion mutations within the TUP1 coding region. Insertion mutations outside of the WD domains were wild type, while insertions within the WD domains induced mutant phenotypes with differential effects on the target genes SUC2, MFA2, RNR2, and HEM13. Deletion mutations confirmed previous findings of two separate repression domains in the N and C termini. The cumulative data suggest that the C-terminal repression domain, located near the first WD repeat, plays the dominant role in repression. Although the N-terminal repression domain is sufficient for partial repression, deletion of this region does not compromise repression. Surprisingly, deletion of the majority of the histone-binding domain of Tup1 also does not significantly reduce repression. The N-terminal region containing potential alpha-helical coiled coils is required for Tup1 oligomerization and association with Cyc8. Association with Cyc8 is required for repression of SUC2, HEM13, and RNR2 but not MFA2 and STE2. PMID:12136003

  5. Analysis of Sir2p domains required for rDNA and telomeric silencing in Saccharomyces cerevisiae.

    PubMed Central

    Cockell, M M; Perrod, S; Gasser, S M

    2000-01-01

    Silent information regulator (Sir) 2 is a limiting component of the Sir2/3/4 complex, which represses transcription at subtelomeric and HM loci. Sir2p also acts independently of Sir3p and Sir4p to influence chromatin organization in the rDNA locus. Deleted and mutated forms of Sir2p have been tested for their ability to complement and/or to disrupt silencing. The highly conserved C-terminal domain of Sir2p (aa 199-562) is insufficient to restore repression at either telomeric or rDNA reporters in a sir2Delta background and fails to nucleate silencing when targeted to an appropriate reporter gene. However, its expression in an otherwise wild-type strain disrupts telomeric repression. Similarly, a point mutation (P394L) within this conserved core inactivates the full-length protein but renders it dominant negative for all types of silencing. Deletion of aa 1-198 from Sir2(394L) eliminates its dominant negative effect. Thus we define two distinct functional domains in Sir2p, both essential for telomeric and rDNA repression: the conserved core domain found within aa 199-562 and a second domain that encompasses aa 94-198. Immunolocalization and two-hybrid studies show that aa 94-198 are required for the binding of Sir2p to Sir4p and for the targeting of Sir2p to the nucleolus through another ligand. The globular core domain provides an essential silencing function distinct from that of targeting or Sir complex formation that may reflect its reported mono-ADP-ribosyl transferase activity. PMID:10757754

  6. Splicing factor SF1 from Drosophila and Caenorhabditis: presence of an N-terminal RS domain and requirement for viability.

    PubMed Central

    Mazroui, R; Puoti, A; Krämer, A

    1999-01-01

    Splicing factor SF1 contributes to the recognition of the 3' splice site by interacting with U2AF65 and binding to the intron branch site during the formation of the early splicing complex E. These interactions and the essential functional domains of SF1 are highly conserved in Saccharomyces cerevisiae. We have isolated cDNAs encoding SF1 from Drosophila (Dm) and Caenorhabditis (Ce). The encoded proteins share the U2AF65 interaction domain, a hnRNP K homology domain, and one or two zinc knuckles required for RNA binding as well as Pro-rich C-terminal sequences with their yeast and mammalian counterparts. In contrast to SF1 in other species, DmSF1 and CeSF1 are characterized by an N-terminal region enriched in Ser, Arg, Lys, and Asp residues with homology to the RS domains of several splicing proteins. These domains mediate protein-protein or protein-RNA interactions, suggesting an additional role for DmSF1 and CeSF1 in pre-mRNA splicing. Human (Hs), fly, and worm SF1 interact equally well with HsU2AF65 or the Drosophila homolog DmU2AF50. Moreover, DmSF1 lacking its N terminus is functional in prespliceosome formation in a HeLa splicing system, emphasizing the conserved nature of interactions at an early step in spliceosome assembly. The Ce-SF1 gene is located in a polycistronic transcription unit downstream of the genes encoding U2AF35 (uaf-2) and a cyclophilin (cyp-13), implying the coordinate transcriptional regulation of these genes. Injection of double-stranded RNA into C. elegans results in embryonic lethality; thus, the SF1 gene is essential not only in yeast but also in at least one metazoan. PMID:10606272

  7. Structural And Functional Studies of ALIX Interactions With YPXnL Late Domains of HIV-1 And EIAV

    SciTech Connect

    Zhai, Q.; Fisher, R.D.; Chung, H.-Y.; Myszka, D.G.; Sundquist, W.I.; Hill, C.P.

    2009-05-28

    Retrovirus budding requires short peptide motifs (late domains) located within the viral Gag protein that function by recruiting cellular factors. The YPX{sub n}L late domains of HIV and other lentiviruses recruit the protein ALIX (also known as AIP1), which also functions in vesicle formation at the multivesicular body and in the abscission stage of cytokinesis. Here, we report the crystal structures of ALIX in complex with the YPX{sub n}L late domains from HIV-1 and EIAV. The two distinct late domains bind at the same site on the ALIX V domain but adopt different conformations that allow them to make equivalent contacts. Binding studies and functional assays verified the importance of key interface residues and revealed that binding affinities are tuned by context-dependent effects. These results reveal how YPX{sub n}L late domains recruit ALIX to facilitate virus budding and how ALIX can bind YPX{sub n}L sequences with both n = 1 and n = 3.

  8. Structural Heterogeneity and Functional Domains of Murine Immunoglobulin G Fc Receptors

    NASA Astrophysics Data System (ADS)

    Ravetch, Jeffrey V.; Luster, Andrew D.; Weinshank, Richard; Kochan, Jarema; Pavlovec, Amalia; Portnoy, Daniel A.; Hulmes, Jeffrey; Pan, Yu-Ching E.; Unkeless, Jay C.

    1986-11-01

    Binding of antibodies to effector cells by way of receptors to their constant regions (Fc receptors) is central to the pathway that leads to clearance of antigens by the immune system. The structure and function of this important class of receptors on immune cells is addressed through the molecular characterization of Fc receptors (FcR) specific for the murine immunoglobulin G isotype. Structural diversity is encoded by two genes that by alternative splicing result in expression of molecules with highly conserved extracellular domains and different transmembrane and intracytoplasmic domains. The proteins encoded by these genes are members of the immunoglobulin supergene family, most homologous to the major histocompatibility complex molecule Eβ. Functional reconstitution of ligand binding by transfection of individual FcR genes demonstrates that the requirements for ligand binding are encoded in a single gene. These studies demonstrate the molecular basis for the functional heterogeneity of FcR's, accounting for the possible transduction of different signals in response to a single ligand.

  9. Insights into Hox Protein Function from a Large Scale Combinatorial Analysis of Protein Domains

    PubMed Central

    Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K.; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

    2011-01-01

    Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences. PMID:22046139

  10. Insights into Hox protein function from a large scale combinatorial analysis of protein domains.

    PubMed

    Merabet, Samir; Litim-Mecheri, Isma; Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

    2011-10-01

    Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences. PMID:22046139

  11. ELMO Domains, Evolutionary and Functional Characterization of a Novel GTPase-activating Protein (GAP) Domain for Arf Protein Family GTPases*

    PubMed Central

    East, Michael P.; Bowzard, J. Bradford; Dacks, Joel B.; Kahn, Richard A.

    2012-01-01

    The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases. PMID:23014990

  12. Identification of zero density of states domains in band gap fibers using a single binary function.

    PubMed

    Li, Guangrui; Schmidt, Markus A

    2016-07-11

    Here we introduce a new calculation method to find the domains of zero density of states for photonic band gap guiding fibers consisting of arrays of high refractive index strands in a low refractive index cladding. We find an analytic expression that associates any combination of geometric parameter, effective index, material and wavelength with a single binary function which allows direct determination whether the density of cladding states is zero or not. The method neither requires the typically used root finding procedure for dispersion tracking nor simulation volume discretization. We verify the validity of our approach on well-established results and reveal as example that band gap regions are mainly determined by the two lowest order Bessel function orders. Our method allows for extensive parameter scans and evaluation of photonic band gap structures against structural and material inaccuracies with substantially reduced simulation effort. PMID:27410888

  13. Function analysis of a new type I PKS-SAT domain by SAT-EAT domain replacement.

    PubMed

    Jiao, Y L; Wang, L H; Jiao, B H; Wang, S J; Fang, Y W; Liu, S

    2010-01-01

    The function of a new starter unit acyltransferase (SAT) domain SAT-EF080951 (GenBank accession number) encoded in a new type I polyketide synthase (PKS) gene cluster EF568935 (GenBank accession number) isolated for this study was analyzed by domain replacement with an extender unit AT (EAT) domain of avermectin PKS. It was shown that the SAT-EF080951 incorporated malonyl-CoA specifically in vivo, which contradicted the specificity that we had previously determined by substrate binding test in vitro. The result of this study indicates that type I PKS-SAT can alter its specificity in vivo and functions well in extender units and proved the feasibility of the SAT-EAT domain replacement in type I PKS. We propose that SAT-EAT replacement strategy could be a novel route for increasing the diversity of new polyketides combinatorially biosynthesized. The new type I PKS-SAT-EF080951 studied herein may be further employed for related studies on enzymology or combinatorial biosynthesis of polyketides. PMID:20391758

  14. Expanding the Landscape of Chromatin Modification (CM)-Related Functional Domains and Genes in Human

    PubMed Central

    Pu, Shuye; Turinsky, Andrei L.; Vlasblom, James; On, Tuan; Xiong, Xuejian; Emili, Andrew; Zhang, Zhaolei; Greenblatt, Jack; Parkinson, John; Wodak, Shoshana J.

    2010-01-01

    Chromatin modification (CM) plays a key role in regulating transcription, DNA replication, repair and recombination. However, our knowledge of these processes in humans remains very limited. Here we use computational approaches to study proteins and functional domains involved in CM in humans. We analyze the abundance and the pair-wise domain-domain co-occurrences of 25 well-documented CM domains in 5 model organisms: yeast, worm, fly, mouse and human. Results show that domains involved in histone methylation, DNA methylation, and histone variants are remarkably expanded in metazoan, reflecting the increased demand for cell type-specific gene regulation. We find that CM domains tend to co-occur with a limited number of partner domains and are hence not promiscuous. This property is exploited to identify 47 potentially novel CM domains, including 24 DNA-binding domains, whose role in CM has received little attention so far. Lastly, we use a consensus Machine Learning approach to predict 379 novel CM genes (coding for 329 proteins) in humans based on domain compositions. Several of these predictions are supported by very recent experimental studies and others are slated for experimental verification. Identification of novel CM genes and domains in humans will aid our understanding of fundamental epigenetic processes that are important for stem cell differentiation and cancer biology. Information on all the candidate CM domains and genes reported here is publicly available. PMID:21124763

  15. Transforming User Needs into Functional Requirements for an Antibiotic Clinical Decision Support System

    PubMed Central

    Bright, T.J.

    2013-01-01

    Summary Background Many informatics studies use content analysis to generate functional requirements for system development. Explication of this translational process from qualitative data to functional requirements can strengthen the understanding and scientific rigor when applying content analysis in informatics studies. Objective To describe a user-centered approach transforming emergent themes derived from focus group data into functional requirements for informatics solutions and to illustrate these methods to the development of an antibiotic clinical decision support system (CDS). Methods The approach consisted of five steps: 1) identify unmet therapeutic planning information needs via Focus Group Study-I, 2) develop a coding framework of therapeutic planning themes to refine the domain scope to antibiotic therapeutic planning, 3) identify functional requirements of an antibiotic CDS system via Focus Group Study-II, 4) discover informatics solutions and functional requirements from coded data, and 5) determine the types of information needed to support the antibiotic CDS system and link with the identified informatics solutions and functional requirements. Results The coding framework for Focus Group Study-I revealed unmet therapeutic planning needs. Twelve subthemes emerged and were clustered into four themes; analysis indicated a need for an antibiotic CDS intervention. Focus Group Study-II included five types of information needs. Comments from the Barrier/Challenge to information access and Function/Feature themes produced three informatics solutions and 13 functional requirements of an antibiotic CDS system. Comments from the Patient, Institution, and Domain themes generated required data elements for each informatics solution. Conclusion This study presents one example explicating content analysis of focus group data and the analysis process to functional requirements from narrative data. Illustration of this 5-step method was used to develop an

  16. Correspondence between immunological and functional domains in the transforming protein of Fujinami sarcoma virus.

    PubMed Central

    Stone, J C; Pawson, T

    1985-01-01

    Monoclonal antibodies reactive with either gag or fps portions of the wild-type Fujinami sarcoma virus transforming protein have been used to probe the structure of proteins encoded by mutant genomes constructed in vitro. The pattern of immunoreactivity suggests that the functional domain defined in genetic studies (Stone et al., Cell 37:549-558, 1984) corresponds to a discrete immunological domain in the native, wild-type Fujinami sarcoma virus protein. At least one mutation affecting both the structure and function of the proposed NH2-terminal fps-specific domain encodes a product with high specific activities in kinase assays. Furthermore, a cell line expressing high levels of this mutant protein is only moderately transformed. The striking correspondence between the immunological domain defined here and the functional domain inferred from the results of transfection experiments suggests that this non-kinase-specifying region constitutes a discrete structural as well as functional component of the viral protein. Images PMID:2991592

  17. Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1

    PubMed Central

    Evans, Chantell S.; He, Zixuan; Bai, Hua; Lou, Xiaochu; Jeggle, Pia; Sutton, R. Bryan; Edwardson, J. Michael; Chapman, Edwin R.

    2016-01-01

    C2 domains are widespread motifs that often serve as Ca2+-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca2+ sensor for neuronal exocytosis. Substitution of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca2+, and shifted the Ca2+ dependency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions between the tandem C2 domains of syt-1 contribute to excitation–secretion coupling. PMID:26792839

  18. CAN A FUNCTION-BASED THERAPY FOR SPOUSALLY BEREAVED SENIORS ACCRUE BENEFITS IN BOTH FUNCTIONAL AND EMOTIONAL DOMAINS?

    PubMed Central

    Pfoff, Marissa K.; Zarotney, Joette R.; Monk, Timothy H.

    2013-01-01

    Late-life spousal bereavement has detrimental effects in daily functioning and emotional distress. This study tested the hypothesis that a therapy based exclusively upon functioning issues (sleep and daily lifestyle regularity) would accrue benefits in both functional and emotional domains. A comparison was made with a control therapy which concentrated on emotional issues, and specifically avoided discussing sleep or lifestyle regularity. Thirty-eight spousally bereaved seniors were randomly assigned to either functional or control therapy. Assessments were made before, during and after a 6-month, 10-session individual therapy. The functional therapy assisted in both functional and emotional domains and the hypothesis was confirmed. PMID:24666144

  19. On the function of chitin synthase extracellular domains in biomineralization.

    PubMed

    Weiss, Ingrid M; Lüke, Florian; Eichner, Norbert; Guth, Christina; Clausen-Schaumann, Hauke

    2013-08-01

    Molluscs with various shell architectures evolved around 542-525 million years ago, as part of a larger phenomenon related to the diversification of metazoan phyla. Molluscs deposit minerals in a chitin matrix. The mollusc chitin is synthesized by transmembrane enzymes that contain several unique extracellular domains. Here we investigate the assembly mechanism of the chitin synthase Ar-CS1 via its extracellular domain ArCS1_E22. The corresponding transmembrane protein ArCS1_E22TM accumulates in membrane fractions of the expression host Dictyostelium discoideum. Soluble recombinant ArCS1_E22 proteins can be purified as monomers only at basic pH. According to confocal fluorescence microscopy experiments, immunolabeled ArCS1_E22 proteins adsorb preferably to aragonitic nacre platelets at pH 7.75. At pH 8.2 or pH 9.0 the fluorescence signal is less intense, indicating that protein-mineral interaction is reduced with increasing pH. Furthermore, ArCS1_E22 forms regular nanostructures on cationic substrates as revealed by atomic force microscopy (AFM) experiments on modified mica cleavage planes. These experiments suggest that the extracellular domain ArCS1_E22 is involved in regulating the multiple enzyme activities of Ar-CS1 such as chitin synthesis and myosin movements by interaction with mineral surfaces and eventually by protein assembly. The protein complexes could locally probe the status of mineralization according to pH unless ions and pCO2 are balanced with suitable buffer substances. Taking into account that the intact enzyme could act as a force sensor, the results presented here provide further evidence that shell formation is coordinated physiologically with precise adjustment of cellular activities to the structure, topography and stiffness at the mineralizing interface. PMID:23643908

  20. Structure and Functional Studies of the CS Domain of the Essential H/ACA Ribonucleoparticle Assembly Protein SHQ1

    SciTech Connect

    Singh, Mahavir; Gonzales, Fernando A.; Cascio, Duilio; Heckmann, Nathanael; Chanfreau, Guillaume; Feigon, Juli

    2009-03-16

    H/ACA ribonucleoprotein particles are essential for ribosomal RNA and telomerase RNA processing and metabolism. Shq1p has been identified as an essential eukaryotic H/ACA small nucleolar (sno) ribonucleoparticle (snoRNP) biogenesis and assembly factor. Shq1p is postulated to be involved in the early biogenesis steps of H/ACA snoRNP complexes, and Shq1p depletion leads to a specific decrease in H/ACA small nucleolar RNA levels and to defects in ribosomal RNA processing. Shq1p contains two predicted domains as follows: an N-terminal CS (named after CHORD-containing proteins and SGT1) or HSP20-like domain, and a C-terminal region of high sequence homology called the Shq1 domain. Here we report the crystal structure and functional studies of the Saccharomyces cerevisiae Shq1p CS domain. The structure consists of a compact anti-parallel {beta}-sandwich fold that is composed of two {beta}-sheets containing four and three {beta}-strands, respectively, and a short {alpha}-helix. Deletion studies showed that the CS domain is required for the essential functions of Shq1p. Point mutations in residues Phe-6, Gln-10, and Lys-80 destabilize Shq1p in vivo and induce a temperature-sensitive phenotype with depletion of H/ACA small nucleolar RNAs and defects in rRNA processing. Although CS domains are frequently found in co-chaperones of the Hsp90 molecular chaperone, no interaction was detected between the Shq1p CS domain and yeast Hsp90 in vitro. These results show that the CS domain is essential for Shq1p function in H/ACA snoRNP biogenesis in vivo, possibly in an Hsp90-independent manner.

  1. Function of steroidogenic factor 1 domains in nuclear localization, transactivation, and interaction with transcription factor TFIIB and c-Jun.

    PubMed

    Li, L A; Chiang, E F; Chen, J C; Hsu, N C; Chen, Y J; Chung, B C

    1999-09-01

    Normal endocrine development and function require nuclear hormone receptor SF-1 (steroidogenic factor 1). To understand the molecular mechanism of SF-1 action, we have investigated its domain function by mutagenesis and functional analyses. Our mutant studies show that the putative AF2 (activation function 2) helix located at the C-terminal end is indispensable for gene activation. SF-1 does not have an N-terminal AF1 domain. Instead, it contains a unique FP region, composed of the Ftz-F1 box and the proline cluster, after the zinc finger motif. The FP region interacts with transcription factor IIB (TFIIB) in vitro. This interaction requires residues 178-201 of TFIIB, a domain capable of binding several transcription factors. The FP region also mediates physical interaction with c-Jun, and this interaction greatly enhances SF-1 activity. The putative SF-1 ligand, 25-hydroxycholesterol, has no effects on these bindings. In addition, the Ftz-F1 box contains a bipartite nuclear localization signal (NLS). Removing the basic residues at either end of the key nuclear localization sequence NLS2.2 abolishes the nuclear transport. Expression of mutants containing only the FP region or lacking the AF2 domain blocks wild-type SF-1 activity in cells. By contrast, the mutant having a truncated nuclear localization signal lacks this dominant negative effect. These results delineate the importance of the FP and AF2 regions in nuclear localization, protein-protein interaction, and transcriptional activation. PMID:10478848

  2. How do disordered regions achieve comparable functions to structured domains?

    PubMed Central

    Latysheva, Natasha S; Flock, Tilman; Weatheritt, Robert J; Chavali, Sreenivas; Babu, M Madan

    2015-01-01

    The traditional structure to function paradigm conceives of a protein's function as emerging from its structure. In recent years, it has been established that unstructured, intrinsically disordered regions (IDRs) in proteins are equally crucial elements for protein function, regulation and homeostasis. In this review, we provide a brief overview of how IDRs can perform similar functions to structured proteins, focusing especially on the formation of protein complexes and assemblies and the mediation of regulated conformational changes. In addition to highlighting instances of such functional equivalence, we explain how differences in the biological and physicochemical properties of IDRs allow them to expand the functional and regulatory repertoire of proteins. We also discuss studies that provide insights into how mutations within functional regions of IDRs can lead to human diseases. PMID:25752799

  3. Hanford tanks initiative test facility functions and requirements

    SciTech Connect

    Krieg, S.A., Fluor Daniel Hanford

    1997-03-01

    This document presents the functions and requirements for a test facility for testing single-shell tank waste retrieval equipment and systems for the Hanford Tanks Initiative (HTI) project. This effort includes review of previous test facility functions and requirements and conducting a workshop to develop specific functions and requirements for HTI testing needs. Functions and requirements for testing future retrieval systems that follow HTI are also identified.

  4. Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia.

    PubMed

    Baldursson, O; Berger, H A; Welsh, M J

    2000-11-01

    The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl(-) current. Mutation of four in vivo phosphorylation sites, Ser(660), Ser(737), Ser(795), and Ser(813) (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser(660) or Ser(813) alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser(660) nor Ser(813) alone increased current to wild-type levels; both residues were required. Changing Ser(737) to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser(737) may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines. PMID:11053017

  5. Domain-specific functional software testing: A progress report

    NASA Technical Reports Server (NTRS)

    Nonnenmann, Uwe

    1992-01-01

    Software Engineering is a knowledge intensive activity that involves defining, designing, developing, and maintaining software systems. In order to build effective systems to support Software Engineering activities, Artificial Intelligence techniques are needed. The application of Artificial Intelligence technology to Software Engineering is called Knowledge-based Software Engineering (KBSE). The goal of KBSE is to change the software life cycle such that software maintenance and evolution occur by modifying the specifications and then rederiving the implementation rather than by directly modifying the implementation. The use of domain knowledge in developing KBSE systems is crucial. Our work is mainly related to one area of KBSE that is called automatic specification acquisition. One example is the WATSON prototype on which our current work is based. WATSON is an automatic programming system for formalizing specifications for telephone switching software mainly restricted to POTS, i.e., plain old telephone service. Our current approach differentiates itself from other approaches in two antagonistic ways. On the one hand, we address a large and complex real-world problem instead of a 'toy domain' as in many research prototypes. On the other hand, to allow such scaling, we had to relax the ambitious goal of complete automatic programming, to the easier task of automatic testing.

  6. On a New Class of p-Valent Meromorphic Functions Defined in Conic Domains

    PubMed Central

    Alamri, Mohammed Ali

    2016-01-01

    We define a new class of multivalent meromorphic functions using the generalised hypergeometric function. We derived this class related to conic domain. It is also shown that this new class of functions, under certain conditions, becomes a class of starlike functions. Some results on inclusion and closure properties are also derived. PMID:27529076

  7. On a New Class of p-Valent Meromorphic Functions Defined in Conic Domains.

    PubMed

    Alamri, Mohammed Ali; Darus, Maslina

    2016-01-01

    We define a new class of multivalent meromorphic functions using the generalised hypergeometric function. We derived this class related to conic domain. It is also shown that this new class of functions, under certain conditions, becomes a class of starlike functions. Some results on inclusion and closure properties are also derived. PMID:27529076

  8. A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution.

    PubMed

    Ostedgaard, L S; Baldursson, O; Vermeer, D W; Welsh, M J; Robertson, A D

    2000-05-01

    Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708-831 of CFTR. Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional. Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis. The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8. These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity. PMID:10792060

  9. A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution

    PubMed Central

    Ostedgaard, Lynda S.; Baldursson, Olafur; Vermeer, Daniel W.; Welsh, Michael J.; Robertson, Andrew D.

    2000-01-01

    Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708–831 of CFTR. Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional. Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis. The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8. These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity. PMID:10792060

  10. Psychosocial functioning in schizophrenia: are some symptoms or demographic characteristics predictors across the functioning domains?

    PubMed Central

    Suttajit, Sirijit; Arunpongpaisal, Suwanna; Srisurapanont, Manit; Thavichachart, Nuntika; Kongsakon, Ronnachai; Chantakarn, Sunanta; Chantarasak, Vasu; Jariyavilas, Apichat; Jaroensook, Piyadit; Kittiwattanagul, Khanogwan; Nerapusee, Osot

    2015-01-01

    This study aimed to examine symptoms/demographic characteristics as predictors for psychosocial functioning among individuals with schizophrenia. The Personal and Social Performance (PSP) scale was used to assess psychosocial functioning. Other measures of interest included were the Clinical Global Impression, Severity scale, and the Marder’s five-factor model of the Positive and Negative Syndrome Scale. This study included 199 participants with non-acute stage schizophrenia. Spearman correlation coefficients and stepwise multiple linear regression analyses were applied to determine the correlates and predictors of PSP domain/total scores. Younger age, earlier age of schizophrenia onset, severe illness, positive symptoms, negative symptoms, disorganized thought, hostility/excitement, and anxiety/depression were found to significantly correlate with poor functioning. Severe illness and negative symptoms are the main predictors of greater impairment of socially useful activities, personal and social relationships, and self-care. Further prospective studies in other settings, which would include an increased number of variables such as neurocognitive function and social support, are warranted. PMID:26491325

  11. The Influence of Domain Knowledge on the Functional Capacity of Working Memory

    ERIC Educational Resources Information Center

    Ricks, Travis Rex; Wiley, Jennifer

    2009-01-01

    Theories of expertise have proposed that superior cognitive performance is in part due to increases in the functional capacity of working memory during domain-related tasks. Consistent with this approach Fincher-Kiefer et al. (1988), found that domain knowledge increased scores on baseball-related reading span tasks. The present studies extended…

  12. Identification of the functional domains of ANT-1, a novel coactivator of the androgen receptor

    SciTech Connect

    Fan Shuli; Goto, Kiminobu; Chen Guangchun; Morinaga, Hidetaka; Nomura, Masatoshi; Okabe, Taijiro; Nawata, Hajime; Yanase, Toshihiko . E-mail: yanase@intmed3.med.kyushu-u.ac.jp

    2006-03-03

    Previously, we identified a transcriptional coactivator for the activation function-1 (AF-1) domain of the human androgen receptor (AR) and designated it androgen receptor N-terminal domain transactivating protein-1 (ANT-1). This coactivator, which contains multiple tetratricopeptide repeat (TPR) motifs from amino acid (aa) 294, is identical to a component of U5 small nuclear ribonucleoprotein particles and binds specifically to the AR or glucocorticoid receptor. Here, we identified four distinct functional domains. The AR-AF-1-binding domain, which bound to either aa 180-360 or 360-532 in AR-AF-1, clearly overlapped with TAU-1 and TAU-5. This domain and the subnuclear speckle formation domain in ANT-1 were assigned within the TPR motifs, while the transactivating and nuclear localization signal domains resided within the N-terminal sequence. The existence of these functional domains may further support the idea that ANT-1 can function as an AR-AF-1-specific coactivator while mediating a transcription-splicing coupling.

  13. The LIM domain-containing Dbm1 GTPase-activating protein is required for normal cellular morphogenesis in Saccharomyces cerevisiae.

    PubMed Central

    Chen, G C; Zheng, L; Chan, C S

    1996-01-01

    Normal cell growth in the yeast Saccharomyces cerevisiae involves the selection of genetically determined bud sites where most growth is localized. Previous studies have shown that BEM2, which encodes a GTPase-activating protein (GAP) that is specific for the Rho-type GTPase Rho1p in vitro, is required for proper bud site selection and bud emergence. We show here that DBM1, which encodes another putative Rho-type GAP with two tandemly arranged cysteine-rich LIM domains, also is needed for proper bud site selection, as haploid cells lacking Dbm1p bud predominantly in a bipolar, rather than the normal axial, manner. Furthermore, yeast cells lacking both Bem2p and Dbm1p are inviable. The nonaxial budding defect of dbm1 mutants can be rescued partially by overproduction of Bem3p and is exacerbated by its absence. Since Bem3p has previously been shown to function as a GAP for Cdc42p, and also less efficiently for Rho1p, our results suggest that Dbm1p, like Bem2p and Bem3p, may function in vivo as a GAP for Cdc42p and/or Rho1p. Both LIM domains of Dbm1p are essential for its normal function. Point mutations that alter single conserved cysteine residues within either LIM domain result in mutant forms of Dbm1p that can no longer function in bud site selection but instead are capable of rescuing the inviability of bem2 mutants at 35 degrees C. PMID:8657111

  14. Critical role of the first transmembrane domain of Cx26 in regulating oligomerization and function

    PubMed Central

    Jara, Oscar; Acuña, Rodrigo; García, Isaac E.; Maripillán, Jaime; Figueroa, Vania; Sáez, Juan C.; Araya-Secchi, Raúl; Lagos, Carlos F.; Pérez-Acle, Tomas; Berthoud, Viviana M.; Beyer, Eric C.; Martínez, Agustín D.

    2012-01-01

    To identify motifs involved in oligomerization of the gap junction protein Cx26, we studied individual transmembrane (TM) domains and the full-length protein. Using the TOXCAT assay for interactions of isolated TM α-helices, we found that TM1, a Cx26 pore domain, had a strong propensity to homodimerize. We identified amino acids Val-37–Ala-40 (VVAA) as the TM1 motif required for homodimerization. Two deafness-associated Cx26 mutations localized in this region, Cx26V37I and Cx26A40G, differentially affected dimerization. TM1-V37I dimerized only weakly, whereas TM1-A40G did not dimerize. When the full-length mutants were expressed in HeLa cells, both Cx26V37I and Cx26A40G formed oligomers less efficiently than wild-type Cx26. A Cx26 cysteine substitution mutant, Cx26V37C formed dithiothreitol-sensitive dimers. Substitution mutants of Val-37 formed intercellular channels with reduced function, while mutants of Ala-40 did not form functional gap junction channels. Unlike wild-type Cx26, neither Cx26V37I nor Cx26A40G formed functional hemichannels in low extracellular calcium. Thus the VVAA motif of Cx26 is critical for TM1 dimerization, hexamer formation, and channel function. The differential effects of VVAA mutants on hemichannels and gap junction channels imply that inter-TM interactions can differ in unapposed and docked hemichannels. Moreover, Cx26 oligomerization appears dependent on transient TM1 dimerization as an intermediate step. PMID:22787277

  15. Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1

    PubMed Central

    Belov, George A.; Kovtunovych, Gennadiy; Jackson, Catherine L.; Ehrenfeld, Ellie

    2010-01-01

    Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the Arf GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to BFA, an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism. PMID:20497182

  16. Structural insights into the functional versatility of WW domain-containing oxidoreductase tumor suppressor.

    PubMed

    Farooq, Amjad

    2015-03-01

    Recent work on WW domain-containing oxidoreductase (WWOX) tumor suppressor is beginning to shed new light on both the molecular mechanism of action of its WW domains as well as the contiguous catalytic domain. Herein, the structural basis underlying the ability of WW1 domain to bind to various physiological ligands and how the orphan WW2 tandem partner synergizes its ligand binding in the context of WW1-WW2 tandem module of WWOX is discussed. Notably, the WW domains within the WW1-WW2 tandem module physically associate so as to adopt a fixed spatial orientation relative to each other. In this manner, the association of WW2 domain with WW1 hinders ligand binding to the latter. Consequently, ligand binding to WW1 domain not only results in the displacement of WW2 lid but also disrupts the fixed orientation of WW domains in the liganded conformation. Equally importantly, structure-guided functional approach suggests that the catalytic domain of WWOX likely serves as a retinal oxidoreductase that catalyzes the reversible oxidation and reduction of all-trans-retinal. Collectively, this review provides structural insights into the functional versatility of a key signaling protein with important implications on its biology. PMID:25662954

  17. PDSS/IMC requirements and functional specifications

    NASA Technical Reports Server (NTRS)

    1983-01-01

    The system (software and hardware) requirements for the Payload Development Support System (PDSS)/Image Motion Compensator (IMC) are provided. The PDSS/IMC system provides the capability for performing Image Motion Compensator Electronics (IMCE) flight software test, checkout, and verification and provides the capability for monitoring the IMC flight computer system during qualification testing for fault detection and fault isolation.

  18. Functional studies of the BTB domain in the Drosophila GAGA and Mod(mdg4) proteins.

    PubMed

    Read, D; Butte, M J; Dernburg, A F; Frasch, M; Kornberg, T B

    2000-10-15

    The BTB/POZ (BTB) domain is an approximately 120 residue sequence that is conserved at the N-terminus of many proteins in both vertebrates and invertebrates. We found that the protein encoded by a lethal allele of the Drosophila modifier of mdg4 [mod(mdg4)] gene has two mutated residues in its BTB domain. The identities of the residues at the positions of these mutations are highly conserved in the BTB domain family of proteins, and when the corresponding mutations were engineered into the BTB domain-containing GAGA protein, the activity of GAGA as a transcription activator in a transient transfection assay was severely reduced. The functional equivalence of the BTB domains was established by showing that the BTB domain of the mod(mdg4) protein can effectively substitute for that of GAGA. PMID:11024164

  19. An intrinsically disordered domain has a dual function coupled to compartment-dependent redox control.

    PubMed

    Banci, Lucia; Bertini, Ivano; Cefaro, Chiara; Ciofi-Baffoni, Simone; Gajda, Karolina; Felli, Isabella C; Gallo, Angelo; Pavelkova, Anna; Kallergi, Emmanouela; Andreadaki, Maria; Katrakili, Nitsa; Pozidis, Charalambos; Tokatlidis, Kostas

    2013-02-01

    The functional role of unstructured protein domains is an emerging field in the frame of intrinsically disordered proteins. The involvement of intrinsically disordered domains (IDDs) in protein targeting and biogenesis processes in mitochondria is so far not known. Here, we have characterized the structural/dynamic and functional properties of an IDD of the sulfhydryl oxidase ALR (augmenter of liver regeneration) located in the intermembrane space of mitochondria. At variance to the unfolded-to-folded structural transition of several intrinsically disordered proteins, neither substrate recognition events nor redox switch of its shuttle cysteine pair is linked to any such structural change. However, this unstructured domain performs a dual function in two cellular compartments: it acts (i) as a mitochondrial targeting signal in the cytosol and (ii) as a crucial recognition site in the disulfide relay system of intermembrane space. This domain provides an exciting new paradigm for IDDs ensuring two distinct functions that are linked to intracellular organelle targeting. PMID:23207295

  20. Tank waste remediation system functions and requirements document

    SciTech Connect

    Carpenter, K.E

    1996-10-03

    This is the Tank Waste Remediation System (TWRS) Functions and Requirements Document derived from the TWRS Technical Baseline. The document consists of several text sections that provide the purpose, scope, background information, and an explanation of how this document assists the application of Systems Engineering to the TWRS. The primary functions identified in the TWRS Functions and Requirements Document are identified in Figure 4.1 (Section 4.0) Currently, this document is part of the overall effort to develop the TWRS Functional Requirements Baseline, and contains the functions and requirements needed to properly define the top three TWRS function levels. TWRS Technical Baseline information (RDD-100 database) included in the appendices of the attached document contain the TWRS functions, requirements, and architecture necessary to define the TWRS Functional Requirements Baseline. Document organization and user directions are provided in the introductory text. This document will continue to be modified during the TWRS life-cycle.

  1. Protein domain of unknown function 3233 is a translocation domain of autotransporter secretory mechanism in gamma proteobacteria.

    PubMed

    Prakash, Ananth; Yogeeshwari, S; Sircar, Sanchari; Agrawal, Shipra

    2011-01-01

    Vibrio cholerae, the enteropathogenic gram negative bacteria is one of the main causative agents of waterborne diseases like cholera. About 1/3(rd) of the organism's genome is uncharacterised with many protein coding genes lacking structure and functional information. These proteins form significant fraction of the genome and are crucial in understanding the organism's complete functional makeup. In this study we report the general structure and function of a family of hypothetical proteins, Domain of Unknown Function 3233 (DUF3233), which are conserved across gram negative gammaproteobacteria (especially in Vibrio sp. and similar bacteria). Profile and HMM based sequence search methods were used to screen homologues of DUF3233. The I-TASSER fold recognition method was used to build a three dimensional structural model of the domain. The structure resembles the transmembrane beta-barrel with an axial N-terminal helix and twelve antiparallel beta-strands. Using a combination of amphipathy and discrimination analysis we analysed the potential transmembrane beta-barrel forming properties of DUF3233. Sequence, structure and phylogenetic analysis of DUF3233 indicates that this gram negative bacterial hypothetical protein resembles the beta-barrel translocation unit of autotransporter Va secretory mechanism with a gene organisation that differs from the conventional Va system. PMID:22073138

  2. Mild Depressive Symptoms and Slowing Across Multiple Functional Domains

    PubMed Central

    Albert, Steven M.; Bear-Lehman, Jane; Burkhardt, Ann

    2016-01-01

    Background Subthreshold depressive symptoms are common in older adults. The threshold for the clinical significance of such symptoms is unclear. Mechanisms linking depressed mood to increased risk of disability need further investigation. Methods Older adults who did not meet criteria for depression were divided into two groups based on the 2-item Patient Health Questionnaire (PHQ-2). Respondents reporting no anhedonia and dysphoria over the past 2 weeks were compared to respondents reporting occasional symptoms on a battery of cognitive, psychomotor, and physical performance tests. Results Of 290 community-resident participants without dementia or neurologic disease, 32% (n=93) reported at least one of the two depressive symptoms “several days” in the past 2 weeks. Older adults with mild depressive symptoms did not differ in ADL or IADL disability but reported more physician-diagnosed medical conditions (1.8 vs. 2.2, p < .01) and balance problems (2.9 vs. 1.8 on 0–7 scale, p < .001). Subthreshold depressive symptoms were associated with slowing in gait (p < .01), chair stand time (p < .01) and performance on Trails A (p < .05) and B (p < .001). Conclusions Mild depressive symptoms in people who do not meet criteria for depression are associated with slowing across multiple domains. PMID:21801471

  3. Textured areas detection and segmentation in circular harmonic functions domain

    NASA Astrophysics Data System (ADS)

    Costantini, Luca; Capodiferro, Licia; Carli, Marco; Neri, Alessandro

    2012-03-01

    In this work a novel technique for detecting and segmenting textured areas in natural images is presented. The method is based on the circular harmonic function, and, in particular, on the Laguerre Gauss functions. The detection of the textured areas is performed by analyzing the mean, the mode, and the skewness of the marginal densities of the Laguerre Gauss coefficients. By using these parameters a classification of the patch and of the pixel, is performed. The feature vectors representing the textures are built using the parameters of the Generalized Gaussian Densities that approximate the marginal densities of the Laguerre Gauss functions computed at three different resolutions. The feature vectors are clustered by using the K-means algorithm in which the symmetric Kullback-Leibler distance is adopted. The experimental results, obtained by using a set of natural images, show the effectiveness of the proposed technique.

  4. Mapping interactions between myosin relay and converter domains that power muscle function.

    PubMed

    Kronert, William A; Melkani, Girish C; Melkani, Anju; Bernstein, Sanford I

    2014-05-01

    Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile(508), Asn(509), and Asp(511)) in communicating with converter domain residue Arg(759). We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle. PMID:24627474

  5. Insulator function and topological domain border strength scale with architectural protein occupancy

    PubMed Central

    2014-01-01

    Background Chromosome conformation capture studies suggest that eukaryotic genomes are organized into structures called topologically associating domains. The borders of these domains are highly enriched for architectural proteins with characterized roles in insulator function. However, a majority of architectural protein binding sites localize within topological domains, suggesting sites associated with domain borders represent a functionally different subclass of these regulatory elements. How topologically associating domains are established and what differentiates border-associated from non-border architectural protein binding sites remain unanswered questions. Results By mapping the genome-wide target sites for several Drosophila architectural proteins, including previously uncharacterized profiles for TFIIIC and SMC-containing condensin complexes, we uncover an extensive pattern of colocalization in which architectural proteins establish dense clusters at the borders of topological domains. Reporter-based enhancer-blocking insulator activity as well as endogenous domain border strength scale with the occupancy level of architectural protein binding sites, suggesting co-binding by architectural proteins underlies the functional potential of these loci. Analyses in mouse and human stem cells suggest that clustering of architectural proteins is a general feature of genome organization, and conserved architectural protein binding sites may underlie the tissue-invariant nature of topologically associating domains observed in mammals. Conclusions We identify a spectrum of architectural protein occupancy that scales with the topological structure of chromosomes and the regulatory potential of these elements. Whereas high occupancy architectural protein binding sites associate with robust partitioning of topologically associating domains and robust insulator function, low occupancy sites appear reserved for gene-specific regulation within topological domains. PMID

  6. Boundary regularity of Nevanlinna domains and univalent functions in model subspaces

    NASA Astrophysics Data System (ADS)

    Baranov, Anton D.; Fedorovskiy, Konstantin Yu

    2011-12-01

    In the paper we study boundary regularity of Nevanlinna domains, which have appeared in problems of uniform approximation by polyanalytic polynomials. A new method for constructing Nevanlinna domains with essentially irregular nonanalytic boundaries is suggested; this method is based on finding appropriate univalent functions in model subspaces, that is, in subspaces of the form K_\\varTheta=H^2\\ominus\\varTheta H^2, where \\varTheta is an inner function. To describe the irregularity of the boundaries of the domains obtained, recent results by Dolzhenko about boundary regularity of conformal mappings are used. Bibliography: 18 titles.

  7. Domain-oriented functional analysis based on expression profiling

    PubMed Central

    Ding, Wei; Wang, Luquan; Qiu, Ping; Kostich, Mitchel; Greene, Jonathan; Hernandez, Marco

    2002-01-01

    Background Co-regulation of genes may imply involvement in similar biological processes or related function. Many clusters of co-regulated genes have been identified using microarray experiments. In this study, we examined co-regulated gene families using large-scale cDNA microarray experiments on the human transcriptome. Results We present a simple model, which, for each probe pair, distills expression changes into binary digits and summarizes the expression of multiple members of a gene family as the Family Regulation Ratio. The set of Family Regulation Ratios for each protein family across multiple experiments is called a Family Regulation Profile. We analyzed these Family Regulation Profiles using Pearson Correlation Coefficients and derived a network diagram portraying relationships between the Family Regulation Profiles of gene families that are well represented on the microarrays. Our strategy was cross-validated with two randomly chosen data subsets and was proven to be a reliable approach. Conclusion This work will help us to understand and identify the functional relationships between gene families and the regulatory pathways in which each family is involved. Concepts presented here may be useful for objective clustering of protein functions and deriving a comprehensive protein interaction map. Functional genomic approaches such as this may also be applicable to the elucidation of complex genetic regulatory networks. PMID:12456268

  8. Insights into common functional domains of tospovirus NSm proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Direct demonstration of tospovirus gene function has been impeded by the absence of reliable reverse genetics systems for this virus genus. Use of a Tobacco mosaic virus (TMV)-based expression system has demonstrated that the Tomato spotted wilt virus (TSWV) NSm protein supports cell-to-cell moveme...

  9. Speech and Language Functions that Require a Functioning Broca's Area

    ERIC Educational Resources Information Center

    Davis, Cameron; Kleinman, Jonathan T.; Newhart, Melissa; Gingis, Leila; Pawlak, Mikolaj; Hillis, Argye E.

    2008-01-01

    A number of previous studies have indicated that Broca's area has an important role in understanding and producing syntactically complex sentences and other language functions. If Broca's area is critical for these functions, then either infarction of Broca's area or temporary hypoperfusion within this region should cause impairment of these…

  10. Invariant domain watermarking using heaviside function of order alpha and fractional Gaussian field.

    PubMed

    Abbasi, Almas; Woo, Chaw Seng; Ibrahim, Rabha Waell; Islam, Saeed

    2015-01-01

    Digital image watermarking is an important technique for the authentication of multimedia content and copyright protection. Conventional digital image watermarking techniques are often vulnerable to geometric distortions such as Rotation, Scaling, and Translation (RST). These distortions desynchronize the watermark information embedded in an image and thus disable watermark detection. To solve this problem, we propose an RST invariant domain watermarking technique based on fractional calculus. We have constructed a domain using Heaviside function of order alpha (HFOA). The HFOA models the signal as a polynomial for watermark embedding. The watermark is embedded in all the coefficients of the image. We have also constructed a fractional variance formula using fractional Gaussian field. A cross correlation method based on the fractional Gaussian field is used for watermark detection. Furthermore the proposed method enables blind watermark detection where the original image is not required during the watermark detection thereby making it more practical than non-blind watermarking techniques. Experimental results confirmed that the proposed technique has a high level of robustness. PMID:25884854

  11. Invariant Domain Watermarking Using Heaviside Function of Order Alpha and Fractional Gaussian Field

    PubMed Central

    Abbasi, Almas; Woo, Chaw Seng; Ibrahim, Rabha Waell; Islam, Saeed

    2015-01-01

    Digital image watermarking is an important technique for the authentication of multimedia content and copyright protection. Conventional digital image watermarking techniques are often vulnerable to geometric distortions such as Rotation, Scaling, and Translation (RST). These distortions desynchronize the watermark information embedded in an image and thus disable watermark detection. To solve this problem, we propose an RST invariant domain watermarking technique based on fractional calculus. We have constructed a domain using Heaviside function of order alpha (HFOA). The HFOA models the signal as a polynomial for watermark embedding. The watermark is embedded in all the coefficients of the image. We have also constructed a fractional variance formula using fractional Gaussian field. A cross correlation method based on the fractional Gaussian field is used for watermark detection. Furthermore the proposed method enables blind watermark detection where the original image is not required during the watermark detection thereby making it more practical than non-blind watermarking techniques. Experimental results confirmed that the proposed technique has a high level of robustness. PMID:25884854

  12. Functional analysis of conserved residues in the putative "finger" domain of telomerase reverse transcriptase.

    PubMed

    Bosoy, D; Lue, N F

    2001-12-01

    Telomerase is a ribonucleoprotein reverse transcriptase (RT) responsible for the maintenance of one strand of telomere terminal repeats. The catalytic protein subunit of telomerase, known generically as telomerase reverse transcriptase (TERT), exhibits significant homology to RTs encoded by retroviruses and retroelements. The polymerization mechanisms of telomerase may therefore be similar to those of the "conventional" RTs. In this study, we explored the extent of mechanistic conservation by analyzing mutations of conserved residues within the putative "finger" domain of TERT. Previous analysis has implicated this domain of retroviral RTs in nucleotide and RNA binding and in processivity control. Our results demonstrate that residues conserved between TERT and human immunodeficiency virus-1 RT are more likely than TERT-specific residues to be required for enzyme activity. In addition, residues presumed to make direct contact with either the RNA or nucleotide substrate appear to be functionally more important. Furthermore, distinct biochemical defects can be observed for alterations in the putative RNA- and nucleotide-binding TERT residues in a manner that can be rationalized by their postulated mechanisms of action. This study thus supports a high degree of mechanistic conservation between telomerase and retroviral RTs and underscores the roles of distinct aspects of telomerase biochemistry in telomere length maintenance. PMID:11581271

  13. Human DNA polymerase. alpha. : Predicted functional domains and relationships with viral DNA polymerases

    SciTech Connect

    Wang, T.S.F.; Wong, S.W.; Korn, D. )

    1989-01-01

    The primary sequence of human DNA polymerase {alpha} deduced from the full-length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage {phi}19, yeast DNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus. The conservation of these homologous regions across this vast phylogenetic expanse indicates that these prokaryotic and eukaryotic DNA polymerases may all have evolved from a common primordial gene. Based on the sequence analysis and genetic results from yeast and herpes simplex virus studies, these consensus sequences are suggested to define potential sites that subserve essential roles in the DNA polymerase reaction. Two of these conserved regions appear to participate directly in the active site required for substrate deoxynucleotide interaction. One region toward the carboxyl-terminus has the potential to be the DNA interacting domain is predicted toward the amino-terminus. The provisional assignment of these domains can be used to identify unique or dissimilar features of functionally homologous catalytic sites in viral DBA polymerases of pathogenetic significance and thereby serve to guide more rational antiviral drug design.

  14. Ecogenomic Perspectives on Domains of Unknown Function: Correlation-Based Exploration of Marine Metagenomes

    PubMed Central

    Buttigieg, Pier Luigi; Hankeln, Wolfgang; Kostadinov, Ivaylo; Kottmann, Renzo; Yilmaz, Pelin; Duhaime, Melissa Beth; Glöckner, Frank Oliver

    2013-01-01

    Background The proportion of conserved DNA sequences with no clear function is steadily growing in bioinformatics databases. Studies of sequence and structural homology have indicated that many uncharacterized protein domain sequences are variants of functionally described domains. If these variants promote an organism's ecological fitness, they are likely to be conserved in the genome of its progeny and the population at large. The genetic composition of microbial communities in their native ecosystems is accessible through metagenomics. We hypothesize the co-variation of protein domain sequences across metagenomes from similar ecosystems will provide insights into their potential roles and aid further investigation. Methodology/Principal findings We calculated the correlation of Pfam protein domain sequences across the Global Ocean Sampling metagenome collection, employing conservative detection and correlation thresholds to limit results to well-supported hits and associations. We then examined intercorrelations between domains of unknown function (DUFs) and domains involved in known metabolic pathways using network visualization and cluster-detection tools. We used a cautious “guilty-by-association” approach, referencing knowledge-level resources to identify and discuss associations that offer insight into DUF function. We observed numerous DUFs associated to photobiologically active domains and prevalent in the Cyanobacteria. Other clusters included DUFs associated with DNA maintenance and repair, inorganic nutrient metabolism, and sodium-translocating transport domains. We also observed a number of clusters reflecting known metabolic associations and cases that predicted functional reclassification of DUFs. Conclusion/Significance Critically examining domain covariation across metagenomic datasets can grant new perspectives on the roles and associations of DUFs in an ecological setting. Targeted attempts at DUF characterization in the laboratory or in

  15. Function-selective domain architecture plasticity potentials in eukaryotic genome evolution

    PubMed Central

    Linkeviciute, Viktorija; Rackham, Owen J.L.; Gough, Julian; Oates, Matt E.; Fang, Hai

    2015-01-01

    To help evaluate how protein function impacts on genome evolution, we introduce a new concept of ‘architecture plasticity potential’ – the capacity to form distinct domain architectures – both for an individual domain, or more generally for a set of domains grouped by shared function. We devise a scoring metric to measure the plasticity potential for these domain sets, and evaluate how function has changed over time for different species. Applying this metric to a phylogenetic tree of eukaryotic genomes, we find that the involvement of each function is not random but highly selective. For certain lineages there is strong bias for evolution to involve domains related to certain functions. In general eukaryotic genomes, particularly animals, expand complex functional activities such as signalling and regulation, but at the cost of reducing metabolic processes. We also observe differential evolution of transcriptional regulation and a unique evolutionary role of channel regulators; crucially this is only observable in terms of the architecture plasticity potential. Our findings provide a new layer of information to understand the significance of function in eukaryotic genome evolution. A web search tool, available at http://supfam.org/Pevo, offers a wide spectrum of options for exploring functional importance in eukaryotic genome evolution. PMID:25980317

  16. Function-selective domain architecture plasticity potentials in eukaryotic genome evolution.

    PubMed

    Linkeviciute, Viktorija; Rackham, Owen J L; Gough, Julian; Oates, Matt E; Fang, Hai

    2015-12-01

    To help evaluate how protein function impacts on genome evolution, we introduce a new concept of 'architecture plasticity potential' - the capacity to form distinct domain architectures - both for an individual domain, or more generally for a set of domains grouped by shared function. We devise a scoring metric to measure the plasticity potential for these domain sets, and evaluate how function has changed over time for different species. Applying this metric to a phylogenetic tree of eukaryotic genomes, we find that the involvement of each function is not random but highly selective. For certain lineages there is strong bias for evolution to involve domains related to certain functions. In general eukaryotic genomes, particularly animals, expand complex functional activities such as signalling and regulation, but at the cost of reducing metabolic processes. We also observe differential evolution of transcriptional regulation and a unique evolutionary role of channel regulators; crucially this is only observable in terms of the architecture plasticity potential. Our findings provide a new layer of information to understand the significance of function in eukaryotic genome evolution. A web search tool, available at http://supfam.org/Pevo, offers a wide spectrum of options for exploring functional importance in eukaryotic genome evolution. PMID:25980317

  17. New Generation Nuclear Plant -- High Level Functions and Requirements

    SciTech Connect

    J. M. Ryskamp; E. J. Gorski; E. A. Harvego; S. T. Khericha; G. A. Beitel

    2003-09-01

    This functions and requirements (F&R) document was prepared for the Next Generation Nuclear Plant (NGNP) Project. The highest-level functions and requirements for the NGNP preconceptual design are identified in this document, which establishes performance definitions for what the NGNP will achieve. NGNP designs will be developed based on these requirements by commercial vendor(s).

  18. Describing functional requirements for knowledge sharing communities

    NASA Technical Reports Server (NTRS)

    Garrett, Sandra; Caldwell, Barrett

    2002-01-01

    Human collaboration in distributed knowledge sharing groups depends on the functionality of information and communication technologies (ICT) to support performance. Since many of these dynamic environments are constrained by time limits, knowledge must be shared efficiently by adapting the level of information detail to the specific situation. This paper focuses on the process of knowledge and context sharing with and without mediation by ICT, as well as issues to be resolved when determining appropriate ICT channels. Both technology-rich and non-technology examples are discussed.

  19. Formation of functional cell membrane domains: the interplay of lipid- and protein-mediated interactions.

    PubMed Central

    Harder, Thomas

    2003-01-01

    Numerous cell membrane associated processes, including signal transduction, membrane sorting, protein processing and virus trafficking take place in membrane subdomains. Protein-protein interactions provide the frameworks necessary to generate biologically functional membrane domains. For example, coat proteins define membrane areas destined for sorting processes, viral proteins self-assemble to generate a budding virus, and adapter molecules organize multimolecular signalling assemblies, which catalyse downstream reactions. The concept of raft lipid-based membrane domains provides a different principle for compartmentalization and segregation of membrane constituents. Accordingly, rafts are defined by the physical properties of the lipid bilayer and function by selective partitioning of membrane lipids and proteins into membrane domains of specific phase behaviour and lipid packing. Here, I will discuss the interplay of these independent principles of protein scaffolds and raft lipid microdomains leading to the generation of biologically functional membrane domains. PMID:12803918

  20. Structure-function relationships in the catalytic and starch binding domains of glucoamylase.

    PubMed

    Coutinho, P M; Reilly, P J

    1994-03-01

    Sixteen primary sequences from five sub-families of fungal, yeast and bacterial glucoamylases were related to structural information from the model of the catalytic domain of Aspergillus awamori var. X100 glucoamylase obtained by protein crystallography. This domain is composed of thirteen alpha-helices, with five conserved regions defining the active site. Interactions between methyl alpha-maltoside and active site residues were modelled, and the importance of these residues on the catalytic action of different glucoamylases was shown by their presence in each primary sequence. The overall structure of the starch binding domain of some fungal glucoamylases was determined based on homology to the C-terminal domains of Bacillus cyclodextrin glucosyl-transferases. Crystallography indicated that this domain contains 6-8 beta-strands and homology allowed the attribution of a disulfide bridge in the glucoamylase starch binding domain. Glucoamylase residues Thr525, Asn530 and Trp560, homologous to Bacillus stearothermophilus cyclodextrin glucosyltransferase residues binding to maltose in the C-terminal domain, could be involved in raw-starch binding. The structure and length of the linker region between the catalytic and starch binding domains in fungal glucoamylases can vary substantially, a further indication of the functional independence of the two domains. PMID:8177888

  1. Towards a minimal generic set of domains of functioning and health

    PubMed Central

    2014-01-01

    Background The World Health Organization (WHO) has argued that functioning, and, more concretely, functioning domains constitute the operationalization that best captures our intuitive notion of health. Functioning is, therefore, a major public-health goal. A great deal of data about functioning is already available. Nonetheless, it is not possible to compare and optimally utilize this information. One potential approach to address this challenge is to propose a generic and minimal set of functioning domains that captures the experience of individuals and populations with respect to functioning and health. The objective of this investigation was to identify a minimal generic set of ICF domains suitable for describing functioning in adults at both the individual and population levels. Methods We performed a psychometric study using data from: 1) the German National Health Interview and Examination Survey 1998, 2) the United States National Health and Nutrition Examination Survey 2007/2008, and 3) the ICF Core Set studies. Random Forests and Group Lasso regression were applied using one self-reported general-health question as a dependent variable. The domains selected were compared to those of the World Health Survey (WHS) developed by the WHO. Results Seven domains of the International Classification of Functioning, Disability and Health (ICF) are proposed as a minimal generic set of functioning and health: energy and drive functions, emotional functions, sensation of pain, carrying out daily routine, walking, moving around, and remunerative employment. The WHS domains of self-care, cognition, interpersonal activities, and vision were not included in our selection. Conclusions The minimal generic set proposed in this study is the starting point to address one of the most important challenges in health measurement – the comparability of data across studies and countries. It also represents the first step in developing a common metric of health to link information

  2. Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain.

    PubMed Central

    Kirkpatrick, D T; Fan, Q; Petes, T D

    1999-01-01

    The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot. Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination. We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of recombination hotspots, alpha (transcription factor dependent) and beta (transcription factor independent). PMID:10224246

  3. Functional brain imaging with a supercontinuum time-domain NIRS system

    NASA Astrophysics Data System (ADS)

    Selb, Juliette; Zimmermann, Bernhard B.; Martino, Mark; Ogden, Tyler; Boas, David A.

    2013-03-01

    We have developed the second generation of our time-domain near-infrared spectroscopy (TD-NIRS) system for baseline and functional brain imaging. The instrument uses a pulsed broadband supercontinuum laser emitting a large spectrum between 650 and 1700 nm, and a gated detection based on an intensified CCD camera. The source laser beam is split into two arms, below and above 776 nm. In each arm, a fast motorized filter wheel enables selection of a bandpass filter at the required wavelength. Each filtered laser beam is then launched into one array of source fibers. The multiplexing through the array of fibers is implemented through a very compact home-made design consisting of two galvanometer mirrors followed by an achromatic doublet. Source fibers are then recombined one-by-one from both arms into the source optodes to be positioned on the head. The detection fibers are all imaged in parallel through a relay lens on an intensified CCD camera. By using detection fibers of different lengths, we introduce optical delays that enable simultaneous recording in different delay windows of the temporal point spread functions. We present the instrumentation and show its preliminary functional imaging capabilities. We also introduce a new probe where we use different fiber lengths on the source and the detector sides in order to record simultaneously both wavelengths from one location through different sets of fibers.

  4. Anti-candidal activity of genetically engineered histatin variants with multiple functional domains.

    PubMed

    Oppenheim, Frank G; Helmerhorst, Eva J; Lendenmann, Urs; Offner, Gwynneth D

    2012-01-01

    The human bodily defense system includes a wide variety of innate antimicrobial proteins. Histatins are small molecular weight proteins produced by the human salivary glands that exhibit antifungal and antibacterial activities. While evolutionarily old salivary proteins such as mucins and proline-rich proteins contain large regions of tandem repeats, relatively young proteins like histatins do not contain such repeated domains. Anticipating that domain duplications have a functional advantage, we genetically engineered variants of histatin 3 with one, two, three, or four copies of the functional domain by PCR and splice overlap. The resulting proteins, designated reHst3 1-mer, reHist3 2-mer, reHis3 3-mer and reHist3 4-mer, exhibited molecular weights of 4,062, 5,919, 7,777, and 9,634 Da, respectively. The biological activities of these constructs were evaluated in fungicidal assays toward Candida albicans blastoconidia and germinated cells. The antifungal activities per mole of protein increased concomitantly with the number of functional domains present. This increase, however, was higher than could be anticipated from the molar concentration of functional domains present in the constructs. The demonstrated increase in antifungal activity may provide an evolutionary explanation why such domain multiplication is a frequent event in human salivary proteins. PMID:23251551

  5. Domain II plays a crucial role in the function of ribosome recycling factor

    PubMed Central

    2005-01-01

    RRF (ribosome recycling factor) consists of two domains, and in concert with EF-G (elongation factor-G), triggers dissociation of the post-termination ribosomal complex. However, the function of the individual domains of RRF remains unclear. To clarify this, two RRF chimaeras, EcoDI/TteDII and TteDI/EcoDII, were created by domain swaps between the proteins from Escherichia coli and Thermoanaerobacter tengcongensis. The ribosome recycling activity of the RRF chimaeras was compared with their wild-type RRFs by using in vivo and in vitro activity assays. Like wild-type TteRRF (T. tengcongensis RRF), the EcoDI/TteDII chimaera is non-functional in E. coli, but both wild-type TteRRF, and EcoDI/TteDII can be activated by coexpression of T. tengcongensis EF-G in E. coli. By contrast, like wild-type E. coli RRF (EcoRRF), TteDI/EcoDII is fully functional in E. coli. These findings suggest that domain II of RRF plays a crucial role in the concerted action of RRF and EF-G for the post-termination complex disassembly, and the specific interaction between RRF and EF-G on ribosomes mainly depends on the interaction between domain II of RRF and EF-G. This study provides direct genetic and biochemical evidence for the function of the individual domains of RRF. PMID:16262604

  6. Functionally distinct NEAT (NEAr Transporter) domains within the Staphylococcus aureus IsdH/HarA protein extract heme from methemoglobin.

    PubMed

    Pilpa, Rosemarie M; Robson, Scott A; Villareal, Valerie A; Wong, Melissa L; Phillips, Martin; Clubb, Robert T

    2009-01-01

    The pathogen Staphylococcus aureus uses iron-regulated surface determinant (Isd) proteins to scavenge the essential nutrient iron from host hemoproteins. The IsdH protein (also known as HarA) is a receptor for hemoglobin (Hb), haptoglobin (Hp), and the Hb-Hp complex. It contains three NEAT (NEAr Transporter) domains: IsdH(N1), IsdH(N2), and IsdH(N3). Here we show that they have different functions; IsdH(N1) binds Hb and Hp, whereas IsdH(N3) captures heme that is released from Hb. The staphylococcal IsdB protein also functions as an Hb receptor. Primary sequence homology to IsdH indicates that it will also employ functionally distinct NEAT domains to bind heme and Hb. We have used site-directed mutagenesis and surface plasmon resonance methods to localize the Hp and Hb binding surface on IsdH(N1). High affinity binding to these structurally unrelated proteins requires residues located within a conserved aromatic motif that is positioned at the end of the beta-barrel structure. Interestingly, this site is quite malleable, as other NEAT domains use it to bind heme. We also demonstrate that the IsdC NEAT domain can capture heme directly from Hb, suggesting that there are multiple pathways for heme transfer across the cell wall. PMID:18984582

  7. Genetic and Biochemical Dissection of a HisKA Domain Identifies Residues Required Exclusively for Kinase and Phosphatase Activities

    PubMed Central

    Willett, Jonathan W.; Kirby, John R.

    2012-01-01

    Two-component signal transduction systems, composed of histidine kinases (HK) and response regulators (RR), allow bacteria to respond to diverse environmental stimuli. The HK can control both phosphorylation and subsequent dephosphorylation of its cognate RR. The majority of HKs utilize the HisKA subfamily of dimerization and histidine phosphotransfer (DHp) domains, which contain the phospho-accepting histidine and directly contact the RR. Extensive genetics, biochemistry, and structural biology on several prototypical TCS systems including NtrB-NtrC and EnvZ-OmpR have provided a solid basis for understanding the function of HK–RR signaling. Recently, work on NarX, a HisKA_3 subfamily protein, indicated that two residues in the highly conserved region of the DHp domain are responsible for phosphatase activity. In this study we have carried out both genetic and biochemical analyses on Myxococcus xanthus CrdS, a member of the HisKA subfamily of bacterial HKs. CrdS is required for the regulation of spore formation in response to environmental stress. Following alanine-scanning mutagenesis of the α1 helix of the DHp domain of CrdS, we determined the role for each mutant protein for both kinase and phosphatase activity. Our results indicate that the conserved acidic residue (E372) immediately adjacent to the site of autophosphorylation (H371) is specifically required for kinase activity but not for phosphatase activity. Conversely, we found that the conserved Thr/Asn residue (N375) was required for phosphatase activity but not for kinase activity. We extended our biochemical analyses to two CrdS homologs from M. xanthus, HK1190 and HK4262, as well as Thermotoga maritima HK853. The results were similar for each HisKA family protein where the conserved acidic residue is required for kinase activity while the conserved Thr/Asn residue is required for phosphatase activity. These data are consistent with conserved mechanisms for kinase and phosphatase activities in the

  8. Structure determination of the functional domain interaction of a chimeric nonribosomal peptide synthetase from a challenging crystal with noncrystallographic translational symmetry

    SciTech Connect

    Sundlov, Jesse A.; Gulick, Andrew M.

    2013-08-01

    The structure of the functional interaction of NRPS adenylation and carrier protein domains, trapped with a mechanism-based inhibitor, is described. Crystals exhibit translational non-crystallographic symmetry, which challenged structure determination and refinement. The nonribosomal peptide synthetases (NRPSs) are a family of modular proteins that contain multiple catalytic domains joined in a single protein. Together, these domains work to produce chemically diverse peptides, including compounds with antibiotic activity or that play a role in iron acquisition. Understanding the structural mechanisms that govern the domain interactions has been a long-standing goal. During NRPS synthesis, amino-acid substrates are loaded onto integrated carrier protein domains through the activity of NRPS adenylation domains. The structures of two adenylation domain–carrier protein domain complexes have recently been determined in an effort that required the use of a mechanism-based inhibitor to trap the domain interaction. Here, the continued analysis of these proteins is presented, including a higher resolution structure of an engineered di-domain protein containing the EntE adenylation domain fused with the carrier protein domain of its partner EntB. The protein crystallized in a novel space group in which molecular replacement and refinement were challenged by noncrystallographic pseudo-translational symmetry. The structure determination and how the molecular packing impacted the diffraction intensities are reported. Importantly, the structure illustrates that in this new crystal form the functional interface between the adenylation domain and the carrier protein domain remains the same as that observed previously. At a resolution that allows inclusion of water molecules, additional interactions are observed between the two protein domains and between the protein and its ligands. In particular, a highly solvated region that surrounds the carrier protein cofactor is described.

  9. The SNW Domain of SKIP Is Required for Its Integration into the Spliceosome and Its Interaction with the Paf1 Complex in Arabidopsis.

    PubMed

    Li, Yan; Xia, Congcong; Feng, Jinlin; Yang, Dong; Wu, Fangming; Cao, Ying; Li, Legong; Ma, Ligeng

    2016-07-01

    SKIP is a conserved protein from yeasts to plants and humans. In plant cells, SKIP is a bifunctional regulator that works in the nucleus as a splicing factor by integrating into the spliceosome and as a transcriptional activator by interacting with the Paf1 complex. In this study, we identified two nuclear localization signals in SKIP and confirmed that each is sufficient to target SKIP to the nucleus. The SNW domain of SKIP is required for both its function as a splicing factor by promoting integration into the spliceosome in response to stress, and its function as a transcriptional activator by controlling its interaction with the Paf1 complex to participate in flowering. Truncated proteins that included the SNW domain and the N- or C-terminus of SKIP were still able to carry out the functions of the full-length protein in gene splicing and transcriptional activation in Arabidopsis. In addition, we found that SKIP undergoes 26S proteasome-mediated degradation, and that the C-terminus of SKIP is required to maintain the stability of the protein in plant cells. Together, our findings demonstrate the structural domain organization of SKIP and reveal the core domains and motifs underlying SKIP function in plants. PMID:27130079

  10. Introducing a domain flexing function in the Jiles-Atherton hysteresis model

    NASA Astrophysics Data System (ADS)

    Miljavec, Damijan; Zidarič, Bogomir

    The Jiles-Atherton hysteresis model (J-A model) exhibits a certain unphysical behavior when magnetic excitation reaches or reverses from the extremity of the hysteresis loop. Introducing a domain flexing function, coherent with the magnetic excitation level, improves accuracy of the J-A hysteresis model and at the same time prevents its unphysical behavior. Moreover, applying this function also improves representation of inner (lower excitation level) hysteresis loops. Implementation of magnetic excitation dependence in the domain flexing function adds a valuable parameter to the J-A original model on the way towards its further optimization. In the proposed hysteresis model, genetic algorithms are used in parameters optimization.

  11. The charged linker of the molecular chaperone Hsp90 modulates domain contacts and biological function

    PubMed Central

    Jahn, Markus; Rehn, Alexandra; Pelz, Benjamin; Hellenkamp, Björn; Richter, Klaus; Rief, Matthias; Buchner, Johannes; Hugel, Thorsten

    2014-01-01

    The heat shock protein 90 (Hsp90) is a dimeric molecular chaperone essential in numerous cellular processes. Its three domains (N, M, and C) are connected via linkers that allow the rearrangement of domains during Hsp90’s chaperone cycle. A unique linker, called charged linker (CL), connects the N- and M-domain of Hsp90. We used an integrated approach, combining single-molecule techniques and biochemical and in vivo methods, to study the unresolved structure and function of this region. Here we show that the CL facilitates intramolecular rearrangements on the milliseconds timescale between a state in which the N-domain is docked to the M-domain and a state in which the N-domain is more flexible. The docked conformation is stabilized by 1.1 kBT (2.7 kJ/mol) through binding of the CL to the N-domain of Hsp90. Docking and undocking of the CL affects the much slower intermolecular domain movement and Hsp90’s chaperone cycle governing client activation, cell viability, and stress tolerance. PMID:25468961

  12. Structural and functional characterizations reveal the importance of a zinc binding domain in Bloom's syndrome helicase

    PubMed Central

    Guo, Rong-bin; Rigolet, Pascal; Zargarian, Loussiné; Fermandjian, Serge; Xi, Xu Guang

    2005-01-01

    Bloom's syndrome (BS) is an autosomal recessive human disorder characterized by genomic instability and a predisposition to a wide variety of cancers. The gene mutated in BS, BLM, encodes a protein containing three domains: an N-terminal domain whose function remains elusive, a helicase domain characterized by seven ‘signature’ motifs conserved in a wide range of helicases and a C-terminal extension that can be further divided into two sub-domains: RecQ-Ct and HRDC. The RecQ-Ct domain appears essential because two point-mutations altering highly conserved cysteine residues within this domain have been found in BS patients. We report herein that BLM contains a zinc ion. Modelling studies suggest that four conserved cysteine residues within the RecQ-Ct domain coordinate this zinc ion and subsequent mutagenesis studies further confirm this prediction. Biochemical and biophysical studies show that the ATPase, helicase and DNA binding activities of the mutants are severely modified. Structural analysis of both wild-type and mutant proteins reveal that alteration of cysteine residues does not significantly change the overall conformation. The observed defects in ATPase and helicase activities were inferred to result from a compromise of DNA binding. Our results implicate an important role of this zinc binding domain in both DNA binding and protein conformation. They could be pivotal for understanding the molecular basis of BS disease. PMID:15930159

  13. Verprolin function in endocytosis and actin organization. Roles of the Las17p (yeast WASP)-binding domain and a novel C-terminal actin-binding domain.

    PubMed

    Thanabalu, Thirumaran; Rajmohan, Rajamuthiah; Meng, Lei; Ren, Gang; Vajjhala, Parimala R; Munn, Alan L

    2007-08-01

    Vrp1p (verprolin, End5p) is the yeast ortholog of human Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP). Vrp1p localizes to the cortical actin cytoskeleton, is necessary for its polarization to sites of growth and is also essential for endocytosis. At elevated temperature, Vrp1p becomes essential for growth. A C-terminal Vrp1p fragment (C-Vrp1p) retains the ability to localize to the cortical actin cytoskeleton and function in actin-cytoskeleton polarization, endocytosis and growth. Here, we demonstrate that two submodules in C-Vrp1p are required for actin-cytoskeleton polarization: a novel C-terminal actin-binding submodule (CABS) that contains a novel G-actin-binding domain, which we call a verprolin homology 2 C-terminal (VH2-C) domain; and a second submodule comprising the Las17p-binding domain (LBD) that binds Las17p (yeast WASP). The LBD localizes C-Vrp1p to membranes and the cortical actin cytoskeleton. Intriguingly, the LBD is sufficient to restore endocytosis and growth at elevated temperature to Vrp1p-deficient cells. The CABS also restores these functions, but only if modified by a lipid anchor to provide membrane association. Our findings highlight the role of Las17p binding for Vrp1p membrane association, suggest general membrane association may be more important than specific targeting to the cortical actin cytoskeleton for Vrp1p function in endocytosis and cell growth, and suggest that Vrp1p binding to individual effectors may alter their physiological activity. PMID:17635585

  14. Structure of the Toxoplasma gondii ROP18 Kinase Domain Reveals a Second Ligand Binding Pocket Required for Acute Virulence*

    PubMed Central

    Lim, Daniel; Gold, Daniel A.; Julien, Lindsay; Rosowski, Emily E.; Niedelman, Wendy; Yaffe, Michael B.; Saeij, Jeroen P. J.

    2013-01-01

    At least a third of the human population is infected with the intracellular parasite Toxoplasma gondii, which contributes significantly to the disease burden in immunocompromised and neutropenic hosts and causes serious congenital complications when vertically transmitted to the fetus. Genetic analyses have identified the Toxoplasma ROP18 Ser/Thr protein kinase as a major factor mediating acute virulence in mice. ROP18 is secreted into the host cell during the invasion process, and its catalytic activity is required for the acute virulence phenotype. However, its precise molecular function and regulation are not fully understood. We have determined the crystal structure of the ROP18 kinase domain, which is inconsistent with a previously proposed autoinhibitory mechanism of regulation. Furthermore, a sucrose molecule bound to our structure identifies an additional ligand-binding pocket outside of the active site cleft. Mutational analysis confirms an important role for this pocket in virulence. PMID:24129568

  15. Evolution of domain-peptide interactions to coadapt specificity and affinity to functional diversity.

    PubMed

    Kelil, Abdellali; Levy, Emmanuel D; Michnick, Stephen W

    2016-07-01

    Evolution of complexity in eukaryotic proteomes has arisen, in part, through emergence of modular independently folded domains mediating protein interactions via binding to short linear peptides in proteins. Over 30 years, structural properties and sequence preferences of these peptides have been extensively characterized. Less successful, however, were efforts to establish relationships between physicochemical properties and functions of domain-peptide interactions. To our knowledge, we have devised the first strategy to exhaustively explore the binding specificity of protein domain-peptide interactions. We applied the strategy to SH3 domains to determine the properties of their binding peptides starting from various experimental data. The strategy identified the majority (∼70%) of experimentally determined SH3 binding sites. We discovered mutual relationships among binding specificity, binding affinity, and structural properties and evolution of linear peptides. Remarkably, we found that these properties are also related to functional diversity, defined by depth of proteins within hierarchies of gene ontologies. Our results revealed that linear peptides evolved to coadapt specificity and affinity to functional diversity of domain-peptide interactions. Thus, domain-peptide interactions follow human-constructed gene ontologies, which suggest that our understanding of biological process hierarchies reflect the way chemical and thermodynamic properties of linear peptides and their interaction networks, in general, have evolved. PMID:27317745

  16. Structure and Function of the SWIRM Domain, a Conserved Protein Module Found in Chromatin Regulatory Complexes

    SciTech Connect

    Da,G.; Lenkart, J.; Zhao, K.; Shiekhattar, R.; Cairns, B.; Marmorstein, R.

    2006-01-01

    The SWIRM domain is a module found in the Swi3 and Rsc8 subunits of SWI/SNF-family chromatin remodeling complexes, and the Ada2 and BHC110/LSD1 subunits of chromatin modification complexes. Here we report the high-resolution crystal structure of the SWIRM domain from Swi3 and characterize the in vitro and in vivo function of the SWIRM domains from Saccharomyces cerevisiae Swi3 and Rsc8. The Swi3 SWIRM forms a four-helix bundle containing a pseudo 2-fold axis and a helix-turn-helix motif commonly found in DNA-binding proteins. We show that the Swi3 SWIRM binds free DNA and mononucleosomes with high and comparable affinity and that a subset of Swi3 substitution mutants that display growth defects in vivo also show impaired DNA-binding activity in vitro, consistent with a nucleosome targeting function of this domain. Genetic and biochemical studies also reveal that the Rsc8 and Swi3 SWIRM domains are essential for the proper assembly and in vivo functions of their respective complexes. Together, these studies identify the SWIRM domain as an essential multifunctional module for the regulation of gene expression.

  17. 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

  18. Functional anatomy of temporal organisation and domain-specificity of episodic memory retrieval

    PubMed Central

    Kwok, Sze Chai; Shallice, Tim; Macaluso, Emiliano

    2013-01-01

    Episodic memory provides information about the “when” of events as well as “what” and “where” they happened. Using functional imaging, we investigated the domain specificity of retrieval-related processes following encoding of complex, naturalistic events. Subjects watched a 42-min TV episode, and 24 h later, made discriminative choices of scenes from the clip during fMRI. Subjects were presented with two scenes and required to either choose the scene that happened earlier in the film (Temporal), or the scene with a correct spatial arrangement (Spatial), or the scene that had been shown (Object). We identified a retrieval network comprising the precuneus, lateral and dorsal parietal cortex, middle frontal and medial temporal areas. The precuneus and angular gyrus are associated with temporal retrieval, with precuneal activity correlating negatively with temporal distance between two happenings at encoding. A dorsal fronto-parietal network engages during spatial retrieval, while antero-medial temporal regions activate during object-related retrieval. We propose that access to episodic memory traces involves different processes depending on task requirements. These include memory-searching within an organised knowledge structure in the precuneus (Temporal task), online maintenance of spatial information in dorsal fronto-parietal cortices (Spatial task) and combining scene-related spatial and non-spatial information in the hippocampus (Object task). Our findings support the proposal of process-specific dissociations of retrieval. PMID:22877840

  19. Functional Roles of Clusters of Hydrophobic and Polar Residues in the Epithelial Na+ Channel Knuckle Domain.

    PubMed

    Chen, Jingxin; Ray, Evan C; Yates, Megan E; Buck, Teresa M; Brodsky, Jeffrey L; Kinlough, Carol L; Winarski, Katie L; Hughey, Rebecca P; Kleyman, Thomas R; Sheng, Shaohu

    2015-10-01

    The extracellular regions of epithelial Na(+) channel subunits are highly ordered structures composed of domains formed by α helices and β strands. Deletion of the peripheral knuckle domain of the α subunit in the αβγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na(+) (Na(+) self-inhibition). In contrast, deletion of either the β or γ subunit knuckle domain within the αβγ trimer dramatically reduces epithelial Na(+) channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na(+) self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na(+) self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the β or γ subunit knuckle domain resulted in little or no change in Na(+) self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na(+) self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs. PMID:26306034

  20. Gingipains from Porphyromonas gingivalis – Complex domain structures confer diverse functions

    PubMed Central

    Li, N.

    2011-01-01

    Gingipains, a group of arginine or lysine specific cysteine proteinases (also known as RgpA, RgpB and Kgp), have been recognized as major virulence factors in Porphyromonas gingivalis. This bacterium is one of a handful of pathogens that cause chronic periodontitis. Gingipains are involved in adherence to and colonization of epithelial cells, haemagglutination and haemolysis of erythrocytes, disruption and manipulation of the inflammatory response, and the degradation of host proteins and tissues. RgpA and Kgp are multi-domain proteins composed of catalytic domains and haemagglutinin/adhesin (HA) regions. The structure of the HA regions have previously been defined by a gingipain domain structure hypothesis which is a set of putative domain boundaries derived from the sequences of fragments of these proteins extracted from the cell surface. However, multiple sequence alignments and hidden Markov models predict an alternative domain architecture for the HA regions of gingipains. In this alternate model, two or three repeats of the so-called “cleaved adhesin” domains (and one other undefined domain in some strains) are the modules which constitute the substructure of the HA regions. Recombinant forms of these putative cleaved adhesin domains are indeed stable folded protein modules and recently determined crystal structures support the hypothesis of a modular organisation of the HA region. Based on the observed K2 and K3 structures as well as multiple sequence alignments, it is proposed that all the cleaved adhesin domains in gingipains will share the same β-sandwich jelly roll fold. The new domain model of the structure for gingipains and the haemagglutinin (HagA) proteins of P. gingivalis will guide future functional studies of these virulence factors. PMID:24466435

  1. The Popeye domain containing 2 (popdc2) gene in zebrafish is required for heart and skeletal muscle development

    PubMed Central

    Kirchmaier, Bettina C.; Poon, Kar Lai; Schwerte, Thorsten; Huisken, Jan; Winkler, Christoph; Jungblut, Benno; Stainier, Didier Y.; Brand, Thomas

    2013-01-01

    The Popeye domain containing (Popdc) genes encode a family of transmembrane proteins with an evolutionary conserved Popeye domain. These genes are abundantly expressed in striated muscle tissue, however their function is not well understood. In this study we have investigated the role of the popdc2 gene in zebrafish. Popdc2 transcripts were detected in the embryonic myocardium and transiently in the craniofacial and tail musculature. Morpholino oligonucleotide-mediated knockdown of popdc2 resulted in aberrant development of skeletal muscle and heart. Muscle segments in the trunk were irregularly shaped and craniofacial muscles were severely reduced or even missing. In the heart, pericardial edema was prevalent in the morphants and heart chambers were elongated and looping was abnormal. These pathologies in muscle and heart were alleviated after reducing the morpholino concentration. However the heart still was abnormal displaying cardiac arrhythmia at later stages of development. Optical recordings of cardiac contractility revealed irregular ventricular contractions with a 2:1, or 3:1 atrial/ventricular conduction ratio, which caused a significant reduction in heart frequency. Recordings of calcium transients with high spatiotemporal resolution using a transgenic calcium indicator line (Tg(cmlc2:gCaMP)s878) and SPIM microscopy confirmed the presence of a severe arrhythmia phenotype. Our results identify popdc2 as a gene important for striated muscle differentiation and cardiac morphogenesis. In addition it is required for the development of the cardiac conduction system. PMID:22290329

  2. Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.

    PubMed

    Laurent, Clémentine; Lekeux, Gilles; Ukuwela, Ashwinie A; Xiao, Zhiguang; Charlier, Jean-Benoit; Bosman, Bernard; Carnol, Monique; Motte, Patrick; Damblon, Christian; Galleni, Moreno; Hanikenne, Marc

    2016-03-01

    PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases. PMID:26797794

  3. 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

  4. Functional Specialization of Domains Tandemly Duplicated Witin 16S rRNA Methyltransferase RsmC

    SciTech Connect

    Sunita,S.; Purta, E.; Durawa, M.; Tkaczuk, K.; Swaathi, J.; Bujnicki, J.; Sivaraman, J.

    2007-01-01

    RNA methyltransferases (MTases) are important players in the biogenesis and regulation of the ribosome, the cellular machine for protein synthesis. RsmC is a MTase that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to G1207 of 16S rRNA. Mutations of G1207 have dominant lethal phenotypes in Escherichia coli, underscoring the significance of this modified nucleotide for ribosome function. Here we report the crystal structure of E. coli RsmC refined to 2.1 Angstroms resolution, which reveals two homologous domains tandemly duplicated within a single polypeptide. We characterized the function of the individual domains and identified key residues involved in binding of rRNA and SAM, and in catalysis. We also discovered that one of the domains is important for the folding of the other. Domain duplication and subfunctionalization by complementary degeneration of redundant functions (in particular substrate binding versus catalysis) has been reported for many enzymes, including those involved in RNA metabolism. Thus, RsmC can be regarded as a model system for functional streamlining of domains accompanied by the development of dependencies concerning folding and stability.

  5. The epigenetic regulator Smchd1 contains a functional GHKL-type ATPase domain.

    PubMed

    Chen, Kelan; Dobson, Renwick C J; Lucet, Isabelle S; Young, Samuel N; Pearce, F Grant; Blewitt, Marnie E; Murphy, James M

    2016-06-15

    Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic regulator that plays critical roles in gene regulation during development. Mutations in SMCHD1 were recently implicated in the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD), although the mechanistic basis remains of outstanding interest. We have previously shown that Smchd1 associates with chromatin via its homodimeric C-terminal hinge domain, yet little is known about the function of the putative GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPase domain at its N-terminus. To formally assess the structure and function of Smchd1's ATPase domain, we have generated recombinant proteins encompassing the predicted ATPase domain and the adjacent region. Here, we show that the Smchd1 N-terminal region exists as a monomer and adopts a conformation resembling that of monomeric full-length heat shock protein 90 (Hsp90) protein in solution, even though the two proteins share only ∼8% overall sequence identity. Despite being monomeric, the N-terminal region of Smchd1 exhibits ATPase activity, which can be antagonized by the reaction product, ADP, or the Hsp90 inhibitor, radicicol, at a nanomolar concentration. Interestingly, introduction of an analogous mutation to that identified in SMCHD1 of an FSHD patient compromised protein stability, suggesting a possible molecular basis for loss of protein function and pathogenesis. Together, these results reveal important structure-function characteristics of Smchd1 that may underpin its mechanistic action at the chromatin level. PMID:27059856

  6. δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function

    PubMed Central

    Arakel, Eric C.; Richter, Kora P.; Clancy, Anne; Schwappach, Blanche

    2016-01-01

    Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the μ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer. PMID:27298352

  7. δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function.

    PubMed

    Arakel, Eric C; Richter, Kora P; Clancy, Anne; Schwappach, Blanche

    2016-06-21

    Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the μ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer. PMID:27298352

  8. Collision Avoidance Functional Requirements for Step 1. Revision 6

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This Functional Requirements Document (FRD) describes the flow of requirements from the high level operational objectives down to the functional requirements specific to cooperative collision avoidance for high altitude, long endurance unmanned aircraft systems. These are further decomposed into performance and safety guidelines that are backed up by analysis or references to various documents or research findings. The FRD should be considered when establishing future policies, procedures, and standards pertaining to cooperative collision avoidance.

  9. Membrane fusion by the GTPase atlastin requires a conserved C-terminal cytoplasmic tail and dimerization through the middle domain

    PubMed Central

    Moss, Tyler J.; Andreazza, Camilla; Verma, Avani; Daga, Andrea; McNew, James A.

    2011-01-01

    The biogenesis and maintenance of the endoplasmic reticulum (ER) requires membrane fusion. ER homotypic fusion is driven by the large GTPase atlastin. Domain analysis of atlastin shows that a conserved region of the C-terminal cytoplasmic tail is absolutely required for fusion activity. Atlastin in adjacent membranes must associate to bring the ER membranes into molecular contact. Drosophila atlastin dimerizes in the presence of GTPγS but is monomeric with GDP or without nucleotide. Oligomerization requires the juxtamembrane middle domain three-helix bundle, as does efficient GTPase activity. A soluble version of the N-terminal cytoplasmic domain that contains the GTPase domain and the middle domain three-helix bundle serves as a potent, concentration-dependent inhibitor of membrane fusion both in vitro and in vivo. However, atlastin domains lacking the middle domain are without effect. GTP-dependent dimerization of atlastin generates an enzymatically active protein that drives membrane fusion after nucleotide hydrolysis and conformational reorganization. PMID:21690399

  10. Amyloid precursor protein dimerization and synaptogenic function depend on copper binding to the growth factor-like domain.

    PubMed

    Baumkötter, Frederik; Schmidt, Nadine; Vargas, Carolyn; Schilling, Sandra; Weber, Rebecca; Wagner, Katja; Fiedler, Sebastian; Klug, Wilfried; Radzimanowski, Jens; Nickolaus, Sebastian; Keller, Sandro; Eggert, Simone; Wild, Klemens; Kins, Stefan

    2014-08-13

    Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cis-orientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or disease-associated changes in copper homeostasis likely go along with altered APP synaptic function. PMID:25122912

  11. De novo design and engineering of functional metal and porphyrin-binding protein domains

    NASA Astrophysics Data System (ADS)

    Everson, Bernard H.

    In this work, I describe an approach to the rational, iterative design and characterization of two functional cofactor-binding protein domains. First, a hybrid computational/experimental method was developed with the aim of algorithmically generating a suite of porphyrin-binding protein sequences with minimal mutual sequence information. This method was explored by generating libraries of sequences, which were then expressed and evaluated for function. One successful sequence is shown to bind a variety of porphyrin-like cofactors, and exhibits light- activated electron transfer in mixed hemin:chlorin e6 and hemin:Zn(II)-protoporphyrin IX complexes. These results imply that many sophisticated functions such as cofactor binding and electron transfer require only a very small number of residue positions in a protein sequence to be fixed. Net charge and hydrophobic content are important in determining protein solubility and stability. Accordingly, rational modifications were made to the aforementioned design procedure in order to improve its overall success rate. The effects of these modifications are explored using two `next-generation' sequence libraries, which were separately expressed and evaluated. Particular modifications to these design parameters are demonstrated to effectively double the purification success rate of the procedure. Finally, I describe the redesign of the artificial di-iron protein DF2 into CDM13, a single chain di-Manganese four-helix bundle. CDM13 acts as a functional model of natural manganese catalase, exhibiting a kcat of 0.08s-1 under steady-state conditions. The bound manganese cofactors have a reduction potential of +805 mV vs NHE, which is too high for efficient dismutation of hydrogen peroxide. These results indicate that as a high-potential manganese complex, CDM13 may represent a promising first step toward a polypeptide model of the Oxygen Evolving Complex of the photosynthetic enzyme Photosystem II.

  12. The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis

    PubMed Central

    Guerrier, Sabrice; Coutinho-Budd, Jaeda; Sassa, Takayuki; Gresset, Aurélie; Jordan, Nicole Vincent; Cheng, Ken; Jin, Wei-Lin; Frost, Adam; Polleux, Franck

    2009-01-01

    SUMMARY During brain development, proper neuronal migration and morphogenesis is critical for the establishment of functional neural circuits. Here we report that srGAP2 negatively regulates neuronal migration and induces neurite outgrowth and branching through the ability of its F-BAR domain to induce filopodia-like membrane protrusions resembling those induced by I-BAR domains in vivo and in vitro. Previous work has suggested that in non-neuronal cells, filopodia dynamics decreases the rate of cell migration and the persistence of leading edge protrusions. srGAP2 knockdown reduces leading process branching and increases the rate of neuronal migration in vivo. Overexpression of srGAP2 or its F-BAR domain has the opposite effects, increasing leading process branching and decreasing migration. These results (1) suggest that F-BAR domains are functionally diverse and (2) highlight the functional importance of proteins directly regulating membrane deformation for proper neuronal migration and morphogenesis. PMID:19737524

  13. Functional requirements for gas characterization system computer software

    SciTech Connect

    Tate, D.D.

    1996-01-01

    This document provides the Functional Requirements for the Computer Software operating the Gas Characterization System (GCS), which monitors the combustible gasses in the vapor space of selected tanks. Necessary computer functions are defined to support design, testing, operation, and change control. The GCS requires several individual computers to address the control and data acquisition functions of instruments and sensors. These computers are networked for communication, and must multi-task to accommodate operation in parallel.

  14. Functional Characterization of Glycoprotein H Chimeras Composed of Conserved Domains of the Pseudorabies Virus and Herpes Simplex Virus 1 Homologs

    PubMed Central

    Böhm, Sebastian W.; Backovic, Marija; Klupp, Barbara G.; Rey, Felix A.; Fuchs, Walter

    2015-01-01

    ABSTRACT Membrane fusion is indispensable for entry of enveloped viruses into host cells. The conserved core fusion machinery of the Herpesviridae consists of glycoprotein B (gB) and the gH/gL complex. Recently, crystal structures of gH/gL of herpes simplex virus 2 (HSV-2) and Epstein-Barr virus and of a core fragment of pseudorabies virus (PrV) gH identified four structurally conserved gH domains. To investigate functional conservation, chimeric genes encoding combinations of individual domains of PrV and herpes simplex virus 1 (HSV-1) gH were expressed in rabbit kidney cells, and their processing and transport to the cell surface, as well as activity in fusion assays including gB, gD, and gL of PrV or HSV-1, were analyzed. Chimeric gH containing domain I of HSV-1 and domains II to IV of PrV exhibited limited fusion activity in the presence of PrV gB and gD and HSV-1 gL, but not of PrV gL. More strikingly, chimeric gH consisting of PrV domains I to III and HSV-1 domain IV exhibited considerable fusion activity together with PrV gB, gD, and gL. Replacing PrV gB with the HSV-1 protein significantly enhanced this activity. A cell line stably expressing this chimeric gH supported replication of gH-deleted PrV. Our results confirm the specificity of domain I for gL binding, demonstrate functional conservation of domain IV in two alphaherpesviruses from different genera, and indicate species-specific interactions of this domain with gB. They also suggest that gH domains II and III might form a structural and functional unit which does not tolerate major substitutions. IMPORTANCE Envelope glycoprotein H (gH) is essential for herpesvirus-induced membrane fusion, which is required for host cell entry and viral spread. Although gH is structurally conserved within the Herpesviridae, its precise role and its interactions with other components of the viral fusion machinery are not fully understood. Chimeric proteins containing domains of gH proteins from different

  15. Delineation of structural domains and identification of functionally important residues in DNA repair enzyme exonuclease VII

    PubMed Central

    Poleszak, Katarzyna; Kaminska, Katarzyna H.; Dunin-Horkawicz, Stanislaw; Lupas, Andrei; Skowronek, Krzysztof J.; Bujnicki, Janusz M.

    2012-01-01

    Exonuclease VII (ExoVII) is a bacterial nuclease involved in DNA repair and recombination that hydrolyses single-stranded DNA. ExoVII is composed of two subunits: large XseA and small XseB. Thus far, little was known about the molecular structure of ExoVII, the interactions between XseA and XseB, the architecture of the nuclease active site or its mechanism of action. We used bioinformatics methods to predict the structure of XseA, which revealed four domains: an N-terminal OB-fold domain, a middle putatively catalytic domain, a coiled-coil domain and a short C-terminal segment. By series of deletion and site-directed mutagenesis experiments on XseA from Escherichia coli, we determined that the OB-fold domain is responsible for DNA binding, the coiled-coil domain is involved in binding multiple copies of the XseB subunit and residues D155, R205, H238 and D241 of the middle domain are important for the catalytic activity but not for DNA binding. Altogether, we propose a model of sequence–structure–function relationships in ExoVII. PMID:22718974

  16. Annotation of Protein Domains Reveals Remarkable Conservation in the Functional Make up of Proteomes Across Superkingdoms

    PubMed Central

    Nasir, Arshan; Naeem, Aisha; Khan, Muhammad Jawad; Lopez-Nicora, Horacio D.; Caetano-Anollés, Gustavo

    2011-01-01

    The functional repertoire of a cell is largely embodied in its proteome, the collection of proteins encoded in the genome of an organism. The molecular functions of proteins are the direct consequence of their structure and structure can be inferred from sequence using hidden Markov models of structural recognition. Here we analyze the functional annotation of protein domain structures in almost a thousand sequenced genomes, exploring the functional and structural diversity of proteomes. We find there is a remarkable conservation in the distribution of domains with respect to the molecular functions they perform in the three superkingdoms of life. In general, most of the protein repertoire is spent in functions related to metabolic processes but there are significant differences in the usage of domains for regulatory and extra-cellular processes both within and between superkingdoms. Our results support the hypotheses that the proteomes of superkingdom Eukarya evolved via genome expansion mechanisms that were directed towards innovating new domain architectures for regulatory and extra/intracellular process functions needed for example to maintain the integrity of multicellular structure or to interact with environmental biotic and abiotic factors (e.g., cell signaling and adhesion, immune responses, and toxin production). Proteomes of microbial superkingdoms Archaea and Bacteria retained fewer numbers of domains and maintained simple and smaller protein repertoires. Viruses appear to play an important role in the evolution of superkingdoms. We finally identify few genomic outliers that deviate significantly from the conserved functional design. These include Nanoarchaeum equitans, proteobacterial symbionts of insects with extremely reduced genomes, Tenericutes and Guillardia theta. These organisms spend most of their domains on information functions, including translation and transcription, rather than on metabolism and harbor a domain repertoire characteristic of

  17. Structures and Functions of the Multiple KOW Domains of Transcription Elongation Factor Spt5

    PubMed Central

    Meyer, Peter A.; Li, Sheng; Zhang, Mincheng; Yamada, Kentaro; Takagi, Yuichiro; Hartzog, Grant A.

    2015-01-01

    The eukaryotic Spt4-Spt5 heterodimer forms a higher-order complex with RNA polymerase II (and I) to regulate transcription elongation. Extensive genetic and functional data have revealed diverse roles of Spt4-Spt5 in coupling elongation with chromatin modification and RNA-processing pathways. A mechanistic understanding of the diverse functions of Spt4-Spt5 is hampered by challenges in resolving the distribution of functions among its structural domains, including the five KOW domains in Spt5, and a lack of their high-resolution structures. We present high-resolution crystallographic results demonstrating that distinct structures are formed by the first through third KOW domains (KOW1-Linker1 [K1L1] and KOW2-KOW3) of Saccharomyces cerevisiae Spt5. The structure reveals that K1L1 displays a positively charged patch (PCP) on its surface, which binds nucleic acids in vitro, as shown in biochemical assays, and is important for in vivo function, as shown in growth assays. Furthermore, assays in yeast have shown that the PCP has a function that partially overlaps that of Spt4. Synthesis of our results with previous evidence suggests a model in which Spt4 and the K1L1 domain of Spt5 form functionally overlapping interactions with nucleic acids upstream of the transcription bubble, and this mechanism may confer robustness on processes associated with transcription elongation. PMID:26217010

  18. LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    LysK is a staphylococcal bacteriophage endolysin composed of three domains, an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain (cleaves between D-alanine of the stem peptide and glycine of the cross-bridge peptide) a mid-protein amidase 2 domain (N-ace...

  19. A folded and functional protein domain in an amyloid-like fibril

    PubMed Central

    Sackewitz, Mirko; von Einem, Sabrina; Hause, Gerd; Wunderlich, Michael; Schmid, Franz-Xaver; Schwarz, Elisabeth

    2008-01-01

    The effect of the polypeptide environment on polyalanine-induced fibril formation was investigated with amyloidogenic fragments from PAPBN1, a nuclear protein controlling polyadenylation. Mutation-caused extensions of the natural 10 alanine sequence up to maximally 17 alanines result in fibril formation of PABPN1 and the development of the disease oculopharyngeal muscular dystrophy (OPMD). We explored the influence of fibril formation on the structure and function of a one-domain protein linked to the fibril-forming part of PABPN1. The well-characterized, stably folded, one-domain protein, cold-shock protein CspB from Bacillus subtilis, was fused either to the C terminus of the entire N-terminal domain of PABPN1 or directly to peptides consisting of 10 or 17 alanine residues. The fusion protein between the N-terminal domain of PABPN1 and CspB formed fibrils in which the structure and activity of CspB were retained. In the fibrils formed by fusions in which the polyalanine sequence was directly linked to CspB, CspB was unfolded. These results indicate that the folded conformation and the function of a protein domain can be maintained in amyloid-like fibrils, and that the distance between this domain and the fibril plays an important role. PMID:18424511

  20. TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain

    PubMed Central

    Reglińska-Matveyev, Natalia; Andersson, Helena M.; Rezende, Suely M.; Dahlbäck, Björn; Crawley, James T. B.; Lane, David A.; Ahnström, Josefin

    2014-01-01

    Protein S is a cofactor for tissue factor pathway inhibitor (TFPI), accelerating the inhibition of activated factor X (FXa). TFPI Kunitz domain 3 residue Glu226 is essential for enhancement of TFPI by protein S. To investigate the complementary functional interaction site on protein S, we screened 44 protein S point, composite or domain swap variants spanning the whole protein S molecule for their TFPI cofactor function using a thrombin generation assay. Of these variants, two protein S/growth arrest–specific 6 chimeras, with either the whole sex hormone–binding globulin (SHBG)-like domain (Val243-Ser635; chimera III) or the SHBG laminin G-type 1 subunit (Ser283-Val459; chimera I), respectively, substituted by the corresponding domain in growth arrest–specific 6, were unable to enhance TFPI. The importance of the protein S SHBG-like domain (and its laminin G-type 1 subunit) for binding and enhancement of TFPI was confirmed in FXa inhibition assays and using surface plasmon resonance. In addition, protein S bound to C4b binding protein showed greatly reduced enhancement of TFPI-mediated inhibition of FXa compared with free protein S. We show that binding of TFPI to the protein S SHBG-like domain enables TFPI to interact optimally with FXa on a phospholipid membrane. PMID:24740810

  1. The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

    NASA Astrophysics Data System (ADS)

    Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

    2015-03-01

    The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

  2. The SH2 domain is crucial for function of Fyn in neuronal migration and cortical lamination

    PubMed Central

    Lu, Xi; Hu, Xinde; Song, Lingzhen; An, Lei; Duan, Minghui; Chen, Shulin; Zhao, Shanting

    2015-01-01

    Neurons in the developing brain form the cortical plate (CP) in an inside-out manner, in which the late-born neurons are located more superficially than the early-born neurons. Fyn, a member of the Src family kinases, plays an important role in neuronal migration by binding to many substrates. However, the role of the Src-homology 2 (SH2) domain in function of Fyn in neuronal migration remains poorly understood. Here, we demonstrate that the SH2 domain is essential for the action of Fyn in neuronal migration and cortical lamination. A point mutation in the Fyn SH2 domain (FynR176A) impaired neuronal migration and their final location in the cerebral cortex, by inducing neuronal aggregation and branching. Thus, we provide the first evidence of the Fyn SH2 domain contributing to neuronal migration and neuronal morphogenesis. [BMB Reports 2015; 48(2): 97-102] PMID:24912779

  3. SET for life: biochemical activities and biological functions of SET domain-containing proteins

    PubMed Central

    Herz, Hans-Martin; Garruss, Alexander; Shilatifard, Ali

    2013-01-01

    SET domain-containing proteins belong to a group of enzymes named after a common domain that utilizes the cofactor S-adenosyl-L-methionine (SAM) to achieve methylation of its substrates. Many SET domain-containing proteins have been shown to display catalytic activity towards particular lysine residues on histones, but emerging evidence also indicates that various non-histone proteins are specifically targeted by this clade of enzymes. Here, we summarize the most recent findings on the biological functions of the major families of SET domain-containing proteins catalyzing the methylation of histones 3 on lysines 4, 9, 27, and 36 (H3K4, H3K9, H3K27, and H3K36) and histone 4 on lysine 20 (H4K20) as well as candidates that have been reported to regulate non-histone substrates. PMID:24148750

  4. The L3MBTL3 Methyl-Lysine Reader Domain Functions As a Dimer.

    PubMed

    Baughman, Brandi M; Pattenden, Samantha G; Norris, Jacqueline L; James, Lindsey I; Frye, Stephen V

    2016-03-18

    L3MBTL3 recognizes mono- and dimethylated lysine residues on histone tails. The recently reported X-ray cocrystal structures of the chemical probe UNC1215 and inhibitor UNC2533 bound to the methyl-lysine reading MBT domains of L3MBTL3 demonstrate a unique and flexible 2:2 dimer mode of recognition. In this study, we describe our in vitro analysis of L3MBTL3 dimerization via its MBT domains and additionally show that this dimerization occurs within a cellular context in the absence of small molecule ligands. Furthermore, mutations to the first and second MBT domains abrogated L3MBTL3 dimerization both in vitro and in cells. These observations are consistent with the hypothesis that L3MBTL3 engages methylated histone tails as a dimer while carrying out its normal function and provides an explanation for the presence of repeated MBT domains within L3MBTL3. PMID:26317848

  5. Nse1 RING-like Domain Supports Functions of the Smc5-Smc6 Holocomplex in Genome Stability

    PubMed Central

    Pebernard, Stephanie; Perry, J. Jefferson P.; Tainer, John A.

    2008-01-01

    The Smc5-Smc6 holocomplex plays essential but largely enigmatic roles in chromosome segregation, and facilitates DNA repair. The Smc5-Smc6 complex contains six conserved non-SMC subunits. One of these, Nse1, contains a RING-like motif that often confers ubiquitin E3 ligase activity. We have functionally characterized the Nse1 RING-like motif, to determine its contribution to the chromosome segregation and DNA repair roles of Smc5-Smc6. Strikingly, whereas a full deletion of nse1 is lethal, the Nse1 RING-like motif is not essential for cellular viability. However, Nse1 RING mutant cells are hypersensitive to a broad spectrum of genotoxic stresses, indicating that the Nse1 RING motif promotes DNA repair functions of Smc5-Smc6. We tested the ability of both human and yeast Nse1 to mediate ubiquitin E3 ligase activity in vitro and found no detectable activity associated with full-length Nse1 or the isolated RING domains. Interestingly, however, the Nse1 RING-like domain is required for normal Nse1-Nse3-Nse4 trimer formation in vitro and for damage-induced recruitment of Nse4 and Smc5 to subnuclear foci in vivo. Thus, we propose that the Nse1 RING-like motif is a protein–protein interaction domain required for Smc5-Smc6 holocomplex integrity and recruitment to, or retention at, DNA lesions. PMID:18667531

  6. Structure-Function Relationships in the HAMP and Proximal Signaling Domains of the Aerotaxis Receptor Aer▿

    PubMed Central

    Watts, Kylie J.; Johnson, Mark S.; Taylor, Barry L.

    2008-01-01

    Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two α-helices separated by a structured loop. The proximal signaling domain consisted of two α-helices separated by a short undetermined structure. The Af1503 HAMP domain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the β-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed. PMID:18203838

  7. Dimerization and Transactivation Domains as Candidates for Functional Modulation and Diversity of Sox9

    PubMed Central

    Geraldo, Marcos Tadeu; Valente, Guilherme Targino; Nakajima, Rafael Takahiro; Martins, Cesar

    2016-01-01

    Sox9 plays an important role in a large variety of developmental pathways in vertebrates. It is composed of three domains: high-mobility group box (HMG box), dimerization (DIM) and transactivation (TAD). One of the main processes for regulation and variability of the pathways involving Sox9 is the self-gene expression regulation of Sox9. However, the subsequent roles of the Sox9 domains can also generate regulatory modulations. Studies have shown that TADs can bind to different types of proteins and its function seems to be influenced by DIM. Therefore, we hypothesized that both domains are directly associated and can be responsible for the functional variability of Sox9. We applied a method based on a broad phylogenetic context, using sequences of the HMG box domain, to ensure the homology of all the Sox9 copies used herein. The data obtained included 4,921 sequences relative to 657 metazoan species. Based on coevolutionary and selective pressure analyses of the Sox9 sequences, we observed coevolutions involving DIM and TADs. These data, along with the experimental data from literature, indicate a functional relationship between these domains. Moreover, DIM and TADs may be responsible for the functional plasticity of Sox9 because they are more tolerant for molecular changes (higher Ka/Ks ratio than the HMG box domain). This tolerance could allow a differential regulation of target genes or promote novel targets during transcriptional activation. In conclusion, we suggest that DIM and TADs functional association may regulate differentially the target genes or even promote novel targets during transcription activation mediated by Sox9 paralogs, contributing to the subfunctionalization of Sox9a and Sox9b in teleosts. PMID:27196604

  8. Organization of the pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM domain protein Mosaic eyes

    PubMed Central

    Kramer-Zucker, Albrecht G.; Wiessner, Stephanie; Jensen, Abbie M.; Drummond, Iain A.

    2010-01-01

    Podocytes are specialized cells of the kidney that form the blood filtration barrier in the kidney glomerulus. The barrier function of podocytes depends upon the development of specialized cell–cell adhesion complexes called slit-diaphragms that form between podocyte foot processes surrounding glomerular blood vessels. Failure of the slit-diaphragm to form results in leakage of high molecular weight proteins into the blood filtrate and urine, a condition called proteinuria. In this work, we test whether the zebrafish pronephros can be used as an assay system for the development of glomerular function with the goal of identifying novel components of the slit-diaphragm. We first characterized the function of the zebrafish homolog of Nephrin, the disease gene associated with the congenital nephritic syndrome of the Finnish type, and Podocin, the gene mutated in autosomal recessive steroid-resistant nephrotic syndrome. Zebrafish nephrin and podocin were specifically expressed in pronephric podocytes and required for the development of pronephric podocyte cell structure. Ultrastructurally, disruption of nephrin or podocin expression resulted in a loss of slit-diaphragms at 72 and 96 h post-fertilization and failure to form normal podocyte foot processes. We also find that expression of the band 4.1/FERM domain gene mosaic eyes in podocytes is required for proper formation of slit-diaphragm cell–cell junctions. A functional assay of glomerular filtration barrier revealed that absence of normal nephrin, podocin or mosaic eyes expression results in loss of glomerular filtration discrimination and aberrant passage of high molecular weight substances into the glomerular filtrate. PMID:16102746

  9. Localization of a Guanylyl Cyclase to Chemosensory Cilia Requires the Novel Ciliary MYND Domain Protein DAF-25

    PubMed Central

    Jensen, Victor L.; Bialas, Nathan J.; Bishop-Hurley, Sharon L.; Molday, Laurie L.; Kida, Katarzyna; Nguyen, Phuong Anh T.; Blacque, Oliver E.; Molday, Robert S.; Leroux, Michel R.; Riddle, Donald L.

    2010-01-01

    In harsh conditions, Caenorhabditis elegans arrests development to enter a non-aging, resistant diapause state called the dauer larva. Olfactory sensation modulates the TGF-β and insulin signaling pathways to control this developmental decision. Four mutant alleles of daf-25 (abnormal DAuer Formation) were isolated from screens for mutants exhibiting constitutive dauer formation and found to be defective in olfaction. The daf-25 dauer phenotype is suppressed by daf-10/IFT122 mutations (which disrupt ciliogenesis), but not by daf-6/PTCHD3 mutations (which prevent environmental exposure of sensory cilia), implying that DAF-25 functions in the cilia themselves. daf-25 encodes the C. elegans ortholog of mammalian Ankmy2, a MYND domain protein of unknown function. Disruption of DAF-25, which localizes to sensory cilia, produces no apparent cilia structure anomalies, as determined by light and electron microscopy. Hinting at its potential function, the dauer phenotype, epistatic order, and expression profile of daf-25 are similar to daf-11, which encodes a cilium-localized guanylyl cyclase. Indeed, we demonstrate that DAF-25 is required for proper DAF-11 ciliary localization. Furthermore, the functional interaction is evolutionarily conserved, as mouse Ankmy2 interacts with guanylyl cyclase GC1 from ciliary photoreceptors. The interaction may be specific because daf-25 mutants have normally-localized OSM-9/TRPV4, TAX-4/CNGA1, CHE-2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8, OSM-5/IFT88, and XBX-1/D2LIC in the cilia. Intraflagellar transport (IFT) (required to build cilia) is not defective in daf-25 mutants, although the ciliary localization of DAF-25 itself is influenced in che-11 mutants, which are defective in retrograde IFT. In summary, we have discovered a novel ciliary protein that plays an important role in cGMP signaling by localizing a guanylyl cyclase to the sensory organelle. PMID:21124868

  10. Time-domain representation of frequency-dependent foundation impedance functions

    USGS Publications Warehouse

    Safak, E.

    2006-01-01

    Foundation impedance functions provide a simple means to account for soil-structure interaction (SSI) when studying seismic response of structures. Impedance functions represent the dynamic stiffness of the soil media surrounding the foundation. The fact that impedance functions are frequency dependent makes it difficult to incorporate SSI in standard time-history analysis software. This paper introduces a simple method to convert frequency-dependent impedance functions into time-domain filters. The method is based on the least-squares approximation of impedance functions by ratios of two complex polynomials. Such ratios are equivalent, in the time-domain, to discrete-time recursive filters, which are simple finite-difference equations giving the relationship between foundation forces and displacements. These filters can easily be incorporated into standard time-history analysis programs. Three examples are presented to show the applications of the method.

  11. The Tetramerization Domain Potentiates Kv4 Channel Function by Suppressing Closed-State Inactivation

    PubMed Central

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

    2014-01-01

    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. PMID:25185545

  12. Inter-Species Complementation of the Translocon Beta Subunit Requires Only Its Transmembrane Domain

    PubMed Central

    Leroux, Alexandre; Rokeach, Luis A.

    2008-01-01

    In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61α, β and γ in mammals. Unlike the other subunits, the β subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61β encoding genes results in different phenotypes in different species. Nevertheless, the β subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61β in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1+ results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23°C. Sec61β homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1+ in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61β is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61β from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species. PMID:19057642

  13. Functions & Requirements for Debris Removal System Project A-2

    SciTech Connect

    PRECECHTEL, D.R.

    1999-12-29

    This revision of the Functions and Requirements Document updates the approved Functions and Requirements for Debris Removal Subproject WHC-SD-SNF-FRD-009, Rev. 0. It has been revised in its entirety to reflect the current scope of work for Debris Removal as canisters and lids under the K Basin Projects work breakdown structure (WBS). In this revision the canisters and lids will be consider debris and a new set of Functions and Requirements have been developed to remove the canisters and lids from the basin.

  14. Functional assessment of perforin C2 domain mutations illustrates the critical role for calcium-dependent lipid binding in perforin cytotoxic function.

    PubMed

    Urrea Moreno, Ramon; Gil, Juana; Rodriguez-Sainz, Carmen; Cela, Elena; LaFay, Victor; Oloizia, Brian; Herr, Andrew B; Sumegi, Janos; Jordan, Michael B; Risma, Kimberly A

    2009-01-01

    Perforin-mediated lymphocyte cytotoxicity is critical for pathogen elimination and immune homeostasis. Perforin disruption of target cell membranes is hypothesized to require binding of a calcium-dependent, lipid-inserting, C2 domain. In a family affected by hemophagocytic lymphohistiocytosis, a severe inflammatory disorder caused by perforin deficiency, we identified 2 amino acid substitutions in the perforin C2 domain: T435M, a previously identified mutant with disputed pathogenicity, and Y438C, a novel substitution. Using biophysical modeling, we predicted that the T435M substitution, but not Y438C, would interfere with calcium binding and thus cytotoxic function. The capacity for cytotoxic function was tested after expression of the variant perforins in rat basophilic leukemia cells and murine cytotoxic T lymphocytes. As predicted, cells transduced with perforin-T435M lacked cytotoxicity, but those expressing perforin-Y438C displayed intact cytotoxic function. Using novel antibody-capture and liposome-binding assays, we found that both mutant perforins were secreted; however, only nonmutated and Y438C-substituted perforins were capable of calcium-dependent lipid binding. In addition, we found that perforin-Y438C was capable of mediating cytotoxicity without apparent proteolytic maturation. This study clearly demonstrates the pathogenicity of the T435M mutation and illustrates, for the first time, the critical role of the human perforin C2 domain for calcium-dependent, cytotoxic function. PMID:18927437

  15. Structure of the Bro1 Domain Protein BROX and Functional Analyses of the ALIX Bro1 Domain in HIV-1 Budding

    SciTech Connect

    Zhai Q.; Robinson H.; Landesman M. B.; Sundquist W. I.; Hill C. P.

    2011-12-01

    Bro1 domains are elongated, banana-shaped domains that were first identified in the yeast ESCRT pathway protein, Bro1p. Humans express three Bro1 domain-containing proteins: ALIX, BROX, and HD-PTP, which function in association with the ESCRT pathway to help mediate intraluminal vesicle formation at multivesicular bodies, the abscission stage of cytokinesis, and/or enveloped virus budding. Human Bro1 domains share the ability to bind the CHMP4 subset of ESCRT-III proteins, associate with the HIV-1 NC{sup Gag} protein, and stimulate the budding of viral Gag proteins. The curved Bro1 domain structure has also been proposed to mediate membrane bending. To date, crystal structures have only been available for the related Bro1 domains from the Bro1p and ALIX proteins, and structures of additional family members should therefore aid in the identification of key structural and functional elements. We report the crystal structure of the human BROX protein, which comprises a single Bro1 domain. The Bro1 domains from BROX, Bro1p and ALIX adopt similar overall structures and share two common exposed hydrophobic surfaces. Surface 1 is located on the concave face and forms the CHMP4 binding site, whereas Surface 2 is located at the narrow end of the domain. The structures differ in that only ALIX has an extended loop that projects away from the convex face to expose the hydrophobic Phe105 side chain at its tip. Functional studies demonstrated that mutations in Surface 1, Surface 2, or Phe105 all impair the ability of ALIX to stimulate HIV-1 budding. Our studies reveal similarities in the overall folds and hydrophobic protein interaction sites of different Bro1 domains, and show that a unique extended loop contributes to the ability of ALIX to function in HIV-1 budding.

  16. Domain boundaries in silicene: Density functional theory calculations on electronic properties

    NASA Astrophysics Data System (ADS)

    Xiao, Hong-Jun; Zhang, Li-Zhi; Du, Shi-Xuan; Gao, Hong-Jun

    2015-08-01

    By using density functional theory (DFT)-based first-principles calculations, the structural stability and electronic properties for two kinds of silicene domain boundaries, forming along armchair edge and zigzag edge, have been investigated. The results indicate that a linkage of tetragonal and octagonal rings (4|8) appears along the armchair edge, while a linkage of paired pentagonal and octagonal rings (5|5|8) appears along the zigzag edge. Different from graphene, the buckling properties of silicene lead to two mirror symmetrical edges of silicene line-defect. The formation energies indicate that the 5|5|8 domain boundary is more stable than the 4|8 domain boundary. Similar to graphene, the calculated electronic properties show that the 5|5|8 domain boundaries exhibit metallic properties and the 4|8 domain boundaries are half-metal. Both domain boundaries create the perfect one-dimensional (1D) metallic wires. Due to the metallic properties, these two kinds of nanowires can be used to build the silicene-based devices. Project supported by the National Natural Science Foundation of China (Grant Nos. 61390501 and 51325204), the National Basic Research Program of China (Grant Nos. 2011CB808401 and 2011CB921702), and the Tainjin Supercomputing Center, Chinese Academy of Sciences.

  17. The Aspartate-Less Receiver (ALR) Domains: Distribution, Structure and Function

    PubMed Central

    Weiner, Joshua J.; Han, Lanlan; Peterson, Francis C.; Volkman, Brian F.; Silvaggi, Nicholas R.; Ulijasz, Andrew T.

    2015-01-01

    Two-component signaling systems are ubiquitous in bacteria, Archaea and plants and play important roles in sensing and responding to environmental stimuli. To propagate a signaling response the typical system employs a sensory histidine kinase that phosphorylates a Receiver (REC) domain on a conserved aspartate (Asp) residue. Although it is known that some REC domains are missing this Asp residue, it remains unclear as to how many of these divergent REC domains exist, what their functional roles are and how they are regulated in the absence of the conserved Asp. Here we have compiled all deposited REC domains missing their phosphorylatable Asp residue, renamed here as the Aspartate-Less Receiver (ALR) domains. Our data show that ALRs are surprisingly common and are enriched for when attached to more rare effector outputs. Analysis of our informatics and the available ALR atomic structures, combined with structural, biochemical and genetic data of the ALR archetype RitR from Streptococcus pneumoniae presented here suggest that ALRs have reorganized their active pockets to instead take on a constitutive regulatory role or accommodate input signals other than Asp phosphorylation, while largely retaining the canonical post-phosphorylation mechanisms and dimeric interface. This work defines ALRs as an atypical REC subclass and provides insights into shared mechanisms of activation between ALR and REC domains. PMID:25875291

  18. School-Aged Children Born Preterm: Review of Functioning across Multiple Domains and Guidelines for Assessment

    ERIC Educational Resources Information Center

    Dempsey, Allison G.; Keller-Margulis, Milena; Mire, Sarah; Abrahamson, Catherine; Dutt, Sonia; Llorens, Ashlie; Payan, Anita

    2015-01-01

    Children born preterm are at risk for developmental deficits across multiple functional domains. As the rate of survival for preterm infants increases due to medical advancements, a greater understanding is needed for how to meet the needs of this growing population in schools. Because approximately 50-70% of children born preterm require…

  19. Collegiate Mathematics Students' Misconceptions of Domain and Zeros of Rational Functions

    ERIC Educational Resources Information Center

    Dotson, Geraldine Ting

    2009-01-01

    A new 12 item research questionnaire was developed specifically to assess collegiate mathematics students' concept image of domain and zeros of rational functions. The study was designed to validate Tall and Vinner's (1981) cognitive model. Support was found for the hypothesis that students' mathematical experience influences their growth, with…

  20. Frequency-Domain Green's Functions for Radar Waves in Heterogeneous 2.5D Media

    EPA Science Inventory

    Green’s functions for radar waves propagating in heterogeneous media may be calculated in the frequency domain using a hybrid of two numerical methods. The model is defined in the Cartesian coordinate system, and its electromagnetic properties may vary in the x and z directions, ...

  1. Direct Injection of Functional Single-Domain Antibodies from E. coli into Human Cells

    PubMed Central

    Blanco-Toribio, Ana; Muyldermans, Serge; Frankel, Gad; Fernández, Luis Ángel

    2010-01-01

    Intracellular proteins have a great potential as targets for therapeutic antibodies (Abs) but the plasma membrane prevents access to these antigens. Ab fragments and IgGs are selected and engineered in E. coli and this microorganism may be also an ideal vector for their intracellular delivery. In this work we demonstrate that single-domain Ab (sdAbs) can be engineered to be injected into human cells by E. coli bacteria carrying molecular syringes assembled by a type III protein secretion system (T3SS). The injected sdAbs accumulate in the cytoplasm of HeLa cells at levels ca. 105–106 molecules per cell and their functionality is shown by the isolation of sdAb-antigen complexes. Injection of sdAbs does not require bacterial invasion or the transfer of genetic material. These results are proof-of-principle for the capacity of E. coli bacteria to directly deliver intracellular sdAbs (intrabodies) into human cells for analytical and therapeutic purposes. PMID:21170340

  2. Sustaining multiple ecosystem functions in grassland communities requires higher biodiversity.

    PubMed

    Zavaleta, Erika S; Pasari, Jae R; Hulvey, Kristin B; Tilman, G David

    2010-01-26

    Society places value on the multiple functions of ecosystems from soil fertility to erosion control to wildlife-carrying capacity, and these functions are potentially threatened by ongoing biodiversity losses. Recent empirically based models using individual species' traits suggest that higher species richness is required to provide multiple ecosystem functions. However, no study to date has analyzed the observed functionality of communities of interacting species over multiple temporal scales to assess the relationship between biodiversity and multifunctionality. We use data from the longest-running biodiversity-functioning field experiment to date to test how species diversity affects the ability of grassland ecosystems to provide threshold levels of up to eight ecosystem functions simultaneously. Across years and every combination of ecosystem functions, minimum-required species richness consistently increases with the number of functions considered. Moreover, tradeoffs between functions and variability among years prevent any one community type from providing high levels of multiple functions, regardless of its diversity. Sustained multifunctionality, therefore, likely requires both higher species richness than single ecosystem functionality and a diversity of species assemblages across the landscape. PMID:20080690

  3. Domain Engineering

    NASA Astrophysics Data System (ADS)

    Bjørner, Dines

    Before software can be designed we must know its requirements. Before requirements can be expressed we must understand the domain. So it follows, from our dogma, that we must first establish precise descriptions of domains; then, from such descriptions, “derive” at least domain and interface requirements; and from those and machine requirements design the software, or, more generally, the computing systems.

  4. Comprehensive analysis and identification of the human STIM1 domains for structural and functional studies.

    PubMed

    How, Jonathan; Zhang, Ai; Phillips, Margaret; Reynaud, Aline; Lu, Si Yan; Pan, Lucy Xin; Ho, Hai Ting; Yau, Yin Hoe; Guskov, Albert; Pervushin, Konstantin; Shochat, Susana Geifman; Eshaghi, Said

    2013-01-01

    STIM1 is a Ca(2+) sensor within the ER membrane known to activate the plasma membrane store-operated Ca(2+) channel upon depletion of its target ion in the ER lumen. This activation is a crucial step to initiate the Ca(2+) signaling cascades within various cell types. Human STIM1 is a 77.4 kDa protein consisting of various domains that are involved in Ca(2+) sensing, oligomerization, and channel activation and deactivation. In this study, we identify the domains and boundaries in which functional and stable recombinant human STIM1 can be produced in large quantities. To achieve this goal, we cloned nearly 200 constructs that vary in their initial and terminal residues, length and presence of the transmembrane domain, and we conducted expression and purification analyses using these constructs. The results revealed that nearly half of the constructs could be expressed and purified with high quality, out of which 25% contained the integral membrane domain. Further analyses using surface plasmon resonance, nuclear magnetic resonance and a thermostability assay verified the functionality and integrity of these constructs. Thus, we have been able to identify the most stable and well-behaved domains of the hSTIM1 protein, which can be used for future in vitro biochemical and biophysical studies. PMID:23320111

  5. MscCG from Corynebacterium glutamicum: functional significance of the C-terminal domain.

    PubMed

    Becker, Michael; Krämer, Reinhard

    2015-10-01

    Corynebacterium glutamicum is used in microbial biotechnology for the production of amino acids, e.g., glutamate and lysine. Excretion of glutamate into the surrounding medium under production conditions is mediated by MscCG, an MscS-type mechanosensitive channel. In difference to most other MscS-type channel proteins, MscCG carries, in addition to the N-terminal pore domain, a long C-terminal domain that amounts to about half of the size of the protein and harbors an additional transmembrane segment. Here we study the impact of the C-terminal domain on both functions of MscCG as mechanosensitive channel and as glutamate exporter. Sequential truncations of the C-terminal domain were applied, as well as deletion of particular subdomains, replacement of these segments by other amino acid sequences, and sequence randomization. Several parameters of cell physiology and bioenergetics of the obtained mutants related to both glutamate excretion and response to osmotic stress were quantified. All three subdomains of the C-terminal domain, i.e., the periplasmic loop, the fourth transmembrane segment, and the cytoplasmic loop, proved to be of core significance for MscCG function, in particular for glutamate excretion. PMID:26033538

  6. Structure-Function Analysis of the Mcl-1 Protein Identifies a Novel Senescence-regulating Domain.

    PubMed

    Demelash, Abeba; Pfannenstiel, Lukas W; Tannenbaum, Charles S; Li, Xiaoxia; Kalady, Matthew F; DeVecchio, Jennifer; Gastman, Brian R

    2015-09-01

    Unlike other antiapoptotic Bcl-2 family members, Mcl-1 also mediates resistance to cancer therapy by uniquely inhibiting chemotherapy-induced senescence (CIS). In general, Bcl-2 family members regulate apoptosis at the level of the mitochondria through a common prosurvival binding groove. Through mutagenesis, we determined that Mcl-1 can inhibit CIS even in the absence of its apoptotically important mitochondrion-localizing domains. This finding prompted us to generate a series of Mcl-1 deletion mutants from both the N and C termini of the protein, including one that contained a deletion of all of the Bcl-2 homology domains, none of which impacted anti-CIS capabilities. Through subsequent structure-function analyses of Mcl-1, we identified a previously uncharacterized loop domain responsible for the anti-CIS activity of Mcl-1. The importance of the loop domain was confirmed in multiple tumor types, two in vivo models of senescence, and by demonstrating that a peptide mimetic of the loop domain can effectively inhibit the anti-CIS function of Mcl-1. The results from our studies appear to be highly translatable because we discerned an inverse relationship between the expression of Mcl-1 and of various senescence markers in cancerous human tissues. In summary, our findings regarding the unique structural properties of Mcl-1 provide new approaches for targeted cancer therapy. PMID:26205817

  7. The charged region of Hsp90 modulates the function of the N-terminal domain

    PubMed Central

    Scheibel, Thomas; Siegmund, Heiko Ingo; Jaenicke, Rainer; Ganz, Peter; Lilie, Hauke; Buchner, Johannes

    1999-01-01

    Hsp90, an abundant heat shock protein that is highly expressed even under physiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors. It seems to contain two chaperone sites differing in substrate specificity. Binding of ATP or the antitumor drug geldanamycin alters the substrate affinity of the N-terminal chaperone site, whereas both substances show no influence on the C-terminal one. In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various lengths in different organisms. As this linker region represents the most striking difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s. Here, we have analyzed a fragment of yeast Hsp90 consisting of the N-terminal domain and the charged region (N272) in comparison with the isolated N-terminal domain (N210). We show that the charged region causes an increase in the affinity of the N-terminal domain for nonnative protein and establishes a crosstalk between peptide and ATP binding. Thus, the binding of peptide to N272 decreases its affinity for ATP and geldanamycin, whereas the ATP-binding properties of the monomeric N-terminal domain N210 are not influenced by peptide binding. We propose that the charged region connecting the two chaperone domains plays an important role in regulating chaperone function of Hsp90. PMID:9990018

  8. Function of the CysD domain of the gel-forming MUC2 mucin

    PubMed Central

    Ambort, Daniel; van der Post, Sjoerd; Johansson, Malin E. V.; MacKenzie, Jenny; Thomsson, Elisabeth; Krengel, Ute; Hansson, Gunnar C.

    2011-01-01

    The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its N- and C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by MS revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel, thereby determining the pore sizes of the mucus. PMID:21338337

  9. The conserved glycine residues in the transmembrane domain of the Semliki Forest virus fusion protein are not required for assembly and fusion

    SciTech Connect

    Liao Maofu; Kielian, Margaret . E-mail: kielian@aecom.yu.edu

    2005-02-05

    The alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered fusion reaction mediated by the viral E1 protein. Both the E1 fusion peptide and transmembrane (TM) domain are essential for membrane fusion, but the functional requirements for the TM domain are poorly understood. Here we explored the role of the five TM domain glycine residues, including the highly conserved glycine pair at E1 residues 415/416. SFV mutants with alanine substitutions for individual or all five glycine residues (5G/A) showed growth kinetics and fusion pH dependence similar to those of wild-type SFV. Mutants with increasing substitution of glycine residues showed an increasingly more stringent requirement for cholesterol during fusion. The 5G/A mutant showed decreased fusion kinetics and extent in fluorescent lipid mixing assays. TM domain glycine residues thus are not required for efficient SFV fusion or assembly but can cause subtle effects on the properties of membrane fusion.

  10. Functional characterization of heat-shock protein 90 from Oryza sativa and crystal structure of its N-terminal domain.

    PubMed

    Raman, Swetha; Suguna, Kaza

    2015-06-01

    Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is essential for the normal functioning of eukaryotic cells. It plays crucial roles in cell signalling, cell-cycle control and in maintaining proteome integrity and protein homeostasis. In plants, Hsp90s are required for normal plant growth and development. Hsp90s are observed to be upregulated in response to various abiotic and biotic stresses and are also involved in immune responses in plants. Although there are several studies elucidating the physiological role of Hsp90s in plants, their molecular mechanism of action is still unclear. In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 Å resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 from Dictyostelium discoideum, which shares 70% sequence identity with OsHsp90-NTD. This is the second reported structure of a domain of Hsp90 from a plant source. PMID:26057797

  11. BIM-Mediated Membrane Insertion of the BAK Pore Domain Is an Essential Requirement for Apoptosis

    PubMed Central

    Weber, Kathrin; Harper, Nicholas; Schwabe, John; Cohen, Gerald M.

    2013-01-01

    Summary BAK activation represents a key step during apoptosis, but how it converts into a mitochondria-permeabilizing pore remains unclear. By further delineating the structural rearrangements involved, we reveal that BAK activation progresses through a series of independent steps: BH3-domain exposure, N-terminal change, oligomerization, and membrane insertion. Employing a “BCL-XL-addiction” model, we show that neutralization of BCL-XL by the BH3 mimetic ABT-737 resulted in death only when cells were reconstituted with BCL-XL:BAK, but not BCL-2/ BCL-XL:BIM complexes. Although this resembles the indirect model, release of BAK from BCL-XL did not result in spontaneous adoption of the pore conformation. Commitment to apoptosis required association of the direct activator BIM with oligomeric BAK promoting its conversion to a membrane-inserted pore. The sequential nature of this cascade provides multiple opportunities for other BCL-2 proteins to interfere with or promote BAK activation and unites aspects of the indirect and direct activation models. PMID:24120870

  12. Homogalacturonan synthesis in Arabidopsis thaliana requires a Golgi-localized protein with a putative methyltransferase domain.

    PubMed

    Mouille, Grégory; Ralet, Marie-Christine; Cavelier, Céline; Eland, Cathlene; Effroy, Delphine; Hématy, Kian; McCartney, Lesley; Truong, Hoai Nam; Gaudon, Virginie; Thibault, Jean-François; Marchant, Alan; Höfte, Herman

    2007-05-01

    Pectins are a family of complex cell-wall polysaccharides, the biosynthesis of which remains poorly understood. We identified dwarf mutants with reduced cell adhesion at a novel locus, QUASIMODO2 (QUA2). qua2-1 showed a 50% reduction in homogalacturonan (HG) content compared with the wild type, without affecting other cell-wall polysaccharides. The remaining HG in qua2-1 showed an unaltered degree of methylesterification. Positional cloning and GFP fusions showed that QUA2, consistent with a role in HG synthesis, encodes a Golgi-localized protein. In contrast to QUA1, another Golgi-localized protein required for HG-synthesis, QUA2 does not show sequence similarity to glycosyltransferases, but instead contains a putative methyltransferase (MT) domain. The Arabidopsis genome encodes 29 QUA2-related proteins. Interestingly, the transcript profiles of QUA1 and QUA2 are correlated and other pairs of QUA1 and QUA2 homologues with correlated transcript profiles can be identified. Together, the results lead to the hypothesis that QUA2 is a pectin MT, and that polymerization and methylation of homogalacturonan are interdependent reactions. PMID:17425712

  13. Securin can have a separase cleavage site by substitution mutations in the domain required for stabilization and inhibition of separase.

    PubMed

    Nagao, Koji; Yanagida, Mitsuhiro

    2006-03-01

    Securin-separase complex is required for sister chromatid separation. Securin degrades in an APC/cyclosome dependent manner. Separase is activated on the destruction of securin and cleaves cohesin. Fission yeast securin/Cut2 required for proper separase localization has the motifs for destruction and separase-binding at the N- and C-termini, respectively. We report here the third essential domain, which becomes toxic when the 76-amino acid fragment (81-156) in the middle is overproduced. The fragment inhibits separase, while separase is recruited normally and securin is destroyed. It may interfere with separase activation after destruction of securin. If the 127DIE129 stretch is substituted for AIA, the fragment toxicity and the full-length function are abolished. Interestingly, Cut2 is cleaved in a separase dependent manner if the cleavage consensus is introduced following the DIE sequence. This finding is consistent with the proposed model that the DIE region may mimic the cleavage site of separase and inhibit the activation of separase. Evidence for physical interaction between the fragment and separase is provided. A temperature sensitive mutation cut1-K73 isolated by its specific resistance to the fragment toxicity resides in the superhelical region of separase, suggesting that the catalytic site and the helical region in separase may cooperate for activation. PMID:16483313

  14. Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

    NASA Technical Reports Server (NTRS)

    Ramachandiran, S.; Takezawa, D.; Wang, W.; Poovaiah, B. W.

    1997-01-01

    A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

  15. FEDERAL FACILITY COMPLIANCE AGREEMENT (FFCA) STACK ISOLATION PROJECT FUNCTIONS & REQUIREMENTS

    SciTech Connect

    TRANBARGER, R.K.

    2003-12-16

    This document delineates the functions and requirements for the FFCA Stack Isolation Project for the 244-A, 244-BX, 244-5, and 244-TX DCRTs. The isolation of each ventilation system and stack includes the electrical, instrumentation, and mechanical isolation of the ventilation system and the installation of primary and annulus breather filters to provide passive ventilation to meet the FFCA requirements.

  16. Convolutions of Rayleigh functions and their application to semi-linear equations in circular domains

    NASA Astrophysics Data System (ADS)

    Varlamov, Vladimir

    2007-03-01

    Rayleigh functions [sigma]l([nu]) are defined as series in inverse powers of the Bessel function zeros [lambda][nu],n[not equal to]0, where ; [nu] is the index of the Bessel function J[nu](x) and n=1,2,... is the number of the zeros. Convolutions of Rayleigh functions with respect to the Bessel index, are needed for constructing global-in-time solutions of semi-linear evolution equations in circular domains [V. Varlamov, On the spatially two-dimensional Boussinesq equation in a circular domain, Nonlinear Anal. 46 (2001) 699-725; V. Varlamov, Convolution of Rayleigh functions with respect to the Bessel index, J. Math. Anal. Appl. 306 (2005) 413-424]. The study of this new family of special functions was initiated in [V. Varlamov, Convolution of Rayleigh functions with respect to the Bessel index, J. Math. Anal. Appl. 306 (2005) 413-424], where the properties of R1(m) were investigated. In the present work a general representation of Rl(m) in terms of [sigma]l([nu]) is deduced. On the basis of this a representation for the function R2(m) is obtained in terms of the [psi]-function. An asymptotic expansion is computed for R2(m) as m-->[infinity]. Such asymptotics are needed for establishing function spaces for solutions of semi-linear equations in bounded domains with periodicity conditions in one coordinate. As an example of application of Rl(m) a forced Boussinesq equationutt-2b[Delta]ut=-[alpha][Delta]2u+[Delta]u+[beta][Delta](u2)+f with [alpha],b=const>0 and [beta]=const[set membership, variant]R is considered in a unit disc with homogeneous boundary and initial data. Construction of its global-in-time solutions involves the use of the functions R1(m) and R2(m) which are responsible for the nonlinear smoothing effect.

  17. The Neuronal PAS Domain Protein 4 (Npas4) Is Required for New and Reactivated Fear Memories

    PubMed Central

    Nguyen, Tam; DiLeone, Ralph J.; Schafe, Glenn E.

    2011-01-01

    The Neuronal PAS domain protein 4 (Npas4) is a neuronal activity-dependent immediate early gene that has recently been identified as a transcription factor which regulates the transcription of genes that control inhibitory synapse development and synaptic plasticity. The role Npas4 in learning and memory, however, is currently unknown. Here, we systematically examine the role of Npas4 in auditory Pavlovian fear conditioning, an amygdala-dependent form of emotional learning. In our first series of experiments, we show that Npas4 mRNA and protein are regulated in the rat lateral nucleus of the amygdala (LA) in a learning-dependent manner. Further, knockdown of Npas4 protein in the LA via adeno-associated viral (AAV) mediated gene delivery of RNAi was observed to impair fear memory formation, while innate fear and the expression of fear memory were not affected. In our second series of experiments, we show that Npas4 protein is regulated in the LA by retrieval of an auditory fear memory and that knockdown of Npas4 in the LA impairs retention of a reactivated, but not a non-reactivated, fear memory. Collectively, our findings provide the first comprehensive look at the functional role of Npas4 in learning and memory. PMID:21887312

  18. Identification of plakoglobin domains required for association with N-cadherin and alpha-catenin.

    PubMed

    Sacco, P A; McGranahan, T M; Wheelock, M J; Johnson, K R

    1995-08-25

    Cadherins are calcium-dependent, cell surface glycoproteins involved in cell-cell adhesion. To function in cell-cell adhesion, the transmembrane cadherin molecule must be associated with the cytoskeleton via cytoplasmic proteins known as catenins. Three catenins, alpha-catenin, beta-catenin, and gamma-catenin (also known as plakoglobin), have been identified. The domain of the cadherin molecule important for its interaction with the catenins has been mapped to the COOH-terminal 70 amino acids, but less is known about regions of the catenins that allow them to associate with one another or with the cadherin molecule. In this study we have transfected carboxyl-terminal deletions of plakoglobin into the human fibrosarcoma HT-1080 and used immunofluorescence localization and co-immunoprecipitation to map the regions of plakoglobin that allow it to associate with N-cadherin and with alpha-catenin. Plakoglobin is an armadillo family member containing 13 weakly similar internal repeats. These data show that the alpha-catenin-binding region maps within the first repeat and the N-cadherin-binding region maps within repeats 7 and 8. PMID:7650039

  19. VPS10P-domain receptors - regulators of neuronal viability and function.

    PubMed

    Willnow, Thomas E; Petersen, Claus M; Nykjaer, Anders

    2008-12-01

    VPS10P-domain receptors, such as SORLA and sortilin, constitute a recently identified class of type-1 receptors that are expressed in neurons. Family members are multifunctional proteins that target a range of ligands, including trophic factors and neuropeptides but also other transmembrane proteins. New findings have revealed unexpected roles for VPS10P-domain receptors as regulators of neuronal viability and function through the regulation of both protein transport and signal transduction. Loss of these activities might contribute to the pathophysiology of devastating disorders of the nervous system, including Alzheimer's disease, affective disorders and post-traumatic neuronal cell death. PMID:19002190

  20. Disgust trait modulates frontal-posterior coupling as a function of disgust domain

    PubMed Central

    de Jong, Peter J.; Renken, Remco J.; Georgiadis, Janniko R.

    2013-01-01

    Following the two-stage model of disgust, ‘core disgust’ (e.g. elicited by rotten food) is extended to stimuli that remind us of our animal nature ‘AR disgust’ (e.g. mutilations, animalistic instincts). There is ample evidence that core and AR represent distinct domains of disgust elicitors. Moreover, people show large differences in their tendency to respond with disgust to potential disgust elicitors (propensity), as well as in their appraisal of experiencing disgust (sensitivity). Thus these traits may be important moderators of people's response patterns. Here, we aimed to find brain mechanisms associated with these distinct disgust domains and traits, as well as the interaction between them. The right ventrolateral occipitotemporal cortex, which preferentially responded to visual AR, was functionally coupled to the middle cingulate cortex (MCC), thalamus and prefrontal cortex (medial, dorsolateral), as a function of disgust domain. Coupling with the anterior part of MCC was modulated by disgust ‘propensity’, which was strongest during AR. Coupling with anterior insula and ventral premotor cortex was weaker, but relied fully on this domain–trait interaction. Disgust ‘sensitivity’ modulated left anterior insula activity irrespective of domain, and did not affect functional connectivity. Thus a frontal-posterior network that interacts with disgust ‘propensity’ dissects AR and core disgust. PMID:22258801

  1. Coupled Site-Directed Mutagenesis/Transgenesis Identifies Important Functional Domains of the Mouse Agouti Protein

    PubMed Central

    Perry, W. L.; Nakamura, T.; Swing, D. A.; Secrest, L.; Eagleson, B.; Hustad, C. M.; Copeland, N. G.; Jenkins, N. A.

    1996-01-01

    The agouti locus encodes a novel paracrine signaling molecule containing a signal sequence, an N-linked glycosylation site, a central lysine-rich basic domain, and a C-terminal tail containing 10 cysteine (Cys) residues capable of forming five disulfide bonds. When overexpressed, agouti causes a number of pleiotropic effects including yellow coat and adult-onset obesity. Numerous studies suggest that agouti causes yellow coat color by antagonizing the binding of α-melanocyte-stimulating hormone (α-MSH) to the α-MSH-(melanocortin-1) receptor. With the goal of identifying functional domains of agouti important for its diverse biological activities, we have generated 14 agouti mutations by in vitro site-directed mutagenesis and analyzed these mutations in transgenic mice for their effects on coat color and obesity. These studies demonstrate that the signal sequence, the N-linked glycosylation site, and the C-terminal Cys residues are important for full biological activity, while at least a portion of the lysine-rich basic domain is dispensable for normal function. They also show that the same functional domains of agouti important in coat color determination are important for inducing obesity, consistent with the hypothesis that agouti induces obesity by antagonizing melanocortin binding to other melanocortin receptors. PMID:8878691

  2. THE PSTD ALGORITHM: A TIME-DOMAIN METHOD REQUIRING ONLY TWO CELLS PER WAVELENGTH. (R825225)

    EPA Science Inventory

    A pseudospectral time-domain (PSTD) method is developed for solutions of Maxwell's equations. It uses the fast Fourier transform (FFT), instead of finite differences on conventional finite-difference-time-domain (FDTD) methods, to represent spatial derivatives. Because the Fourie...

  3. Arsenic at very low concentrations alters glucocorticoid receptor (GR)-mediated gene activation but not GR-mediated gene repression: complex dose-response effects are closely correlated with levels of activated GR and require a functional GR DNA binding domain.

    PubMed

    Bodwell, Jack E; Kingsley, Lauren A; Hamilton, Joshua W

    2004-08-01

    Arsenic (As) contamination of drinking water is considered a principal environmental health threat throughout the world. Chronic intake is associated with an increased risk of cancer, diabetes, and cardiovascular disease, and recent studies suggest increased health risks at levels as low as 5-10 ppb. We report here that 0.05-1 microM (6-120 ppb) As showed stimulatory effects on glucocorticoid receptor (GR)-mediated gene activation in rat EDR3 hepatoma cells of both the endogenous tyrosine aminotransferase (TAT) gene and the reporter genes containing TAT glucocorticoid response elements. At slightly higher concentrations (1-3 microM), the effects of As became inhibitory. Thus, over this narrow concentration range, the effects of As changed from a 2- to 4-fold stimulation to a greater than 2-fold suppression in activity. Interestingly, the inhibitory effect of GR on both AP1- and NF-kappa B-mediated gene activation was not affected by As. The magnitude of GR stimulation and inhibition by As was highly dependent on the cellular level of hormone-activated GR. Mutational deletion studies indicated that the central DNA binding domain (DBD) of GR is the minimal region required for the As effect and does not require free sulfhydryls. Point mutations located within the DBD that have known structural consequences significantly altered the GR response to As. In particular, point mutations in the DBD that confer a DNA-bound GR confirmation abolished the low dose As stimulatory effect but enhanced the inhibitory response, further indicating that the DBD is important for mediating these As effects. PMID:15310238

  4. Comparison of misfit functions for phase-only inversion in the frequency domain

    NASA Astrophysics Data System (ADS)

    Jeong, G.; Jeong, W.; Min, D. J.

    2014-12-01

    Full waveform inversion suffers from non-uniqueness and non-linearity problems. By using kinematic property of wavefield rather than dynamic property, we can mitigate such problems because the phase is linear and robust (Kamei et al. 2013). For the phase-only inversion, several misfit functions were suggested. Bednar et al. (2007) compared the logarithmic phase-only inversion proposed by Shin and Min (2006) with the conventional phase-only inversion. On the other hand, Kamei et al. (2014) introduced another method that uses the exponential of phase by normalizing the wavefield with respect to the amplitude. In this study, we compare the aforementioned three phase-only inversion methods in the frequency domain: i) the logarithmic phase-only inversion, ii) the conventional phase-only inversion I (briefly conventional I method) that normalizes wavefield with respect to the amplitude variation, and iii) the conventional phase-only inversion II (briefly conventional II method) that replaces the amplitude of the modeled data with that of field data. In the cases of the logarithmic and conventional I methods, if the modeled signal function is close to 0 or becomes large, the gradients of the misfit function diverge to infinity or converge to 0, respectively. In contrast, the conventional II method does not suffer from these problems. For fair comparison, we removed extremely small or large values with Gaussian filtering to avoid the instability problem in the logarithmic and conventional I methods. In addition, we assumed that the phase of the field data is unwrapped to the same degree as the phase of the modeled data in all the cases. On the other hand, the logarithmic and conventional II methods require the additional assumption that amplitudes of the field data are the same as those of the modeled data. However, the conventional I method does not require such an assumption. Our numerical examples show that the conventional I method yields more robust and accurate

  5. Axon-glia interactions and the domain organization of myelinated axons requires neurexin IV/Caspr/Paranodin.

    PubMed

    Bhat, M A; Rios, J C; Lu, Y; Garcia-Fresco, G P; Ching, W; St Martin, M; Li, J; Einheber, S; Chesler, M; Rosenbluth, J; Salzer, J L; Bellen, H J

    2001-05-01

    Myelinated fibers are organized into distinct domains that are necessary for saltatory conduction. These domains include the nodes of Ranvier and the flanking paranodal regions where glial cells closely appose and form specialized septate-like junctions with axons. These junctions contain a Drosophila Neurexin IV-related protein, Caspr/Paranodin (NCP1). Mice that lack NCP1 exhibit tremor, ataxia, and significant motor paresis. In the absence of NCP1, normal paranodal junctions fail to form, and the organization of the paranodal loops is disrupted. Contactin is undetectable in the paranodes, and K(+) channels are displaced from the juxtaparanodal into the paranodal domains. Loss of NCP1 also results in a severe decrease in peripheral nerve conduction velocity. These results show a critical role for NCP1 in the delineation of specific axonal domains and the axon-glia interactions required for normal saltatory conduction. PMID:11395000

  6. Mediating effects of the ICF domain of function and the gross motor function measure on the ICF domains of activity, and participation in children with cerebral palsy

    PubMed Central

    Lee, Byoung-Hee; Kim, Yu-Mi; Jeong, Goo-Churl

    2015-01-01

    [Purpose] This study aimed to evaluate the mediating effect of gross motor function, measured using the Gross Motor Function Measure (GMFM) and of general function, measured using the International Classification of Functioning, Disability and Health-Child and Youth Check List (ICF-CY), on the ICF domains of activity and participation in children with cerebral palsy (CP). [Subjects] Ninety-five children with CP, from Seoul, Korea, participated in the study. [Methods] The GMFM was administered in its entirety to patients without orthoses or mobility aids. The ICF-CY was used to evaluate the degree of disability and health of subjects. [Results] GMFM score and ICF-CY function were negatively correlated to ICF-CY activity and participation. ICF-CY partially mediated the effects of the GMFM on activity and participation. [Conclusion] When establishing a treatment plan for a child with CP, limitations in activity and participation, as described by the ICF-CY, should be considered in addition to the child’s physical abilities and development. In addition, the treatment plan should focus on increasing the child’s activity and participation level, as well as his/her physical level. PMID:26644643

  7. Mediating effects of the ICF domain of function and the gross motor function measure on the ICF domains of activity, and participation in children with cerebral palsy.

    PubMed

    Lee, Byoung-Hee; Kim, Yu-Mi; Jeong, Goo-Churl

    2015-10-01

    [Purpose] This study aimed to evaluate the mediating effect of gross motor function, measured using the Gross Motor Function Measure (GMFM) and of general function, measured using the International Classification of Functioning, Disability and Health-Child and Youth Check List (ICF-CY), on the ICF domains of activity and participation in children with cerebral palsy (CP). [Subjects] Ninety-five children with CP, from Seoul, Korea, participated in the study. [Methods] The GMFM was administered in its entirety to patients without orthoses or mobility aids. The ICF-CY was used to evaluate the degree of disability and health of subjects. [Results] GMFM score and ICF-CY function were negatively correlated to ICF-CY activity and participation. ICF-CY partially mediated the effects of the GMFM on activity and participation. [Conclusion] When establishing a treatment plan for a child with CP, limitations in activity and participation, as described by the ICF-CY, should be considered in addition to the child's physical abilities and development. In addition, the treatment plan should focus on increasing the child's activity and participation level, as well as his/her physical level. PMID:26644643

  8. UML Profiles for Design Decisions and Non-Functional Requirements

    SciTech Connect

    Zhu, Liming; Gorton, Ian

    2007-06-30

    A software architecture is composed of a collection of design decisions. Each design decision helps or hinders certain Non-Functional Requirements (NFR). Current software architecture views focus on expressing components and connectors in the system. Design decisions and their relationships with non-functional requirements are often captured in separate design documentation, not explicitly expressed in any views. This disassociation makes architecture comprehension and architecture evolution harder. In this paper, we propose a UML profile for modeling design decisions and an associated UML profile for modeling non-functional requirements in a generic way. The two UML profiles treat design decisions and nonfunctional requirements as first-class elements. Modeled design decisions always refer to existing architectural elements and thus maintain traceability between the two. We provide a mechanism for checking consistency over this traceability. An exemplar is given as

  9. Using the Structure-Function Linkage Database to Characterize Functional Domains in Enzymes

    PubMed Central

    Brown, Shoshana; Babbitt, Patricia

    2015-01-01

    The Structure-Function Linkage Database (SFLD; http://sfld.rbvi.ucsf.edu/) is a web-accessible database designed to link enzyme sequence, structure and functional information. This unit describes the protocol by which a user may query the database to predict the function of uncharacterized enzymes and to correct misannotated functional assignments. It is especially useful in helping a user discriminate functional capabilities of a sequence that is only distantly related to characterized sequences in publicly available databases. PMID:25501940

  10. Structural and functional characterization of an atypical activation domain in erythroid Krüppel-like factor (EKLF)

    PubMed Central

    Mas, Caroline; Lussier-Price, Mathieu; Soni, Shefali; Morse, Thomas; Arseneault, Geneviève; Di Lello, Paola; Lafrance-Vanasse, Julien; Bieker, James J.; Omichinski, James G.

    2011-01-01

    Erythroid Krüppel-like factor (EKLF) plays an important role in erythroid development by stimulating β-globin gene expression. We have examined the details by which the minimal transactivation domain (TAD) of EKLF (EKLFTAD) interacts with several transcriptional regulatory factors. We report that EKLFTAD displays homology to the p53TAD and, like the p53TAD, can be divided into two functional subdomains (EKLFTAD1 and EKLFTAD2). Based on sequence analysis, we found that EKLFTAD2 is conserved in KLF2, KLF4, KLF5, and KLF15. In addition, we demonstrate that EKLFTAD2 binds the amino-terminal PH domain of the Tfb1/p62 subunit of TFIIH (Tfb1PH/p62PH) and four domains of CREB-binding protein/p300. The solution structure of the EKLFTAD2/Tfb1PH complex indicates that EKLFTAD2 binds Tfb1PH in an extended conformation, which is in contrast to the α-helical conformation seen for p53TAD2 in complex with Tfb1PH. These studies provide detailed mechanistic information into EKLFTAD functions as well as insights into potential interactions of the TADs of other KLF proteins. In addition, they suggest that not only have acidic TADs evolved so that they bind using different conformations on a common target, but that transitioning from a disordered to a more ordered state is not a requirement for their ability to bind multiple partners. PMID:21670263

  11. Distinct Domains of Yeast Cortical Tag Proteins Bud8p and Bud9p Confer Polar Localization and Functionality

    PubMed Central

    Krappmann, Anne-Brit; Taheri, Naimeh; Heinrich, Melanie

    2007-01-01

    In Saccharomyces cerevisiae, diploid yeast cells follow a bipolar budding program, which depends on the two transmembrane glycoproteins Bud8p and Bud9p that potentially act as cortical tags to mark the cell poles. Here, we have performed systematic structure-function analyses of Bud8p and Bud9p to identify functional domains. We find that polar transport of Bud8p and Bud9p does not depend on N-terminal sequences but instead on sequences in the median part of the proteins and on the C-terminal parts that contain the transmembrane domains. We show that the guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange factor Bud5p, which is essential for bud site selection and physically interacts with Bud8p, also interacts with Bud9p. Regions of Bud8p and Bud9p predicted to reside in the extracellular space are likely to confer interaction with the N-terminal region of Bud5p, implicating indirect interactions between the cortical tags and the GDP/GTP exchange factor. Finally, we have identified regions of Bud8p and Bud9p that are required for interaction with the cortical tag protein Rax1p. In summary, our study suggests that Bud8p and Bud9p carry distinct domains for delivery of the proteins to the cell poles, for interaction with the general budding machinery and for association with other cortical tag proteins. PMID:17581861

  12. Stability of a pinned magnetic domain wall as a function of its internal configuration

    SciTech Connect

    Montaigne, F.; Duluard, A.; Briones, J.; Lacour, D.; Hehn, M.; Childress, J. R.

    2015-01-14

    It is shown that there are many stable configurations for a domain wall pinned by a notch along a magnetic stripe. The stability of several of these configurations is investigated numerically as a function of the thickness of the magnetic film. The depinning mechanism depends on the structure of the domain wall and on the thickness of the magnetic film. In the case of a spin-valve structure, it appears that the stray fields emerging from the hard layer at the notch location influence the stability of the micromagnetic configuration. Different depinning mechanisms are thus observed for the same film thickness depending on the magnetization orientation of the propagating domain. This conclusion qualitatively explains experimental magnetoresistance measurements.

  13. Engineered allosteric mutants of the integrin alphaMbeta2 I domain: structural and functional studies.

    PubMed Central

    McCleverty, Clare J; Liddington, Robert C

    2003-01-01

    The alpha-I domain, found in the alpha-subunit of the leucocyte integrins such as alphaMbeta2 and alphaLbeta2, switches between the open and closed tertiary conformations, reflecting the high- and low-affinity ligand-binding states of the integrin that are required for regulated cell adhesion and migration. In the present study we show, by using point mutations and engineered disulphide bonds, that ligand affinity can be reduced or increased allosterically by altering the equilibrium between the closed and open states. We determined equilibrium constants for the binding of two ligands, fibrinogen and intercellular cell-adhesion molecule 1, to the alphaM-I domain by surface plasmon resonance, and determined crystal structures of a low-affinity mutant. Locking the domain in the open conformation increases affinity by a factor of no greater than 10, consistent with a closely balanced equilibrium between the two conformations in the absence of ligand. This behaviour contrasts with that of the unliganded alphaL-I domain, for which the equilibrium lies strongly in favour of the closed conformation. These results suggest significant differences in the way the parent integrins regulate I domain conformation and hence ligand affinity. PMID:12611591

  14. Separate domains in GCN1 for binding protein kinase GCN2 and ribosomes are required for GCN2 activation in amino acid-starved cells

    PubMed Central

    Sattlegger, Evelyn; Hinnebusch, Alan G.

    2000-01-01

    GCN2 stimulates GCN4 translation in amino acid-starved cells by phosphorylating the α-subunit of translation initiation factor 2. GCN2 function in vivo requires the GCN1/GCN20 complex, which binds to the N-terminal domain of GCN2. A C-terminal segment of GCN1 (residues 2052–2428) was found to be necessary and sufficient for binding GCN2 in vivo and in vitro. Overexpression of this fragment in wild-type cells impaired association of GCN2 with native GCN1 and had a dominant Gcn– phenotype, dependent on Arg2259 in the GCN1 fragment. Substitution of Arg2259 with Ala in full-length GCN1 abolished complex formation with native GCN2 and destroyed GCN1 regulatory function. Consistently, the Gcn– phenotype of gcn1-R2259A, but not that of gcn1Δ, was suppressed by overexpressing GCN2. These findings prove that GCN2 binding to the C-terminal domain of GCN1, dependent on Arg2259, is required for high level GCN2 function in vivo. GCN1 expression conferred sensitivity to paromomycin in a manner dependent on its ribosome binding domain, supporting the idea that GCN1 binds near the ribosomal acceptor site to promote GCN2 activation by uncharged tRNA. PMID:11101534

  15. Separate domains in GCN1 for binding protein kinase GCN2 and ribosomes are required for GCN2 activation in amino acid-starved cells.

    PubMed

    Sattlegger, E; Hinnebusch, A G

    2000-12-01

    GCN2 stimulates GCN4 translation in amino acid-starved cells by phosphorylating the alpha-subunit of translation initiation factor 2. GCN2 function in vivo requires the GCN1/GCN20 complex, which binds to the N-terminal domain of GCN2. A C-terminal segment of GCN1 (residues 2052-2428) was found to be necessary and sufficient for binding GCN2 in vivo and in vitro. Overexpression of this fragment in wild-type cells impaired association of GCN2 with native GCN1 and had a dominant Gcn(-) phenotype, dependent on Arg2259 in the GCN1 fragment. Substitution of Arg2259 with Ala in full-length GCN1 abolished complex formation with native GCN2 and destroyed GCN1 regulatory function. Consistently, the Gcn(-) phenotype of gcn1-R2259A, but not that of gcn1Delta, was suppressed by overexpressing GCN2. These findings prove that GCN2 binding to the C-terminal domain of GCN1, dependent on Arg2259, is required for high level GCN2 function in vivo. GCN1 expression conferred sensitivity to paromomycin in a manner dependent on its ribosome binding domain, supporting the idea that GCN1 binds near the ribosomal acceptor site to promote GCN2 activation by uncharged tRNA. PMID:11101534

  16. The functional integrity of the serpin domain of C1-inhibitor depends on the unique N-terminal domain, as revealed by a pathological mutant.

    PubMed

    Bos, Ineke G A; Lubbers, Yvonne T P; Roem, Dorina; Abrahams, Jan Pieter; Hack, C Erik; Eldering, Eric

    2003-08-01

    C1-inhibitor (C1-Inh) is a serine protease inhibitor (serpin) with a unique, non-conserved N-terminal domain of unknown function. Genetic deficiency of C1-Inh causes hereditary angioedema. A novel type of mutation (Delta 3) in exon 3 of the C1-Inh gene, resulting in deletion of Asp62-Thr116 in this unique domain, was encountered in a hereditary angioedema pedigree. Because the domain is supposedly not essential for inhibitory activity, the unexpected loss-of-function of this deletion mutant was further investigated. The Delta 3 mutant and three additional mutants starting at Pro76, Gly98, and Ser115, lacking increasing parts of the N-terminal domain, were produced recombinantly. C1-Inh76 and C1-Inh98 retained normal conformation and interaction kinetics with target proteases. In contrast, C1-Inh115 and Delta 3, which both lack the connection between the serpin and the non-serpin domain via two disulfide bridges, were completely non-functional because of a complex-like and multimeric conformation, as demonstrated by several criteria. The Delta 3 mutant also circulated in multimeric form in plasma from affected family members. The C1-Inh mutant reported here is unique in that deletion of an entire amino acid stretch from a domain not shared by other serpins leads to a loss-of-function. The deletion in the unique N-terminal domain results in a "multimerization phenotype" of C1-Inh, because of diminished stability of the central beta-sheet. This phenotype, as well as the location of the disulfide bridges between the serpin and the non-serpin domain of C1-Inh, suggests that the function of the N-terminal region may be similar to one of the effects of heparin in antithrombin III, maintenance of the metastable serpin conformation. PMID:12773530

  17. The Dc-Module of Doublecortin: Dynamics, Domain Boundaries, and Functional Implications

    SciTech Connect

    Cierpicki,T.; Kim, M.; Cooper, D.; Derewenda, U.; Bushweller, J.; Derwenda, Z.

    2007-01-01

    The doublecortin-like (DC) domains, which usually occur in tandem, constitute novel microtubule-binding modules. They were first identified in doublecortin (DCX), a protein expressed in migrating neurons, and in the doublecortin-like kinase (DCLK). They are also found in other proteins, including the RP1 gene product which-when mutated-causes a form of inherited blindness. We previously reported an X-ray structure of the N-terminal DC domain of DCLK (N-DCLK), and a solution structure of an analogous module of human doublecortin (N-DCX). These studies showed that the DC domain has a tertiary fold closely reminiscent of ubiquitin and similar to several GTPase-binding domains. We now report an X-ray structure of a mutant of N-DCX, in which the C-terminal fragment (residues 139-147) unexpectedly shows an altered, 'open' conformation. However, heteronuclear NMR data show that this C-terminal fragment is only transiently open in solution, and assumes a predominantly 'closed' conformation. While the 'open' conformation may be artificially stabilized by crystal packing interactions, the observed switching between the 'open' and 'closed' conformations, which shortens the linker between the two DC-domains by {approx}20 A, is likely to be of functional importance in the control of tubulin polymerization and microtubule bundling by doublecortin.

  18. Development of Spectral Domain Optical Coherence Tomography for in vivo Functional Imaging of Biological Tissues

    NASA Astrophysics Data System (ADS)

    An, Lin

    Optical coherence tomography is a rapidly developing optical imaging modality capable of noninvasively providing depth resolved information of biological tissue at micrometer scale. In this thesis, we described several OCT technologies that can be used to double the imaging depth, realize functional vasculature imaging of biological tissue and increase the imaging speed of OCT system. Aim 1: Use of a scanner to introduce spatial frequency modulation to OCT spectral interferograms for in vivo full-range Fourier-domain optical coherence tomography. A novel method was developed that could easily introduce a modulation frequency onto the X-direction (i.e., B-scan) of the FDOCT scanning system, enabling full-range Fourier-domain Optical Coherence Tomography (frFDOCT). Compared to the conventional FDOCT system, the newly developed frFDOCT system can provide increased system sensitivity and deeper imaging depth. The previous technology that can achieve frFDOCT either needed multiple steps for data capturing, which is time consuming, or required additional components which increased the system's complexity. The newly developed method generates a modulation spatial frequency in the spectral interferogram by simply offsetting the probe beam at the X-scanner. Aim 2: Using optical micro-angiography to achieve in vivo volumetric imaging of vascular perfusion within human retina and choroids. Optical Micro-Angiography (OMAG) is a functional extension of FDOCT technology. It can achieve visualization of vasculature network of biological tissue. In order to apply the OMAG method to image vasculature map of human retina and choroid, a phase compensation algorithm was developed, which could minimize the motion artifacts generated by the movements of human eye and head. Aim 3: Developing ultrahigh sensitive optical micro-angiography to achieve micro vasculature imaging of biological tissue. To improve the vasculature image quality, we developed ultrahigh sensitive OMAG (UHS

  19. Functional analysis of TPM domain containing Rv2345 of Mycobacterium tuberculosis identifies its phosphatase activity.

    PubMed

    Sinha, Avni; Eniyan, Kandasamy; Sinha, Swati; Lynn, Andrew Michael; Bajpai, Urmi

    2015-07-01

    Mycobacterium tuberculosis (Mtb) is the causal agent of tuberculosis, the second largest infectious disease. With the rise of multi-drug resistant strains of M. tuberculosis, serious challenge lies ahead of us in treating the disease. The availability of complete genome sequence of Mtb has improved the scope for identifying new proteins that would not only further our understanding of biology of the organism but could also serve to discover new drug targets. In this study, Rv2345, a hypothetical membrane protein of M. tuberculosis H37Rv, which is reported to be a putative ortholog of ZipA cell division protein has been assigned function through functional annotation using bioinformatics tools followed by experimental validation. Sequence analysis showed Rv2345 to have a TPM domain at its N-terminal region and predicted it to have phosphatase activity. The TPM domain containing region of Rv2345 was cloned and expressed using pET28a vector in Escherichia coli and purified by Nickel affinity chromatography. The purified TPM domain was tested in vitro and our results confirmed it to have phosphatase activity. The enzyme activity was first checked and optimized with pNPP as substrate, followed by using ATP, which was also found to be used as substrate by the purified protein. Hence sequence analysis followed by in vitro studies characterizes TPM domain of Rv2345 to contain phosphatase activity. PMID:25782739

  20. The rod domain is not essential for the function of plectin in maintaining tissue integrity

    PubMed Central

    Ketema, Mirjam; Secades, Pablo; Kreft, Maaike; Nahidiazar, Leila; Janssen, Hans; Jalink, Kees; de Pereda, Jose M.; Sonnenberg, Arnoud

    2015-01-01

    Epidermolysis bullosa simplex associated with late-onset muscular dystrophy (EBS-MD) is an autosomal recessive disorder resulting from mutations in the plectin gene. The majority of these mutations occur within the large exon 31 encoding the central rod domain and leave the production of a low-level rodless plectin splice variant unaffected. To investigate the function of the rod domain, we generated rodless plectin mice through conditional deletion of exon 31. Rodless plectin mice develop normally without signs of skin blistering or muscular dystrophy. Plectin localization and hemidesmosome organization are unaffected in rodless plectin mice. However, superresolution microscopy revealed a closer juxtaposition of the C-terminus of plectin to the integrin β4 subunit in rodless plectin keratinocytes. Wound healing occurred slightly faster in rodless plectin mice than in wild-type mice, and keratinocytes migration was increased in the absence of the rod domain. The faster migration of rodless plectin keratinocytes is not due to altered biochemical properties because, like full-length plectin, rodless plectin is a dimeric protein. Our data demonstrate that rodless plectin can functionally compensate for the loss of full-length plectin in mice. Thus the low expression level of plectin rather than the absence of the rod domain dictates the development of EBS-MD. PMID:25971800

  1. Syntaxin Opening by the MUN Domain Underlies the Function of Munc13 in Synaptic Vesicle Priming

    PubMed Central

    Yang, Xiaoyu; Wang, Shen; Sheng, Yi; Zhang, Mingshu; Zou, Wenjuan; Wu, Lijie; Kang, Lijun; Rizo, Josep; Zhang, Rongguang; Xu, Tao; Ma, Cong

    2016-01-01

    UNC-13-Munc13s play a central function in synaptic vesicle priming through their MUN domains. However, it is unclear whether this function arises from the ability of the MUN domain to mediate the transition from the Munc18-1–closed syntaxin-1 complex to the SNARE complex in vitro. The crystal structure of rat Munc13-1 MUN domain now reveals an elongated, arch-shaped architecture formed by α-helical bundles, with a highly conserved hydrophobic pocket in the middle. Mutation of two residues (NF) in this pocket abolishes the stimulation caused by the Munc13-1 MUN domain on SNARE complex assembly and on SNARE-dependent proteoliposome fusion in vitro. Moreover, the same mutation in UNC-13 abrogates synaptic vesicle priming in C. elegans neuromuscular junctions. These results strongly support the notion that orchestration of syntaxin-1 opening and SNARE complex assembly underlies the central role of UNC-13-Munc13s in synaptic vesicle priming. PMID:26030875

  2. Crystal Structure of the Human, FIC-Domain Containing Protein HYPE and Implications for Its Functions

    PubMed Central

    Bunney, Tom D.; Cole, Ambrose R.; Broncel, Malgorzata; Esposito, Diego; Tate, Edward W.; Katan, Matilda

    2014-01-01

    Summary Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein, HYPE, which has remained poorly characterized. Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of autoAMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition. PMID:25435325

  3. The selenocysteine-specific elongation factor contains a novel and multi-functional domain.

    PubMed

    Gonzalez-Flores, Jonathan N; Gupta, Nirupama; DeMong, Louise W; Copeland, Paul R

    2012-11-01

    The selenocysteine (Sec)-specific eukaryotic elongation factor (eEFSec) delivers the aminoacylated selenocysteine-tRNA (Sec-tRNA(Sec)) to the ribosome and suppresses UGA codons that are upstream of Sec insertion sequence (SECIS) elements bound by SECIS-binding protein 2 (SBP2). Multiple studies have highlighted the importance of SBP2 forming a complex with the SECIS element, but it is not clear how this regulates eEFSec during Sec incorporation. Compared with the canonical elongation factor eEF1A, eEFSec has a unique C-terminal extension called Domain IV. To understand the role of Domain IV in Sec incorporation, we examined a series of mutant proteins for all of the known molecular functions for eEFSec: GTP hydrolysis, Sec-tRNA(Sec) binding, and SBP2/SECIS binding. In addition, wild-type and mutant versions of eEFSec were analyzed for Sec incorporation activity in a novel eEFSec-dependent translation extract. We have found that Domain IV is essential for both tRNA and SBP2 binding as well as regulating GTPase activity. We propose a model where the SBP2/SECIS complex activates eEFSec by directing functional interactions between Domain IV and the ribosome to promote Sec-tRNA(Sec) binding and accommodation into the ribosomal A-site. PMID:22992746

  4. Native mass spectrometry and ion mobility characterize the orange carotenoid protein functional domains.

    PubMed

    Zhang, Hao; Liu, Haijun; Lu, Yue; Wolf, Nathan R; Gross, Michael L; Blankenship, Robert E

    2016-06-01

    Orange Carotenoid Protein (OCP) plays a unique role in protecting many cyanobacteria from light-induced damage. The active form of OCP is directly involved in energy dissipation by binding to the phycobilisome (PBS), the major light-harvesting complex in cyanobacteria. There are two structural modules in OCP, an N-terminal domain (NTD), and a C-terminal domain (CTD), which play different functional roles during the OCP-PBS quenching cycle. Because of the quasi-stable nature of active OCP, structural analysis of active OCP has been lacking compared to its inactive form. In this report, partial proteolysis was used to generate two structural domains, NTD and CTD, from active OCP. We used multiple native mass spectrometry (MS) based approaches to interrogate the structural features of the NTD and the CTD. Collisional activation and ion mobility analysis indicated that the NTD releases its bound carotenoid without forming any intermediates and the CTD is resistant to unfolding upon collisional energy ramping. The unfolding intermediates observed in inactive intact OCP suggest that it is the N-terminal extension and the NTD-CTD loop that lead to the observed unfolding intermediates. These combined approaches extend the knowledge of OCP photo-activation and structural features of OCP functional domains. Combining native MS, ion mobility, and collisional activation promises to be a sensitive new approach for studies of photosynthetic protein-pigment complexes. PMID:26921809

  5. Localized axial Green's function method for the convection-diffusion equations in arbitrary domains

    NASA Astrophysics Data System (ADS)

    Lee, Wanho; Kim, Do Wan

    2014-10-01

    A localized axial Green's function method (LAGM) is proposed for the convection-diffusion equation. The axial Green's function method (AGM) enables us to calculate the numerical solution of a multi-dimensional problem using only one-dimensional Green's functions for the axially split differential operators. This AGM has been developed not only for the elliptic boundary value problems but also for the steady Stokes flows, however, this paper is concerned with the localization of the AGM. This localization of the method is needed for practical purpose when computing the axial Green's function, specifically for the convection-diffusion equation on a line segment that we call the local axial line. Although our focus is mainly on the convection-dominated cases in arbitrary domains, this method can solve other cases in a unified way. Numerical results show that, despite irregular types of discretization on an arbitrary domain, we can calculate the numerical solutions using the LAGM without loss of accuracy even in cases of large convection. In particular, it is also shown that randomly distributed axial lines are available in our LAGM and complicated domains are not a burden.

  6. Phosphatidylserine exposure is required for ADAM17 sheddase function

    PubMed Central

    Sommer, Anselm; Kordowski, Felix; Büch, Joscha; Maretzky, Thorsten; Evers, Astrid; Andrä, Jörg; Düsterhöft, Stefan; Michalek, Matthias; Lorenzen, Inken; Somasundaram, Prasath; Tholey, Andreas; Sönnichsen, Frank D.; Kunzelmann, Karl; Heinbockel, Lena; Nehls, Christian; Gutsmann, Thomas; Grötzinger, Joachim; Bhakdi, Sucharit; Reiss, Karina

    2016-01-01

    ADAM17, a prominent member of the ‘Disintegrin and Metalloproteinase' (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates. Here we present evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. PS exposure is tightly coupled to substrate shedding provoked by diverse ADAM17 activators. PS dependency is demonstrated in the following: (a) in Raji cells undergoing apoptosis; (b) in mutant PSA-3 cells with manipulatable PS content; and (c) in Scott syndrome lymphocytes genetically defunct in their capacity to externalize PS in response to intracellular Ca2+ elevation. Soluble phosphorylserine but not phosphorylcholine inhibits substrate cleavage. The isolated membrane proximal domain (MPD) of ADAM17 binds to PS but not to phosphatidylcholine liposomes. A cationic PS-binding motif is identified in this domain, replacement of which abrogates liposome-binding and renders the protease incapable of cleaving its substrates in cells. We speculate that surface-exposed PS directs the protease to its targets where it then executes its shedding function. PMID:27161080

  7. Phosphatidylserine exposure is required for ADAM17 sheddase function.

    PubMed

    Sommer, Anselm; Kordowski, Felix; Büch, Joscha; Maretzky, Thorsten; Evers, Astrid; Andrä, Jörg; Düsterhöft, Stefan; Michalek, Matthias; Lorenzen, Inken; Somasundaram, Prasath; Tholey, Andreas; Sönnichsen, Frank D; Kunzelmann, Karl; Heinbockel, Lena; Nehls, Christian; Gutsmann, Thomas; Grötzinger, Joachim; Bhakdi, Sucharit; Reiss, Karina

    2016-01-01

    ADAM17, a prominent member of the 'Disintegrin and Metalloproteinase' (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates. Here we present evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. PS exposure is tightly coupled to substrate shedding provoked by diverse ADAM17 activators. PS dependency is demonstrated in the following: (a) in Raji cells undergoing apoptosis; (b) in mutant PSA-3 cells with manipulatable PS content; and (c) in Scott syndrome lymphocytes genetically defunct in their capacity to externalize PS in response to intracellular Ca(2+) elevation. Soluble phosphorylserine but not phosphorylcholine inhibits substrate cleavage. The isolated membrane proximal domain (MPD) of ADAM17 binds to PS but not to phosphatidylcholine liposomes. A cationic PS-binding motif is identified in this domain, replacement of which abrogates liposome-binding and renders the protease incapable of cleaving its substrates in cells. We speculate that surface-exposed PS directs the protease to its targets where it then executes its shedding function. PMID:27161080

  8. NMR characterization of the conformational fluctuations of the human lymphocyte function-associated antigen-1 I-domain

    PubMed Central

    Leung, Hoi Tik Alvin; Kukic, Predrag; Camilloni, Carlo; Bemporad, Francesco; De Simone, Alfonso; Aprile, Francesco A; Kumita, Janet R; Vendruscolo, Michele

    2014-01-01

    Lymphocyte function-associated antigen-1 (LFA-1) is an integrin protein that transmits information across the plasma membrane through the so-called inside-out and outside-in signaling mechanisms. To investigate these mechanisms, we carried out an NMR analysis of the dynamics of the LFA-1 I-domain, which has enabled us to characterize the motions of this domain on a broad range of timescales. We studied first the internal motions on the nanosecond timescale by spin relaxation measurements and model-free analysis. We then extended this analysis to the millisecond timescale motions by measuring 15N-1H residual dipolar couplings of the backbone amide groups. We analyzed these results in the context of the three major conformational states of the I-domain using their corresponding X-ray crystallographic structures. Our results highlight the importance of the low-frequency motions of the LFA-1 I-domain in the inactive apo-state. We found in particular that α-helix 7 is in a position in the apo-closed state that cannot be fully described by any of the existing X-ray structures, as it appears to be in dynamic exchange between different conformations. This type of motion seems to represent an inherent property of the LFA-1 I-domain and might be relevant for controlling the access to the allosteric binding pocket, as well as for the downward displacement of α-helix 7 that is required for the activation of LFA-1. PMID:25147050

  9. Interplay of two functionally and structurally distinct domains of the c-fos AU-rich element specifies its mRNA-destabilizing function.

    PubMed Central

    Chen, C Y; Chen, T M; Shyu, A B

    1994-01-01

    AU-rich elements (ARE) in the 3' untranslated region of many highly labile mRNAs for proto-oncogenes, lymphokines, and cytokines can act as an RNA-destabilizing element. The absence of a clear understanding of the key sequence and structural features of the ARE that are required for its destabilizing function has precluded the further elucidation of its mode of action and the basis of its specificity. Combining extensive mutagenesis of the c-fos ARE with in vivo analysis of mRNA stability, we were able to identify mutations that exhibited kinetic phenotypes consistent with the biphasic decay characteristic of a two-step mechanism: accelerated poly(A) shortening and subsequent decay of the transcribed portion of the mRNA. These mutations, which affected either an individual step or both steps, all changed the mRNA stability. Our experiments further revealed the existence of two structurally distinct and functionally interdependent domains that constitute the c-fos ARE. Domain I, which is located within the 5' 49-nucleotide segment of the ARE and contains the three AUUUA motifs, can function as an RNA destabilizer by itself. It forms the essential core unit necessary for the ARE-destabilizing function. Domain II is a 20-nucleotide U-rich sequence which is located within the 3' part of the c-fos ARE. Although it alone can not act as an RNA destabilizer, this domain serves two critical roles: (i) its presence enhances the destabilizing ability of domain I by accelerating the deadenylation step, and (ii) it has a novel capacity of buffering decay-impeding effects exerted by mutations introduced within domain I. A model is proposed to explain how these critical structural features may be involved in the c-fos ARE-directed mRNA decay pathway. These findings have important implications for furthering our understanding of the molecular basis of differential mRNA decay mediated by different AREs. Images PMID:7903419

  10. The CD225 Domain of IFITM3 Is Required for both IFITM Protein Association and Inhibition of Influenza A Virus and Dengue Virus Replication

    PubMed Central

    John, Sinu P.; Chin, Christopher R.; Perreira, Jill M.; Feeley, Eric M.; Aker, Aaron M.; Savidis, George; Smith, Sarah E.; Elia, Andrew E. H.; Everitt, Aaron R.; Vora, Mehul; Pertel, Thomas; Elledge, Stephen J.; Kellam, Paul

    2013-01-01

    The interferon-induced transmembrane protein 3 (IFITM3) gene is an interferon-stimulated gene that inhibits the replication of multiple pathogenic viruses in vitro and in vivo. IFITM3 is a member of a large protein superfamily, whose members share a functionally undefined area of high amino acid conservation, the CD225 domain. We performed mutational analyses of IFITM3 and identified multiple residues within the CD225 domain, consisting of the first intramembrane domain (intramembrane domain 1 [IM1]) and a conserved intracellular loop (CIL), that are required for restriction of both influenza A virus (IAV) and dengue virus (DENV) infection in vitro. Two phenylalanines within IM1 (F75 and F78) also mediate a physical association between IFITM proteins, and the loss of this interaction decreases IFITM3-mediated restriction. By extension, similar IM1-mediated associations may contribute to the functions of additional members of the CD225 domain family. IFITM3's distal N-terminal domain is also needed for full antiviral activity, including a tyrosine (Y20), whose alteration results in mislocalization of a portion of IFITM3 to the cell periphery and surface. Comparative analyses demonstrate that similar molecular determinants are needed for IFITM3's restriction of both IAV and DENV. However, a portion of the CIL including Y99 and R87 is preferentially needed for inhibition of the orthomyxovirus. Several IFITM3 proteins engineered with rare single-nucleotide polymorphisms demonstrated reduced expression or mislocalization, and these events were associated with enhanced viral replication in vitro, suggesting that possessing such alleles may impact an individual's risk for viral infection. On the basis of this and other data, we propose a model for IFITM3-mediated restriction. PMID:23658454

  11. What is the minimal vestibular function required for compensation?

    NASA Technical Reports Server (NTRS)

    Black, F. O.; Wade, S. W.; Nashner, L. M.

    1996-01-01

    Living with an uncompensated, abnormal vestibular system requires oppressive modification of life style and often prevents return to work and activities of daily living. Patients with vestibular abnormalities were studied to determine the minimal residual vestibular function required to achieve compensation. Three groups of patients with (a) complete unilateral loss of vestibular function with normal horizontal canal-vestibulo-ocular (HCVOR) function in the opposite ear, (b) complete unilateral loss with abnormal HCVOR function in the opposite ear, and (c) bilateral reduction of vestibular function from aminoglycoside toxicity underwent vestibuloocular (VOR), optokinetic (OKN), visual-VOR (VVOR), and computerized dynamic posturography (CDP) tests before and after therapeutic procedures. Results suggest that a minimal VOR response amplitude must be present for compensation of VVOR function to occur. The roles of VOR and OKN phase shifts in vestibular compensation are more complicated and require further study. Compensation of vestibulospinal function does not necessarily accompany VOR or VVOR compensation. Ascending and descending vestibular compensatory mechanisms may involve different spatial sensory inputs. Results of these studies have important implications for the diagnosis and treatment of patients with vestibular disorders, including selection and monitoring of patients for therapeutic regimens such as vestibular nerve section and streptomycin therapy.

  12. Distinct centromere domain structures with separate functions demonstrated in live fission yeast cells.

    PubMed

    Appelgren, Henrik; Kniola, Barbara; Ekwall, Karl

    2003-10-01

    Fission yeast (Saccharomyces pombe) centromere DNA is organized in a central core region flanked on either side by a region of outer repeat (otr) sequences. The otr region is known to be heterochromatic and bound by the Swi6 protein whereas the central core region contains an unusual chromatin structure involving the histone H3 variant Cnp1 (S. pombe CENP-A). The central core is the base for formation of the kinetochore structure whereas the flanking region is important for sister centromere cohesion. We have previously shown that the ultrastructural domain structure of S. pombe centromeres in interphase is similar to that of human centromeres. Here we demonstrate that S. pombe centromeres are organized in cytologically distinct domains even in mitosis. Fluorescence in situ hybridization of fixed metaphase cells revealed that the otr regions of the centromere were still held together by cohesion even after the sister kinetochores had separated. In live cells, the central cores and kinetochores of sister chromosomes could be distinguished from one another when they were subjected to mitotic tension. The function of the different centromeric domains was addressed. Transacting mutations affecting the kinetochore (nuf2) central core domain (mis6) and the heterochromatin domain (rik1) were analyzed in live cells. In interphase, both nuf2 and mis6 caused declustering of centromeres from the spindle pole body whereas centromere clustering was normal in rik1 despite an apparent decondensation defect. The declustering of centromeres in mis6 cells correlated with loss the Ndc80 kinetochore marker protein from the centromeres. Interestingly the declustered centromeres were still restricted to the nuclear periphery thus revealing a kinetochore-independent peripheral localization mechanism for heterochromatin. Time-lapse microscopy of live mis6 and nuf2-1 mutant cells in mitosis showed similar severe misaggregation phenotypes whereas the rik1 mutants showed a mild cohesion

  13. Structural, biochemical, and functional characterization of the cyclic nucleotide binding homology domain from the mouse EAG1 potassium channel.

    PubMed

    Marques-Carvalho, Maria J; Sahoo, Nirakar; Muskett, Frederick W; Vieira-Pires, Ricardo S; Gabant, Guillaume; Cadene, Martine; Schönherr, Roland; Morais-Cabral, João H

    2012-10-12

    KCNH channels are voltage-gated potassium channels with important physiological functions. In these channels, a C-terminal cytoplasmic region, known as the cyclic nucleotide binding homology (CNB-homology) domain displays strong sequence similarity to cyclic nucleotide binding (CNB) domains. However, the isolated domain does not bind cyclic nucleotides. Here, we report the X-ray structure of the CNB-homology domain from the mouse EAG1 channel. Through comparison with the recently determined structure of the CNB-homology domain from the zebrafish ELK (eag-like K(+)) channel and the CNB domains from the MlotiK1 and HCN (hyperpolarization-activated cyclic nucleotide-gated) potassium channels, we establish the structural features of CNB-homology domains that explain the low affinity for cyclic nucleotides. Our structure establishes that the "self-liganded" conformation, where two residues of the C-terminus of the domain are bound in an equivalent position to cyclic nucleotides in CNB domains, is a conserved feature of CNB-homology domains. Importantly, we provide biochemical evidence that suggests that there is also an unliganded conformation where the C-terminus of the domain peels away from its bound position. A functional characterization of this unliganded conformation reveals a role of the CNB-homology domain in channel gating. PMID:22732247

  14. Characterization of the AXH domain of Ataxin-1 using enhanced sampling and functional mode analysis.

    PubMed

    Deriu, Marco A; Grasso, Gianvito; Tuszynski, Jack A; Massai, Diana; Gallo, Diego; Morbiducci, Umberto; Danani, Andrea

    2016-05-01

    Ataxin-1 is the protein responsible for the Spinocerebellar ataxia type 1, an incurable neurodegenerative disease caused by polyglutamine expansion. The AXH domain plays a pivotal role in physiological functions of Ataxin-1. In Spinocerebellar ataxia 1, the AXH domain is involved in the misfolding and aggregation pathway. Here molecular modeling is applied to investigate the protein-protein interactions contributing to the AXH dimer stability. Particular attention is focused on: (i) the characterization of AXH monomer-monomer interface; (ii) the molecular description of the AXH monomer-monomer interaction dynamics. Technically, an approach based on functional mode analysis, here applied to replica exchange molecular dynamics trajectories, was employed. The findings of this study are consistent with previous experimental results and elucidate the pivotal role of the I580 residue in mediating the AXH monomer-monomer interaction dynamics. Proteins 2016; 84:666-673. © 2016 Wiley Periodicals, Inc. PMID:26879337

  15. Axial Green’s function method for steady Stokes flow in geometrically complex domains

    NASA Astrophysics Data System (ADS)

    Jun, Sukky; Kim, Do Wan

    2011-03-01

    Axial Green's function method (AGM) is developed for the simulation of Stokes flow in geometrically complex solution domains. The AGM formulation systematically decomposes the multidimensional steady-state Stokes equations into 1D forms. The representation formula for the solution variables can then be derived using the 1D Green's functions only, from which a system of 1D integral equations is obtained. Furthermore, the explicit representation formula for the pressure variable enable the unique AGM approach to facilitating the stabilization issue caused by the saddle structure between velocity and pressure. The convergence of numerical solutions, the simple axial discretization of complex solution domains, and the nature of integral schemes are demonstrated through a variety of numerical examples.

  16. Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors

    PubMed Central

    Hopf, Thomas A.; Morinaga, Satoshi; Ihara, Sayoko; Touhara, Kazushige; Marks, Debora S.; Benton, Richard

    2015-01-01

    Insect Odorant Receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection. PMID:25584517

  17. Boundary values of functions in a Sobolev space with Muckenhoupt weight on some non-Lipschitz domains

    SciTech Connect

    Tyulenev, A I

    2014-08-01

    This paper gives an explicit description of the traces of functions in a weighted Sobolev space (with local Muckenhoupt weight) on the domain lying between two graphs of Lipschitz functions and on the complement of the closure of this domain. Bibliography: 11 titles.

  18. Functional dissection of the multi-domain di-heme cytochrome c(550) from Thermus thermophilus.

    PubMed

    Robin, Sylvain; Arese, Marzia; Forte, Elena; Sarti, Paolo; Kolaj-Robin, Olga; Giuffrè, Alessandro; Soulimane, Tewfik

    2013-01-01

    In bacteria, oxidation of sulfite to sulfate, the most common strategy for sulfite detoxification, is mainly accomplished by the molybdenum-containing sulfite:acceptor oxidoreductases (SORs). Bacterial SORs are very diverse proteins; they can exist as monomers or homodimers of their core subunit, as well as heterodimers with an additional cytochrome c subunit. We have previously described the homodimeric SOR from Thermus thermophilus HB8 (SOR(TTHB8)), identified its physiological electron acceptor, cytochrome c(550), and demonstrated the key role of the latter in coupling sulfite oxidation to aerobic respiration. Herein, the role of this di-heme cytochrome c was further investigated. The cytochrome was shown to be composed of two conformationally independent domains, each containing one heme moiety. Each domain was separately cloned, expressed in E. coli and purified to homogeneity. Stopped-flow experiments showed that: i) the N-terminal domain is the only one accepting electrons from SOR(TTHB8); ii) the N- and C-terminal domains are in rapid redox equilibrium and iii) both domains are able to transfer electrons further to cytochrome c(552), the physiological substrate of the ba(3) and caa(3) terminal oxidases. These findings show that cytochrome c(550) functions as a electron shuttle, without working as an electron wire with one heme acting as the electron entry and the other as the electron exit site. Although contribution of the cytochrome c(550) C-terminal domain to T. thermophilus sulfur respiration seems to be dispensable, we suggest that di-heme composition of the cytochrome physiologically enables storage of the two electrons generated from sulfite oxidation, thereof ensuring efficient contribution of sulfite detoxification to the respiratory chain-mediated energy generation. PMID:23383080

  19. Intracellular Domain Fragment of CD44 Alters CD44 Function in Chondrocytes*

    PubMed Central

    Mellor, Liliana; Knudson, Cheryl B.; Hida, Daisuke; Askew, Emily B.; Knudson, Warren

    2013-01-01

    The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44. PMID:23884413

  20. Domain organization of phytochelatin synthase: functional properties of truncated enzyme species identified by limited proteolysis.

    PubMed

    Ruotolo, Roberta; Peracchi, Alessio; Bolchi, Angelo; Infusini, Giuseppe; Amoresano, Angela; Ottonello, Simone

    2004-04-01

    Phytochelatin synthase (PCS) is a major determinant of heavy metal tolerance in plants and other organisms. No structural information on this enzyme is as yet available. It is generally believed, however, that the active site region is located in the more conserved N-terminal portion of PCS, whereas various, as yet unidentified (but supposedly less critical) roles have been proposed for the C-terminal region. To gain insight into the structural/functional organization of PCS, we have conducted a limited proteolysis analysis of the enzyme from Arabidopsis (AtPCS1), followed by functional characterization of the resulting polypeptide fragments. Two N-terminal fragments ending at positions 372 (PCS_Nt1) and 283 (PCS_Nt2) were produced sequentially upon V8 protease digestion, without any detectable accumulation of the corresponding C-terminal fragments. As revealed by the results of in vivo and in vitro functional assays, the core PCS_Nt2 fragment is biosynthetically active in the presence of cadmium ions and supports phytochelatin formation at a rate that is only approximately 5-fold lower than that of full-length AtPCS1. The loss of the C-terminal region, however, substantially decreases the thermal stability of the enzyme and impairs phytochelatin formation in the presence of certain heavy metals (e.g. mercury and zinc, but not cadmium or copper). The latter phenotype was shared by PCS_Nt2 and by its precursor fragment PCS_Nt1, which, on the other hand, was almost as stable and biosynthetically active (in the presence of cadmium) as the full-length enzyme. AtPCS1 thus appears to be composed of a protease-resistant (and hence presumably highly structured) N-terminal domain, flanked by an intrinsically unstable C-terminal region. The most upstream part of such a region (positions 284-372) is important for enzyme stabilization, whereas its most terminal part (positions 373-485) appears to be required to determine enzyme responsiveness to a broader range of heavy metals

  1. Space Tug avionics definition study. Volume 2: Avionics functional requirements

    NASA Technical Reports Server (NTRS)

    1975-01-01

    Flight and ground operational phases of the tug/shuttle system are analyzed to determine the general avionics support functions that are needed during each of the mission phases and sub-phases. Each of these general support functions is then expanded into specific avionics system requirements, which are then allocated to the appropriate avionics subsystems. This process is then repeated at the next lower level of detail where these subsystem requirements are allocated to each of the major components that comprise a subsystem.

  2. Domain Organization in Candida glabrata THI6, a Bifunctional Enzyme Required for Thiamin Biosynthesis in Eukaryotes

    SciTech Connect

    Paul, Debamita; Chatterjee, Abhishek; Begley, Tadhg P.; Ealick, Steven E.

    2010-11-15

    THI6 is a bifunctional enzyme found in the thiamin biosynthetic pathway in eukaryotes. The N-terminal domain of THI6 catalyzes the ligation of the thiamin thiazole and pyrimidine moieties to form thiamin phosphate, and the C-terminal domain catalyzes the phosphorylation of 4-methyl-5-hydroxyethylthiazole in a salvage pathway. In prokaryotes, thiamin phosphate synthase and 4-methyl-5-hydroxyethylthiazole kinase are separate gene products. Here we report the first crystal structure of a eukaryotic THI6 along with several complexes that characterize the active sites responsible for the two chemical reactions. THI6 from Candida glabrata is a homohexamer in which the six protomers form a cage-like structure. Each protomer is composed of two domains, which are structurally homologous to their monofunctional bacterial counterparts. Two loop regions not found in the bacterial enzymes provide interactions between the two domains. The structures of different protein-ligand complexes define the thiazole and ATP binding sites of the 4-methyl-5-hydroxyethylthiazole kinase domain and the thiazole phosphate and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate binding sites of the thiamin phosphate synthase domain. Our structural studies reveal that the active sites of the two domains are 40 {angstrom} apart and are not connected by an obvious channel. Biochemical studies show 4-methyl-5-hydroxyethylthiazole phosphate is a substrate for THI6; however, adenosine diphospho-5{beta}-ethyl-4-methylthiazole-2-carboxylic acid, the product of THI4, is not a substrate for THI6. This suggests that an unidentified enzyme is necessary to produce the substrate for THI6 from the THI4 product.

  3. Differential Contributions of Nonmuscle Myosin II Isoforms and Functional Domains to Stress Fiber Mechanics.

    PubMed

    Chang, Ching-Wei; Kumar, Sanjay

    2015-01-01

    While is widely acknowledged that nonmuscle myosin II (NMMII) enables stress fibers (SFs) to generate traction forces against the extracellular matrix, little is known about how specific NMMII isoforms and functional domains contribute to SF mechanics. Here we combine biophotonic and genetic approaches to address these open questions. First, we suppress the NMMII isoforms MIIA and MIIB and apply femtosecond laser nanosurgery to ablate and investigate the viscoelastic retraction of individual SFs. SF retraction dynamics associated with MIIA and MIIB suppression qualitatively phenocopy our earlier measurements in the setting of Rho kinase (ROCK) and myosin light chain kinase (MLCK) inhibition, respectively. Furthermore, fluorescence imaging and photobleaching recovery reveal that MIIA and MIIB are enriched in and more stably localize to ROCK- and MLCK-controlled central and peripheral SFs, respectively. Additional domain-mapping studies surprisingly reveal that deletion of the head domain speeds SF retraction, which we ascribe to reduced drag from actomyosin crosslinking and frictional losses. We propose a model in which ROCK/MIIA and MLCK/MIIB functionally regulate common pools of SFs, with MIIA crosslinking and motor functions jointly contributing to SF retraction dynamics and cellular traction forces. PMID:26336830

  4. Correlating structure and function during the evolution of fibrinogen-related domains.

    PubMed

    Doolittle, Russell F; McNamara, Kyle; Lin, Kevin

    2012-12-01

    Fibrinogen-related domains (FReDs) are found in a variety of animal proteins with widely different functions, ranging from non-self recognition to clot formation. All appear to have a common surface where binding of one sort or other occurs. An examination of 19 completed animal genomes--including a sponge and sea anemone, six protostomes, and 11 deuterostomes--has allowed phylogenies to be constructed that show where various types of FReP (proteins containing FReDs) first made their appearance. Comparisons of sequences and structures also reveal particular features that correlate with function, including the influence of neighbor-domains. A particular set of insertions in the carboxyl-terminal subdomain was involved in the transition from structures known to bind sugars to those known to bind amino-terminal peptides. Perhaps not unexpectedly, FReDs with different functions have changed at different rates, with ficolins by far the fastest changing group. Significantly, the greatest amount of change in ficolin FReDs occurs in the third subdomain ("P domain"), the very opposite of the situation in most other vertebrate FReDs. The unbalanced style of change was also observed in FReDs from non-chordates, many of which have been implicated in innate immunity. PMID:23076991

  5. Molecular Genetic Analysis of Suppressor 2 of zeste Identifies Key Functional Domains

    PubMed Central

    Emmons, Richard B.; Genetti, Heather; Filandrinos, Stephen; Lokere, Jillian; Wu, Chao-ting

    2009-01-01

    The Su(z)2 complex contains Posterior sex combs (Psc) and Suppressor 2 of zeste [Su(z)2], two paralogous genes that likely arose by gene duplication. Psc encodes a Polycomb group protein that functions as a central component of the PRC1 complex, which maintains transcriptional repression of a wide array of genes. Although much is known about Psc, very little is known about Su(z)2, the analysis of which has been hampered by a dearth of alleles. We have generated new alleles of Su(z)2 and analyzed them at the genetic and molecular levels. Some of these alleles display negative complementation in that they cause lethality when heterozygous with the gain-of-function Su(z)21 allele but are hemizygous and, in some cases, homozygous viable. Interestingly, alleles of this class identify protein domains within Su(z)2 that are highly conserved in Psc and the mammalian Bmi-1 and Mel-18 proteins. We also find several domains of intrinsic disorder in the C-terminal regions of both Psc and Su(z)2 and suggest that these domains may contribute to the essential functions of both proteins. PMID:19528329

  6. Differential Contributions of Nonmuscle Myosin II Isoforms and Functional Domains to Stress Fiber Mechanics

    PubMed Central

    Chang, Ching-Wei; Kumar, Sanjay

    2015-01-01

    While is widely acknowledged that nonmuscle myosin II (NMMII) enables stress fibers (SFs) to generate traction forces against the extracellular matrix, little is known about how specific NMMII isoforms and functional domains contribute to SF mechanics. Here we combine biophotonic and genetic approaches to address these open questions. First, we suppress the NMMII isoforms MIIA and MIIB and apply femtosecond laser nanosurgery to ablate and investigate the viscoelastic retraction of individual SFs. SF retraction dynamics associated with MIIA and MIIB suppression qualitatively phenocopy our earlier measurements in the setting of Rho kinase (ROCK) and myosin light chain kinase (MLCK) inhibition, respectively. Furthermore, fluorescence imaging and photobleaching recovery reveal that MIIA and MIIB are enriched in and more stably localize to ROCK- and MLCK-controlled central and peripheral SFs, respectively. Additional domain-mapping studies surprisingly reveal that deletion of the head domain speeds SF retraction, which we ascribe to reduced drag from actomyosin crosslinking and frictional losses. We propose a model in which ROCK/MIIA and MLCK/MIIB functionally regulate common pools of SFs, with MIIA crosslinking and motor functions jointly contributing to SF retraction dynamics and cellular traction forces. PMID:26336830

  7. Orbit targeting specialist function: Level C formulation requirements

    NASA Technical Reports Server (NTRS)

    Dupont, A.; Mcadoo, S.; Jones, H.; Jones, A. K.; Pearson, D.

    1978-01-01

    A definition of the level C requirements for onboard maneuver targeting software is provided. Included are revisions of the level C software requirements delineated in JSC IN 78-FM-27, Proximity Operations Software; Level C Requirements, dated May 1978. The software supports the terminal phase midcourse (TPM) maneuver, braking and close-in operations as well as supporting computation of the rendezvous corrective combination maneuver (NCC), and the terminal phase initiation (TPI). Specific formulation is contained here for the orbit targeting specialist function including the processing logic, linkage, and data base definitions for all modules. The crew interface with the software is through the keyboard and the ORBIT-TGT display.

  8. Determination of the functional domains involved in nucleolar targeting of nucleolin.

    PubMed Central

    Créancier, L; Prats, H; Zanibellato, C; Amalric, F; Bugler, B

    1993-01-01

    Nucleolin (713 aa), a major nucleolar protein, presents two structural domains: a N-terminus implicated in interaction with chromatin and a C-terminus containing four RNA-binding domains (RRMs) and a glycine/arginine-rich domain mainly involved in pre-rRNA packaging. Furthermore, nucleolin was shown to shuttle between cytoplasm and nucleolus. To get an insight on the nature of nuclear and nucleolar localization signals, a set of nucleolin deletion mutants in fusion with the prokaryotic chloramphenicol acetyltransferase (CAT) were constructed, and the resulting chimeric proteins were recognized by anti-CAT antibodies. First, a nuclear location signal bipartite and composed of two short basic stretches separated by eleven residues was characterized. Deletion of either motifs renders the protein cytoplasmic. Second, by deleting one or more domains implicated in nucleolin association either with DNA, RNA, or proteins, we demonstrated that nucleolar accumulation requires, in addition to the nuclear localization sequence, at least two of the five RRMs in presence or absence of N-terminus. However, in presence of only one RRM the N-terminus allowed a partial targeting of the chimeric protein to the nucleolus. Images PMID:8167407

  9. The highly conserved domain of unknown function 1792 has a distinct glycosyltransferase fold

    PubMed Central

    Zhang, Hua; Zhu, Fan; Yang, Tiandi; Ding, Lei; Zhou, Meixian; Li, Jingzhi; Haslam, Stuart M; Dell, Anne; Erlandsen, Heidi; Wu, Hui

    2014-01-01

    More than 33,000 glycosyltransferases have been identified. Structural studies, however, have only revealed two distinct glycosyltransferase (GT) folds, GT-A and GT-B. Here we report a 1.34 Å resolution X-ray crystallographic structure of a previously uncharacterized “domain of unknown function” 1792 (DUF1792) and show that the domain adopts a new fold and is required for glycosylation of a family of serine-rich repeat streptococcal adhesins. Biochemical studies reveal that the domain is a glucosyltransferase, and it catalyzes the transfer of glucose to the branch point of the hexasaccharide O-linked to the serine-rich repeat of the bacterial adhesin, Fap1 of Streptococcus parasanguinis. DUF1792 homologs from both Gram-positive and Gram-negative bacteria also exhibit the activity. Thus DUF1792 represents a new family of glycosyltransferases, so we designate it as a GT-D glycosyltransferase fold. As the domain is highly conserved in bacteria and not found in eukaryotes, it can be explored as a new antibacterial target. PMID:25023666

  10. The N-terminal unstructured domain of yeast ODC functions as a transplantable and replaceable ubiquitin-independent degron.

    PubMed

    Gödderz, Daniela; Schäfer, Ekaterine; Palanimurugan, R; Dohmen, R Jürgen

    2011-04-01

    Ornithine decarboxylase (ODC), a homodimeric enzyme with a rate-limiting function in polyamine biosynthesis, is subject to a feedback control involving its selective proteolysis. Targeting of ODC monomers to the proteasome is mediated by ODC antizyme (OAZ), the expression of which is induced by high levels of polyamines. Here, we report our analysis of the N-terminal degron in Saccharomyces cerevisiae ODC and the mechanism of its antizyme-dependent targeting. This ∼45-residue domain of ODC [termed ODC degradation signal (ODS)] is essential for degradation of ODC. Extensive mutagenesis indicated that it is not a specific sequence within ODS that is important but, rather, its unstructured nature. Consistent with this conclusion, ODS could be functionally replaced by an unrelated unstructured domain. We show that increasing the distance of ODS to the rest of the ODC protein reduced the dependence on Oaz1 for targeting, indicating that exposure of ODS is critical for its function. Disruption of ODC dimers by introducing interface mutations, in contrast, was insufficient for targeting. Binding of Oaz1 to ODC monomers is thus required to activate ODS. Fusion of ODS to the N terminus of Ura3 was sufficient to convert it into a ubiquitin-independent substrate of the proteasome. By contrast, ODS failed to destabilize maltose-binding protein or dihydrofolate reductase, indicating that this degron only operates in an appropriate structural context that enables rapid unfolding. PMID:21295581

  11. Domain structure and functional analysis of the carboxyl-terminal polyacidic sequence of the RAD6 protein of Saccharomyces cerevisiae.

    PubMed Central

    Morrison, A; Miller, E J; Prakash, L

    1988-01-01

    The RAD6 gene of Saccharomyces cerevisiae, which is required for normal tolerance of DNA damage and for sporulation, encodes a 172-residue protein whose 23 carboxyl-terminal residues are almost all acidic. We show that this polyacidic sequence appends to RAD6 protein as a polyanionic tail and that its function in vivo does not require stoichiometry of length. RAD6 protein was purified to near homogeneity from a yeast strain carrying a RAD6 overproducing plasmid. Approximately the first 150 residues of RAD6 protein composed a structural domain that was resistant to proteinase K and had a Stokes radius typical of a globular protein of its calculated mass. The carboxyl-terminal polyacidic sequence was sensitive to proteinase K, and it endowed RAD6 protein with an aberrantly large Stokes radius that indicates an asymmetric shape. We deduce that RAD6 protein is monomeric and comprises a globular domain with a freely extending polyacidic tail. We tested the phenotypic effects of partial or complete deletion of the polyacidic sequence, demonstrating the presence of the shortened proteins in the cell by using antibody to RAD6 protein. Removal of the entire polyacidic sequence severely reduced sporulation but only slightly affected survival after UV irradiation or UV-induced mutagenesis. Strains with deletions of all but the first 4 or 15 residues of the polyacidic sequence were phenotypically almost wild type or wild type, respectively. We conclude that the intrinsic activity of RAD6 protein resides in the globular domain, that the polyacidic sequence has a stimulatory or modifying role evident primarily in sporulation, and that only a short section apparently functions as effectively as the entire polyacidic sequence. Images PMID:3285176

  12. Scaffold-Assisted Catalysis: A Novel Domain In the Ste5 Scaffold Protein is Required to Unlock the MAPK Fus3 for Phosphorylation by the MAPKK Ste7

    PubMed Central

    Good, Matthew; Tang, Grace; Singleton, Julie; Remenyi, Attila; Lim, Wendell A.

    2009-01-01

    The scaffold protein Ste5 is required to properly direct signaling through the yeast mating pathway to the mitogen-activated protein kinase (MAPK), Fus3. Scaffolds are thought to function by tethering kinase and substrate in proximity. We find, however, that the previously identified Fus3-binding site on Ste5 is not required for signaling, suggesting an alternative mechanism controls Fus3’s activation by the MAPKK Ste7. Reconstituting MAPK signaling in vitro, we find that Fus3 is an intrinsically poor substrate for Ste7, although the related filamentation MAPK, Kss1, is an excellent substrate. We identify and structurally characterize a novel domain in Ste5 that catalytically unlocks Fus3 for phosphorylation by Ste7. This domain selectively increases the kcat of Ste7➔Fus3 phosphorylation by 5000-fold but has no effect on Ste7➔Kss1 phosphorylation. The dual requirement for both Ste7 and this Ste5 domain in Fus3 activation explains why Fus3 is selectively activated by the mating pathway, and not by other pathways that also utilize Ste7. PMID:19303851

  13. An engineering time-domain model for curve squeal: Tangential point-contact model and Green's functions approach

    NASA Astrophysics Data System (ADS)

    Zenzerovic, I.; Kropp, W.; Pieringer, A.

    2016-08-01

    Curve squeal is a strong tonal sound that may arise when a railway vehicle negotiates a tight curve. In contrast to frequency-domain models, time-domain models are able to capture the nonlinear and transient nature of curve squeal. However, these models are computationally expensive due to requirements for fine spatial and time discretization. In this paper, a computationally efficient engineering model for curve squeal in the time-domain is proposed. It is based on a steady-state point-contact model for the tangential wheel/rail contact and a Green's functions approach for wheel and rail dynamics. The squeal model also includes a simple model of sound radiation from the railway wheel from the literature. A validation of the tangential point-contact model against Kalker's transient variational contact model reveals that the point-contact model performs well within the squeal model up to at least 5 kHz. The proposed squeal model is applied to investigate the influence of lateral creepage, friction and wheel/rail contact position on squeal occurrence and amplitude. The study indicates a significant influence of the wheel/rail contact position on squeal frequencies and amplitudes. Friction and lateral creepage show an influence on squeal occurrence and amplitudes, but this is only secondary to the influence of the contact position.

  14. Structure and function of interacting IcmR-IcmQ domains from a Type IVb secretion system in Legionella pneumophila

    PubMed Central

    Raychaudhury, Suchismita; Farelli, Jeremiah D.; Montminy, Timothy P.; Matthews, Miguelina; Ménétret, Jean-François; Duménil, Guillaume; Roy, Craig R.; Head, James F.; Isberg, Ralph R.; Akey, Christopher W.

    2009-01-01

    Summary During infection, Legionella pneumophila creates a replication vacuole within eukaryotic cells and this process requires a Type IVb secretion system (T4bSS). IcmQ is a critical component of this translocase and associates with IcmR. In this report, we show that the N-terminal domain of IcmQ (Qn) mediates IcmQ dimerization, while the C-terminal domain with a linker region promotes a stable membrane association of IcmQ. We then determined crystal structures of Qn with the interacting domain of IcmR. In this complex, each protein forms an α-helical hairpin within a parallel 4-helix bundle. We find that IcmR binding to IcmQ prevents dimerization of IcmQ and blocks membrane permeabilization. However, IcmR does not completely block membrane association of IcmQ. The amphipathic nature of Qn suggests two models for how IcmQ may permeabilize membranes. The Rm-Qn structure also suggests how hyper-variable IcmR-like proteins in other Legionellae may interact with their IcmQ partners to regulate IcmQ function. PMID:19368892

  15. High mobility group protein 2 functionally interacts with the POU domains of octamer transcription factors.

    PubMed Central

    Zwilling, S; König, H; Wirth, T

    1995-01-01

    The octamer transcription factors Oct1 and Oct2 are involved in the transcriptional regulation of both lymphoid-specific and ubiquitously expressed genes. Their activity depends critically on their interaction with distinct cellular cofactors. Therefore, we have isolated cDNAs encoding proteins that physically interact with Oct2. Here we describe the analysis of one such clone, representing the murine homologue of high mobility group (HMG) protein 2. We have mapped the interaction domains for both proteins and have shown that HMG2 and Oct2 interact via their HMG domains and POU homeodomains, respectively. This interaction is not restricted to Oct2, as other members of the octamer transcription factor family like Oct1 and Oct6 also interact with HMG2. The interaction with HMG2 results in a marked increase in the sequence-specific DNA binding activity of the Oct proteins. Interestingly, the HMG2 protein is not present in the protein-DNA complex detected by an electrophoretic mobility shift assay. The Oct and HMG2 proteins also interact in vivo. A chimeric protein, in which the strong transactivation domain of VP16 was fused directly to the HMG domains of HMG2, stimulated the activity of an octamer-dependent reporter construct upon cotransfection. Furthermore, the expression of antisense RNA for HMG2 specifically reduces octamer-dependent transcription. These results suggest that one of the functions of HMG2 is to support the octamer transcription factors in their role as transcriptional activators. Images PMID:7720710

  16. The intracellular domains of Notch1 and Notch2 are functionally equivalent during development and carcinogenesis.

    PubMed

    Liu, Zhenyi; Brunskill, Eric; Varnum-Finney, Barbara; Zhang, Chi; Zhang, Andrew; Jay, Patrick Y; Bernstein, Irv; Morimoto, Mitsuru; Kopan, Raphael

    2015-07-15

    Although Notch1 and Notch2 are closely related paralogs and function through the same canonical signaling pathway, they contribute to different outcomes in some cell and disease contexts. To understand the basis for these differences, we examined in detail mice in which the Notch intracellular domains (N1ICD and N2ICD) were swapped. Our data indicate that strength (defined here as the ultimate number of intracellular domain molecules reaching the nucleus, integrating ligand-mediated release and nuclear translocation) and duration (half-life of NICD-RBPjk-MAML-DNA complexes, integrating cooperativity and stability dependent on shared sequence elements) are the factors that underlie many of the differences between Notch1 and Notch2 in all the contexts we examined, including T-cell development, skin differentiation and carcinogenesis, the inner ear, the lung and the retina. We were able to show that phenotypes in the heart, endothelium, and marginal zone B cells are attributed to haploinsufficiency but not to intracellular domain composition. Tissue-specific differences in NICD stability were most likely caused by alternative scissile bond choices by tissue-specific γ-secretase complexes following the intracellular domain swap. Reinterpretation of clinical findings based on our analyses suggests that differences in outcome segregating with Notch1 or Notch2 are likely to reflect outcomes dependent on the overall strength of Notch signals. PMID:26062937

  17. The intracellular domains of Notch1 and Notch2 are functionally equivalent during development and carcinogenesis

    PubMed Central

    Liu, Zhenyi; Brunskill, Eric; Varnum-Finney, Barbara; Zhang, Chi; Zhang, Andrew; Jay, Patrick Y.; Bernstein, Irv; Morimoto, Mitsuru; Kopan, Raphael

    2015-01-01

    Although Notch1 and Notch2 are closely related paralogs and function through the same canonical signaling pathway, they contribute to different outcomes in some cell and disease contexts. To understand the basis for these differences, we examined in detail mice in which the Notch intracellular domains (N1ICD and N2ICD) were swapped. Our data indicate that strength (defined here as the ultimate number of intracellular domain molecules reaching the nucleus, integrating ligand-mediated release and nuclear translocation) and duration (half-life of NICD-RBPjk-MAML-DNA complexes, integrating cooperativity and stability dependent on shared sequence elements) are the factors that underlie many of the differences between Notch1 and Notch2 in all the contexts we examined, including T-cell development, skin differentiation and carcinogenesis, the inner ear, the lung and the retina. We were able to show that phenotypes in the heart, endothelium, and marginal zone B cells are attributed to haploinsufficiency but not to intracellular domain composition. Tissue-specific differences in NICD stability were most likely caused by alternative scissile bond choices by tissue-specific γ-secretase complexes following the intracellular domain swap. Reinterpretation of clinical findings based on our analyses suggests that differences in outcome segregating with Notch1 or Notch2 are likely to reflect outcomes dependent on the overall strength of Notch signals. PMID:26062937

  18. Enhanced functionality in magnonics by domain walls and inhomogeneous spin configurations.

    PubMed

    Duerr, G; Huber, R; Grundler, D

    2012-01-18

    This paper discusses nanomagnetic structures enabling the manipulation of propagating spin waves. We address in particular how domain walls, or more generally speaking inhomogeneous spin configurations, enhance the control of spin-wave transmission and thereby the functionality of magnonic devices. Three different microscopic mechanisms are outlined, considering an interference device, a spin-wave bus and a magnonic crystal. Inhomogeneous spin configurations are argued to shift the spin-wave phase, guide spin waves in nanochannels and allow for reprogrammable spin-wave band structures in periodic nanostructures, respectively. Such devices and functionalities are relevant for further developments in magnonics. PMID:22172683

  19. Enhanced functionality in magnonics by domain walls and inhomogeneous spin configurations

    NASA Astrophysics Data System (ADS)

    Duerr, G.; Huber, R.; Grundler, D.

    2012-01-01

    This paper discusses nanomagnetic structures enabling the manipulation of propagating spin waves. We address in particular how domain walls, or more generally speaking inhomogeneous spin configurations, enhance the control of spin-wave transmission and thereby the functionality of magnonic devices. Three different microscopic mechanisms are outlined, considering an interference device, a spin-wave bus and a magnonic crystal. Inhomogeneous spin configurations are argued to shift the spin-wave phase, guide spin waves in nanochannels and allow for reprogrammable spin-wave band structures in periodic nanostructures, respectively. Such devices and functionalities are relevant for further developments in magnonics.

  20. A flavin-mononucleotide-binding site in Hansenula anomala nicked flavocytochrome b2, requiring the association of two domains.

    PubMed

    Gervais, M; Labeyrie, F; Risler, Y; Vergnes, O

    1980-10-01

    Previous experiments in our laboratory with Saccharomyces cervisiae flavocytochrom b2 indicated that both fragments alpha and beta of the enzyme after cleavage by yeast proteases are required to form the flavin site. More detailed experiments have not been carried out on the nicked Hansenula anomala enzyme obtained by tryptic cleavage. A method has been devised that gives a quantitative separation in 4 M urea of beta, and alpha with its heme still bound. The characteristics of the various species: isolated alpha and beta and mixed alpha + beta were studied in 4 M urea and after elimination of this reagent by dialysis in the presence of FMN and 2-mercaptoethanol. Several methods, including heme spectroscopy, tryptophan fluorescence, sedimentation studies, and titration of bound flavin, were used. The results indicate that isolated alpha and beta have a folded globular structure after renaturation. The flavin binding to the alpha + beta mixture was important (50-100%) with recovery of the flavodehydrogenase activity. In contrast, binding was not detectable (< 0.5%, Kf > 10 mM) for isolated alpha and beta. As far as mononucleotide binding is concerned, such a cooperative requirement for two folding domains has never been reported in other enzymes. The present results are discussed together with others obtained in our laboratory which demonstrate that, as deduced from their sensitivity to trypsin, the structure of S. cerevisiae and H. anomala flavocytochrome b2 protomers is triglobular 'n-x-beta' (n and x combined within alpha). The tetramer assembly, which remains intact as a nicked enzyme (alpha beta)4 after the first trypsin cleavage, is broken down following a second cleavage of the chain into four cytochrome cores (n) and a functional T-flavodehydrogenase entity, a tetramer of the type (x beta)4. PMID:7439181

  1. Functional Requirements for Bibliographic Records: An Investigation of Two Prototypes

    ERIC Educational Resources Information Center

    Pisanski, Jan; Zumer, Maja

    2007-01-01

    Purpose: This paper aims to establish how the Functional Requirements for Bibliographic Records (FRBR) conceptual model, which holds a lot of potential in theory, works in practice. It also aims to identify, and if possible, give solutions to problems found in two of the existing prototypes. Design/methodology/approach: An independent evaluation…

  2. Regulatory T cells require TCR signaling for their suppressive function

    PubMed Central

    Schmidt, Amanda M.; Lu, Wen; Sindhava, Vishal J.; Huang, Yanping; Burkhardt, Janis K.; Yang, Enjun; Riese, Matthew J.; Maltzman, Jonathan S.; Jordan, Martha S.; Kambayashi, Taku

    2015-01-01

    Regulatory T cells (Tregs) are a subset of CD4+ T cells that maintain immune tolerance in part by their ability to inhibit the proliferation of conventional CD4+ T cells (Tconvs). The role of the T cell receptor (TCR) and the downstream signaling pathways required for this suppressive function of Tregs are not fully understood. To yield insight into how TCR-mediated signals influence Treg suppressive function, we assessed the ability of Tregs with altered TCR-mediated signaling capacity to inhibit Tconv proliferation. Mature Tregs deficient in SLP-76, an adaptor protein that nucleates the proximal signaling complex downstream of the TCR, were unable to inhibit Tconv proliferation, suggesting that TCR signaling is required for Treg suppressive function. Moreover, Tregs with defective PLCγ activation due to a Y145F mutation of SLP-76 were also defective in their suppressive function. Conversely, enhancement of diacylglycerol-mediated signaling downstream of PLCγ by genetic ablation of a negative regulator of diacylglycerol kinase ζ increased the suppressive ability of Tregs. Since SLP-76 is also important for integrin activation and signaling, we tested the role of integrin activation in Treg-mediated suppression. Tregs lacking the adaptor proteins ADAP or Crk/CrkL, which are required for TCR-mediated integrin activation, inhibited Tconv proliferation to a similar extent as wildtype Tregs. Together, these data suggest that TCR-mediated PLCγ activation but not integrin activation is required for Tregs to inhibit Tconv proliferation. PMID:25821220

  3. Multiple Functional Domains of AML1: PU.1 and C/EBPα Synergize with Different Regions of AML1

    PubMed Central

    Petrovick, Martha S.; Hiebert, Scott W.; Friedman, Alan D.; Hetherington, Christopher J.; Tenen, Daniel G.; Zhang, Dong-Er

    1998-01-01

    Control elements of many genes are regulated by multiple activators working in concert to confer the maximal level of expression, but the mechanism of such synergy is not completely understood. The promoter of the human macrophage colony-stimulating factor (M-CSF) receptor presents an excellent model with which we can study synergistic, tissue-specific activation for two reasons. First, myeloid-specific expression of the M-CSF receptor is regulated transcriptionally by three factors which are crucial for normal hematopoiesis: PU.1, AML1, and C/EBPα. Second, these proteins interact in such a way as to demonstrate at least two examples of synergistic activation. We have shown that AML1 and C/EBPα activate the M-CSF receptor promoter in a synergistic manner. As we report here, AML1 also synergizes, and interacts physically, with PU.1. Detailed analysis of the physical and functional interaction of AML1 with PU.1 and C/EBPα has revealed that the proteins contact one another through their DNA-binding domains and that AML1 exhibits cooperative DNA binding with C/EBPα but not with PU.1. This difference in DNA-binding abilities may explain, in part, the differences observed in synergistic activation. Furthermore, the activation domains of all three factors are required for synergistic activation, and the region of AML1 required for synergy with PU.1 is distinct from that required for synergy with C/EBPα. These observations present the possibility that synergistic activation is mediated by secondary proteins contacted through the activation domains of AML1, C/EBPα, and PU.1. PMID:9632776

  4. Functional Consequences of Exchanging Domains Between LacI and PurR are Mediated by the Intervening Linker Sequence

    PubMed Central

    Tungtur, Sudheer; Egan, Susan M.; Swint-Kruse, Liskin

    2007-01-01

    Homologue function can be differentiated by changing residues that affect binding sites or long-range interactions. LacI and PurR are two proteins that represent the LacI/GalR family (>500 members) of bacterial transcription regulators. All members have distinct DNA-binding and regulatory domains linked by ~18 amino acids. Each homologue has specificity for different DNA and regulatory effector ligands; LacI and PurR also exhibit differences in allosteric communication between DNA and effector binding sites. A comparative study of LacI and PurR suggested that alterations in the interface between the regulatory domain and linker are important for differentiating their functions. Four residues (equivalent to LacI positions 48, 55, 58, and 61) appear particularly important for creating a unique interface and were predicted to be necessary for allosteric regulation. However, nearby residues in the linker interact with DNA ligand. Thus, differences observed in interactions between linker and regulatory domain may be the cause of altered function or an effect of the two proteins binding different DNA ligands. To separate these possibilities, we created a chimeric protein with the LacI DNA-binding domain/linker and the PurR regulatory domain (LLhP). If the interface requires homologue-specific interactions in order to propagate the signal from effector binding, then LLhP repression should not be allosterically regulated by effector binding. Experiments show that LLhP is capable of repression from lacO1 and, contrary to expectation, allosteric response is intact. Further, restoring the potential for PurR-like interactions via substitutions in the LLhP linker tends to diminish repression. These effects are especially pronounced for residues 58 and 61. Clearly, binding affinity of LLhP for the lacO1 DNA site is sensitive to long-range changes in the linker. This result also raises the possibility that mutations at positions 58 and 61 co-evolved with changes in the DNA

  5. Functional requirements for a central research imaging data repository.

    PubMed

    Franke, Thomas; Gruetz, Romanus; Dickmann, Frank

    2013-01-01

    The current situation at many university medical centers regarding the management of biomedical research imaging data leaves much to be desired. In contrast to the recommendations of the German Research Foundation (DFG) and the German Council of Sciences and Humanities regarding the professional management of research data, there are commonly many individual data pools for research data in each institute and the management remains the responsibility of the researcher. A possible solution for this situation would be to install local central repositories for biomedical research imaging data. In this paper, we developed a scenario based on abstracted use-cases for institutional research undertakings as well as collaborative biomedical research projects and analyzed the functional requirements that a local repository would have to fulfill. We determined eight generic categories of functional requirements, which can be viewed as a basic guideline for the minimum functionality of a central repository for biomedical research imaging data. PMID:23920564

  6. Spent Nuclear Fuel Project Canister Storage Building Functions and Requirements

    SciTech Connect

    KLEM, M.J.

    2000-10-18

    In 1998, a major change in the technical strategy for managing Multi Canister Overpacks (MCO) while stored within the Canister Storage Building (CSB) occurred. The technical strategy is documented in Baseline Change Request (BCR) No. SNF-98-006, Simplified SNF Project Baseline (MCO Sealing) (FDH 1998). This BCR deleted the hot conditioning process initially adopted for the Spent Nuclear Fuel Project (SNF Project) as documented in WHC-SD-SNF-SP-005, Integrated Process Strategy for K Basins Spent Nuclear Fuel (WHC 199.5). In summary, MCOs containing Spent Nuclear Fuel (SNF) from K Basins would be placed in interim storage following processing through the Cold Vacuum Drying (CVD) facility. With this change, the needs for the Hot Conditioning System (HCS) and inerting/pressure retaining capabilities of the CSB storage tubes and the MCO Handling Machine (MHM) were eliminated. Mechanical seals will be used on the MCOs prior to transport to the CSB. Covers will be welded on the MCOs for the final seal at the CSB. Approval of BCR No. SNF-98-006, imposed the need to review and update the CSB functions and requirements baseline documented herein including changing the document title to ''Spent Nuclear Fuel Project Canister Storage Building Functions and Requirements.'' This revision aligns the functions and requirements baseline with the CSB Simplified SNF Project Baseline (MCO Sealing). This document represents the Canister Storage Building (CSB) Subproject technical baseline. It establishes the functions and requirements baseline for the implementation of the CSB Subproject. The document is organized in eight sections. Sections 1.0 Introduction and 2.0 Overview provide brief introductions to the document and the CSB Subproject. Sections 3.0 Functions, 4.0 Requirements, 5.0 Architecture, and 6.0 Interfaces provide the data described by their titles. Section 7.0 Glossary lists the acronyms and defines the terms used in this document. Section 8.0 References lists the

  7. Regulation of dynamin-2 assembly-disassembly and function through the SH3A domain of intersectin-1s.

    PubMed

    Knezevic, Ivana; Predescu, Dan; Bardita, Cristina; Wang, Minhua; Sharma, Tiffany; Keith, Barbara; Neamu, Radu; Malik, Asrar B; Predescu, Sanda

    2011-11-01

    Intersectin-1s (ITSN-1s), a five Src homology 3 (SH3) domain-containing protein, is critically required for caveolae and clathrin-mediated endocytosis (CME), due to its interactions with dynamin (dyn). Of the five SH3A-E domains, SH3A is unique because of its high affinity for dyn and potent inhibition of CME. However, the molecular mechanism by which SH3A integrates in the overall function of ITSN-1s to regulate the endocytic process is not understood. Using biochemical and functional approaches as well as high-resolution electron microscopy, we show that SH3A exogenously expressed in human lung endothelial cells caused abnormal endocytic structures, distorted caveolae clusters, frequent staining-dense rings around the caveolar necks and 60% inhibition of caveolae internalization. In vitro studies further revealed that SH3A, similar to full-length ITSN-1s stimulates dyn2 oligomerization and guanosine triphosphatase (GTP)ase activity, effects not detected when other SH3 domains of ITSN-1s were used as controls. Strikingly, in the presence of SH3A, dyn2-dyn2 interactions are stabilized and despite continuous GTP hydrolysis, dyn2 oligomers cannot disassemble. SH3A may hold up caveolae release from the plasma membrane and formation of free-transport vesicles, by prolonging the lifetime of assembled dyn2. Altogether, our results indicate that ITSN-1s, via its SH3A has the unique ability to regulate dyn2 assembly-disassembly and function during endocytosis. PMID:21129155

  8. Regulation of dynamin-2 assembly–disassembly and function through the SH3A domain of intersectin-1s

    PubMed Central

    Knezevic, Ivana; Predescu, Dan; Bardita, Cristina; Wang, Minhua; Sharma, Tiffany; Keith, Barbara; Neamu, Radu; Malik, Asrar B; Predescu, Sanda

    2011-01-01

    Abstract Intersectin-1s (ITSN-1s), a five Src homology 3 (SH3) domain-containing protein, is critically required for caveolae and clathrin-mediated endocytosis (CME), due to its interactions with dynamin (dyn). Of the five SH3A-E domains, SH3A is unique because of its high affinity for dyn and potent inhibition of CME. However, the molecular mechanism by which SH3A integrates in the overall function of ITSN-1s to regulate the endocytic process is not understood. Using biochemical and functional approaches as well as high-resolution electron microscopy, we show that SH3A exogenously expressed in human lung endothelial cells caused abnormal endocytic structures, distorted caveolae clusters, frequent staining-dense rings around the caveolar necks and 60% inhibition of caveolae internalization. In vitro studies further revealed that SH3A, similar to full-length ITSN-1s stimulates dyn2 oligomerization and guanosine triphosphatase (GTP)ase activity, effects not detected when other SH3 domains of ITSN-1s were used as controls. Strikingly, in the presence of SH3A, dyn2–dyn2 interactions are stabilized and despite continuous GTP hydrolysis, dyn2 oligomers cannot disassemble. SH3A may hold up caveolae release from the plasma membrane and formation of free-transport vesicles, by prolonging the lifetime of assembled dyn2. Altogether, our results indicate that ITSN-1s, via its SH3A has the unique ability to regulate dyn2 assembly–disassembly and function during endocytosis. PMID:21129155

  9. Approximate analytical time-domain Green's functions for the Caputo fractional wave equation.

    PubMed

    Kelly, James F; McGough, Robert J

    2016-08-01

    The Caputo fractional wave equation [Geophys. J. R. Astron. Soc. 13, 529-539 (1967)] models power-law attenuation and dispersion for both viscoelastic and ultrasound wave propagation. The Caputo model can be derived from an underlying fractional constitutive equation and is causal. In this study, an approximate analytical time-domain Green's function is derived for the Caputo equation in three dimensions (3D) for power law exponents greater than one. The Green's function consists of a shifted and scaled maximally skewed stable distribution multiplied by a spherical spreading factor 1/(4πR). The approximate one dimensional (1D) and two dimensional (2D) Green's functions are also computed in terms of stable distributions. Finally, this Green's function is decomposed into a loss component and a diffraction component, revealing that the Caputo wave equation may be approximated by a coupled lossless wave equation and a fractional diffusion equation. PMID:27586735

  10. Opposing functions of two sub-domains of the SNARE-complex in neurotransmission

    PubMed Central

    Weber, Jens P; Reim, Kerstin; Sørensen, Jakob B

    2010-01-01

    The SNARE-complex consisting of synaptobrevin-2/VAMP-2, SNAP-25 and syntaxin-1 is essential for evoked neurotransmission and also involved in spontaneous release. Here, we used cultured autaptic hippocampal neurons from Snap-25 null mice rescued with mutants challenging the C-terminal, N-terminal and middle domains of the SNARE-bundle to dissect out the involvement of these domains in neurotransmission. We report that the stabilities of two different sub-domains of the SNARE-bundle have opposing functions in setting the probability for both spontaneous and evoked neurotransmission. Destabilizing the C-terminal end of the SNARE-bundle abolishes spontaneous neurotransmitter release and reduces evoked release probability, indicating that the C-terminal end promotes both modes of release. In contrast, destabilizing the middle or deleting the N-terminal end of the SNARE-bundle increases both spontaneous and evoked release probabilities. In both cases, spontaneous release was affected more than evoked neurotransmission. In addition, the N-terminal deletion delays vesicle priming after a high-frequency train. We propose that the stability of N-terminal two-thirds of the SNARE-bundle has a function for vesicle priming and limiting spontaneous release. PMID:20562829

  11. Osmosensing and scaffolding functions of the oligomeric four-transmembrane domain osmosensor Sho1

    PubMed Central

    Tatebayashi, Kazuo; Yamamoto, Katsuyoshi; Nagoya, Miho; Takayama, Tomomi; Nishimura, Akiko; Sakurai, Megumi; Momma, Takashi; Saito, Haruo

    2015-01-01

    The yeast high osmolarity glycerol (HOG) pathway activates the Hog1 MAP kinase, which coordinates adaptation to high osmolarity conditions. Here we demonstrate that the four-transmembrane (TM) domain protein Sho1 is an osmosensor in the HKR1 sub-branch of the HOG pathway. Crosslinking studies indicate that Sho1 forms planar oligomers of the dimers-of-trimers architecture by dimerizing at the TM1/TM4 interface and trimerizing at the TM2/TM3 interface. High external osmolarity induces structural changes in the Sho1 TM domains and Sho1 binding to the cytoplasmic adaptor protein Ste50, which leads to Hog1 activation. Besides its osmosensing function, the Sho1 oligomer serves as a scaffold. By binding to the TM proteins Opy2 and Hkr1 at the TM1/TM4 and TM2/TM3 interface, respectively, Sho1 forms a multi-component signalling complex that is essential for Hog1 activation. Our results illuminate how the four TM domains of Sho1 dictate the oligomer structure as well as its osmosensing and scaffolding functions. PMID:25898136

  12. Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

    SciTech Connect

    Iyanagi, Takashi . E-mail: iyanagi@spring8.or.jp

    2005-12-09

    NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH {sup {center_dot}}/FMNH{sub 2} couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form can function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.

  13. Smurf1-mediated Axin Ubiquitination Requires Smurf1 C2 Domain and Is Cell Cycle-dependent*

    PubMed Central

    Fei, Cong; He, Xiaoli; Xie, Sichun; Miao, Haofei; Zhou, Zhilei; Li, Lin

    2014-01-01

    Previously, Smad ubiquitination regulatory factor 1 (Smurf1)-mediated Lys29 (K29)-linked poly-ubiquitination of Axin has been identified as a novel regulatory process in Wnt/β-catenin signaling. In this work, we discovered that the C2 domain of Smurf1 is critical for targeting Axin for ubiquitination. We found that the C2 domain-mediated plasma membrane localization of Smurf1 is required for Axin ubiquitination, and interfering with that disturbs the co-localization of Smurf1 and Axin around the plasma membrane. Moreover, the C2 domain of Smurf1, rather than its WW domains, is involved in Smurf1's interaction with Axin; and the putative PPXY motifs (PY motif) of Axin are not essential for such an interaction, indicating that Smurf1 binds to Axin in a non-canonical way independent of WW-PY interaction. Further, we found that Smurf1-Axin interaction and Axin ubiquitination are attenuated in the G2/M phase of cell cycle, contributing to an increased cell response to Wnt stimulation at that stage. Collectively, we uncovered a dual role of Smurf1 C2 domain, recruiting Smurf1 to membrane for accessing Axin and mediating its interaction with Axin, and that Smurf1-mediated Axin ubiquitination is subjected to the regulation of cell cycle. PMID:24700460

  14. Trimeric Autotransporters Require Trimerization of the Passenger Domain for Stability and Adhesive Activity

    PubMed Central

    Cotter, Shane E.; Surana, Neeraj K.; Grass, Susan; St. Geme, Joseph W.

    2006-01-01

    In recent years, structural studies have identified a number of bacterial, viral, and eukaryotic adhesive proteins that have a trimeric architecture. The prototype examples in bacteria are the Haemophilus influenzae Hia adhesin and the Yersinia enterocolitica YadA adhesin. Both Hia and YadA are members of the trimeric-autotransporter subfamily and are characterized by an internal passenger domain that harbors adhesive activity and a short C-terminal translocator domain that inserts into the outer membrane and facilitates delivery of the passenger domain to the bacterial surface. In this study, we examined the relationship between trimerization of the Hia and YadA passenger domains and the capacity for adhesive activity. We found that subunit-subunit interactions and stable trimerization are essential for native folding and stability and ultimately for full-level adhesive activity. These results raise the possibility that disruption of the trimeric architecture of trimeric autotransporters, and possibly other trimeric adhesins, may be an effective strategy to eliminate adhesive activity. PMID:16855229

  15. Molecular Details of Olfactomedin Domains Provide Pathway to Structure-Function Studies

    PubMed Central

    Hill, Shannon E.; Donegan, Rebecca K.; Nguyen, Elaine; Desai, Tanay M.; Lieberman, Raquel L.

    2015-01-01

    Olfactomedin (OLF) domains are found within extracellular, multidomain proteins in numerous tissues of multicellular organisms. Even though these proteins have been implicated in human disorders ranging from cancers to attention deficit disorder to glaucoma, little is known about their structure(s) and function(s). Here we biophysically, biochemically, and structurally characterize OLF domains from H. sapiens olfactomedin-1 (npoh-OLF, also called noelin, pancortin, OLFM1, and hOlfA), and M. musculus gliomedin (glio-OLF, also called collomin, collmin, and CRG-L2), and compare them with available structures of myocilin (myoc-OLF) recently reported by us and R. norvegicus glio-OLF and M. musculus latrophilin-3 (lat3-OLF) by others. Although the five-bladed β-propeller architecture remains unchanged, numerous physicochemical characteristics differ among these OLF domains. First, npoh-OLF and glio-OLF exhibit prominent, yet distinct, positive surface charges and copurify with polynucleotides. Second, whereas npoh-OLF and myoc-OLF exhibit thermal stabilities typical of human proteins near 55°C, and most myoc-OLF variants are destabilized and highly prone to aggregation, glio-OLF is nearly 20°C more stable and significantly more resistant to chemical denaturation. Phylogenetically, glio-OLF is most similar to primitive OLFs, and structurally, glio-OLF is missing distinguishing features seen in OLFs such as the disulfide bond formed by N- and C- terminal cysteines, the sequestered Ca2+ ion within the propeller central hydrophilic cavity, and a key loop-stabilizing cation-π interaction on the top face of npoh-OLF and myoc-OLF. While deciphering the explicit biological functions, ligands, and binding partners for OLF domains will likely continue to be a challenging long-term experimental pursuit, we used structural insights gained here to generate a new antibody selective for myoc-OLF over npoh-OLF and glio-OLF as a first step in overcoming the impasse in detailed

  16. Molecular Details of Olfactomedin Domains Provide Pathway to Structure-Function Studies.

    PubMed

    Hill, Shannon E; Donegan, Rebecca K; Nguyen, Elaine; Desai, Tanay M; Lieberman, Raquel L

    2015-01-01

    Olfactomedin (OLF) domains are found within extracellular, multidomain proteins in numerous tissues of multicellular organisms. Even though these proteins have been implicated in human disorders ranging from cancers to attention deficit disorder to glaucoma, little is known about their structure(s) and function(s). Here we biophysically, biochemically, and structurally characterize OLF domains from H. sapiens olfactomedin-1 (npoh-OLF, also called noelin, pancortin, OLFM1, and hOlfA), and M. musculus gliomedin (glio-OLF, also called collomin, collmin, and CRG-L2), and compare them with available structures of myocilin (myoc-OLF) recently reported by us and R. norvegicus glio-OLF and M. musculus latrophilin-3 (lat3-OLF) by others. Although the five-bladed β-propeller architecture remains unchanged, numerous physicochemical characteristics differ among these OLF domains. First, npoh-OLF and glio-OLF exhibit prominent, yet distinct, positive surface charges and copurify with polynucleotides. Second, whereas npoh-OLF and myoc-OLF exhibit thermal stabilities typical of human proteins near 55°C, and most myoc-OLF variants are destabilized and highly prone to aggregation, glio-OLF is nearly 20°C more stable and significantly more resistant to chemical denaturation. Phylogenetically, glio-OLF is most similar to primitive OLFs, and structurally, glio-OLF is missing distinguishing features seen in OLFs such as the disulfide bond formed by N- and C- terminal cysteines, the sequestered Ca2+ ion within the propeller central hydrophilic cavity, and a key loop-stabilizing cation-π interaction on the top face of npoh-OLF and myoc-OLF. While deciphering the explicit biological functions, ligands, and binding partners for OLF domains will likely continue to be a challenging long-term experimental pursuit, we used structural insights gained here to generate a new antibody selective for myoc-OLF over npoh-OLF and glio-OLF as a first step in overcoming the impasse in detailed

  17. Transrepressive function of TLX requires the histone demethylase LSD1.

    PubMed

    Yokoyama, Atsushi; Takezawa, Shinichiro; Schüle, Roland; Kitagawa, Hirochika; Kato, Shigeaki

    2008-06-01

    TLX is an orphan nuclear receptor (also called NR2E1) that regulates the expression of target genes by functioning as a constitutive transrepressor. The physiological significance of TLX in the cytodifferentiation of neural cells in the brain is known. However, the corepressors supporting the transrepressive function of TLX have yet to be identified. In this report, Y79 retinoblastoma cells were subjected to biochemical techniques to purify proteins that interact with TLX, and we identified LSD1 (also called KDM1), which appears to form a complex with CoREST and histone deacetylase 1. LSD1 interacted with TLX directly through its SWIRM and amine oxidase domains. LSD1 potentiated the transrepressive function of TLX through its histone demethylase activity as determined by a luciferase assay using a genomically integrated reporter gene. LSD1 and TLX were recruited to a TLX-binding site in the PTEN gene promoter, accompanied by the demethylation of H3K4me2 and deacetylation of H3. Knockdown of either TLX or LSD1 derepressed expression of the endogenous PTEN gene and inhibited cell proliferation of Y79 cells. Thus, the present study suggests that LSD1 is a prime corepressor for TLX. PMID:18391013

  18. An Amphiphysin-Like Domain in Fus2p Is Required for Rvs161p Interaction and Cortical Localization.

    PubMed

    Stein, Richard A; Smith, Jean A; Rose, Mark D

    2016-02-01

    Cell-cell fusion fulfils essential roles in fertilization, development and tissue repair. In the budding yeast, Saccharomyces cerevisiae, fusion between two haploid cells of opposite mating type generates the diploid zygote. Fus2p is a pheromone-induced protein that regulates cell wall removal during mating. Fus2p shuttles from the nucleus to localize at the shmoo tip, bound to Rvs161p, an amphiphysin. However, Rvs161p independently binds a second amphiphysin, Rvs167p, playing an essential role in endocytosis. To understand the basis of the Fus2p-Rvs161p interaction, we analyzed Fus2p structural domains. A previously described N-terminal domain (NTD) is necessary and sufficient to regulate nuclear/cytoplasmic trafficking of Fus2p. The Dbl homology domain (DBH) binds GTP-bound Cdc42p; binding is required for cell fusion, but not localization. We identified an approximately 200 amino acid region of Fus2p that is both necessary and sufficient for Rvs161p binding. The Rvs161p binding domain (RBD) contains three predicted alpha-helices; structural modeling suggests that the RBD adopts an amphiphysin-like structure. The RBD contains a 13-amino-acid region, conserved with Rvs161p and other amphiphysins, which is essential for binding. Mutations in the RBD, predicted to affect membrane binding, abolish cell fusion without affecting Rvs161p binding. We propose that Fus2p/Rvs161p form a novel heterodimeric amphiphysin required for cell fusion. Rvs161p binding is required but not sufficient for Fus2p localization. Mutations in the C-terminal domain (CTD) of Fus2p block localization, but not Rvs161p binding, causing a significant defect in cell fusion. We conclude that the Fus2p CTD mediates an additional, Rvs161p-independent interaction at the shmoo tip. PMID:26681517

  19. A Fast Method of Transforming Relaxation Functions Into the Frequency Domain

    PubMed Central

    Mopsik, Frederick I.

    1999-01-01

    The limits to the error due to truncation of the numeric integration of the one-sided Laplace transform of a relaxation function in the time domain into its equivalent frequency domain are established. Separate results are given for large and small ω. These results show that, for a given ω, only a restricted range of time samples is needed to perform the computation to a given accuracy. These results are then combined with a known error estimate for integration by cubic splines to give a good estimate for the number of points needed to perform the computation to a given accuracy. For a given data window between t1 and t2, the computation time is shown to be proportional to ln(t1/t2).

  20. SMP-domain proteins at membrane contact sites: Structure and function.

    PubMed

    Reinisch, Karin M; De Camilli, Pietro

    2016-08-01

    SMP-domains are found in proteins that localize to membrane contact sites. Elucidation of the properties of these proteins gives clues as to the molecular bases underlying processes that occur at such sites. Described here are recent discoveries concerning the structure, function, and regulation of the Extended-Synaptotagmin proteins and ERMES complex subunits, SMP-domain proteins at endoplasmic reticulum (ER)-plasma membrane and ER-mitochondrial contacts, respectively. They act as tethers contributing to the architecture of these sites and as lipid transporters that convey glycerolipids between apposed membranes. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon. PMID:26686281

  1. Green function method for the time domain simulation of pulse propagation.

    PubMed

    Huang, Jing; Yao, Jianquan; Xu, Degang; Li, Runhua

    2014-06-01

    Based on the Green function method, the nonlinear Schrödinger equation is directly solved in the time domain (without Fourier transform). Because the dispersion and nonlinear effects are calculated simultaneously, it does not bring any spurious effect such as the split-step method in which the step size has to be carefully controlled by an error estimation. By this time domain solution, the pulse fission is analyzed, and we obtain the relationship between the minimum T₀ (the half-width at 1/e-intensity point of a pulse) and dispersion coefficients (β₂, β₃, and β₄). Thus the concrete dispersion values, which have an impact on ultrashort pulses (the quantity units is femtosecond or attosecond), are listed. It has been demonstrated that pulse fission occurs in the normal and anomalous dispersion regimes, even though fourth-order dispersion and the fifth-order nonlinear effects are not taken into account. PMID:24922431

  2. 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

  3. Cardiovascular functioning, personality, and the social world: the domain of hierarchical power.

    PubMed

    Newton, Tamara L

    2009-02-01

    The present paper considers connections between cardiovascular functioning (i.e., disease status and acute stress responses) and social dominance, and its counterpart, social submissiveness, both of which are part of the broader domain of "hierarchical power" [Bugental, D.B., 2000. Acquisition of the algorithms of social life: a domain-based approach. Psychological Bulletin 126, 187-219]. Empirical research on connections between dominance/submissiveness and cardiovascular morbidity and mortality in humans is reviewed, as is research on dominance/submissiveness and cardiovascular reactivity to, and recovery from, acute stressors. Three general conclusions are established. First, in both cross-sectional and longitudinal investigations, trait and behavioral indicators of dominance have been positively associated with cardiovascular disease severity, incidence, and progression, whereas preliminary evidence from two studies suggests that trait submissiveness may protect against poorer disease outcomes. Second, among men and women, trait dominance is associated with reactivity to and recovery from acute stressors, particularly social challenges. Third, linkages between dominance/submissiveness and cardiovascular functioning, especially cardiovascular reactivity, are characterized by gender-specific patterning, and this patterning emerges as a function of social context. Implications for the next generation of research concerning social dominance, gender, and cardiovascular functioning are discussed. PMID:18706926

  4. The Structurally Plastic CH2 Domain Is Linked to Distinct Functions of Fimbrins/Plastins.

    PubMed

    Zhang, Ruihui; Chang, Ming; Zhang, Meng; Wu, Youjun; Qu, Xiaolu; Huang, Shanjin

    2016-08-19

    Fimbrins/plastins have been implicated in the generation of distinct actin structures, which are linked to different cellular processes. Historically, fimbrins/plastins were mainly considered as generating tight actin bundles. Here, we demonstrate that different members of the fimbrin/plastin family have diverged biochemically during evolution to generate either tight actin bundles or loose networks with distinct biochemical and biophysical properties. Using the phylogenetically and functionally distinct Arabidopsis fimbrins FIM4 and FIM5 we found that FIM4 generates both actin bundles and cross-linked actin filaments, whereas FIM5 only generates actin bundles. The distinct functions of FIM4 and FIM5 are clearly observed at single-filament resolution. Domain swapping experiments showed that cooperation between the conformationally plastic calponin-homology domain 2 (CH2) and the N-terminal headpiece determines the function of the full-length protein. Our study suggests that the structural plasticity of fimbrins/plastins has biologically meaningful consequences, and provides novel insights into the structure-function relationship of fimbrins/plastins as well as shedding light on how cells generate distinct actin structures. PMID:27261463

  5. Site-directed mutants of human RECQ1 reveal functional importance of the zinc binding domain.

    PubMed

    Sami, Furqan; Gary, Ronald K; Fang, Yayin; Sharma, Sudha

    2016-08-01

    RecQ helicases are a highly conserved family of ATP-dependent DNA-unwinding enzymes with key roles in DNA replication and repair in all kingdoms of life. The RECQ1 gene encodes the most abundant RecQ homolog in humans. We engineered full-length RECQ1 harboring point mutations in the zinc-binding motif (amino acids 419-480) within the conserved RecQ-specific-C-terminal (RQC) domain known to be critical for diverse biochemical and cellular functions of RecQ helicases. Wild-type RECQ1 contains a zinc ion. Substitution of three of the four conserved cysteine residues that coordinate zinc severely impaired the ATPase and DNA unwinding activities but retained DNA binding and single strand DNA annealing activities. Furthermore, alteration of these residues attenuated zinc binding and significantly changed the overall conformation of full-length RECQ1 protein. In contrast, substitution of cysteine residue at position 471 resulted in a wild-type like RECQ1 protein. Differential contribution of the conserved cysteine residues to the structure and functions of the RECQ1 protein is also inferred by homology modeling. Overall, our results indicate that the zinc binding motif in the RQC domain of RECQ1 is a key structural element that is essential for the structure-functions of RECQ1. Given the recent association of RECQ1 mutations with breast cancer, these results will contribute to understanding the molecular basis of RECQ1 functions in cancer etiology. PMID:27248010

  6. Functional Difference between N Domain and C Domain of hEGF and hTGF-alpha.

    PubMed

    Yuan, Yu; Zhang, Qian; Huang, Pei-Yong; Wang, Yi-Fei; Zhou, Qing-Wei; Gan, Ren-Bao; Li, Zai-Ping

    1999-01-01

    By exchanging the N domain and C domain of hEGF and hTGF-alpha genes by PCR, two chimeras E-TGF(EGF(1-32)-TGF-alpha(34-50))and T-EGF(TGF-alpha(1-33)-EGF(33-53))were constructed. The wild and chimeric molecules were expressed in E.coli under phoA system. The expressed hEGF, hTGF-alpha and two chimeras were purified. The EGF receptor competitive binding affinity of the four molecules was hEGF > hTGF-alpha and E-TGF > T-EGF and the cell proliferation stimulating activity of them was hTGF-alpha and E-TGF > T-EGF > hEGF. The result suggests that the N domain of hEGF and hTGF-alpha may play a major role in receptor binding activity and C domain of them may be responsible for stimulating cell proliferation. PMID:12114963

  7. Systematic analysis of genes required for synapse structure and function.

    PubMed

    Sieburth, Derek; Ch'ng, QueeLim; Dybbs, Michael; Tavazoie, Masoud; Kennedy, Scott; Wang, Duo; Dupuy, Denis; Rual, Jean-François; Hill, David E; Vidal, Marc; Ruvkun, Gary; Kaplan, Joshua M

    2005-07-28

    Chemical synapses are complex structures that mediate rapid intercellular signalling in the nervous system. Proteomic studies suggest that several hundred proteins will be found at synaptic specializations. Here we describe a systematic screen to identify genes required for the function or development of Caenorhabditis elegans neuromuscular junctions. A total of 185 genes were identified in an RNA interference screen for decreased acetylcholine secretion; 132 of these genes had not previously been implicated in synaptic transmission. Functional profiles for these genes were determined by comparing secretion defects observed after RNA interference under a variety of conditions. Hierarchical clustering identified groups of functionally related genes, including those involved in the synaptic vesicle cycle, neuropeptide signalling and responsiveness to phorbol esters. Twenty-four genes encoded proteins that were localized to presynaptic specializations. Loss-of-function mutations in 12 genes caused defects in presynaptic structure. PMID:16049479

  8. Functional domains of a pore-forming cardiotoxic protein, volvatoxin A2.

    PubMed

    Weng, Yui-Ping; Lin, Ya-Ping; Hsu, Chyong-Ing; Lin, Jung-Yaw

    2004-02-20

    Volvatoxin A2 (VVA2), a novel pore-forming cardiotoxic protein was isolated from the mushroom Volvariella volvacea. We identified an N-terminal fragment (NTF) (1-127 residues) of VVA2 as a domain for oligomerization by limited tryptic digestion. On preincubation of NTF with VVA2, NTF was found to inhibit VVA2 hemolytic activity by inducing VVA2 oligomerization in the solution in the same manner as liposomes. By site-directed mutagenesis, the amphipathic alpha-helix B of NTF or VVA2 was shown to be indispensable for its biological functions. Interestingly, at a molar ratio of recombinant NTF (reNTF)/VVA2 as low as 0.01, reNTF was able to inhibit VVA2 hemolytic activity and induce VVA2 oligomerization. This indicates that reNTF can trigger VVA2 oligomerization by a seeding effect. Furthermore, the recombinant C-terminal fragment (128-199 residues) was found to be a functional domain that mediates the membrane binding of VVA2. We found a fragment localized at the C-terminal half of VVA2 containing beta6, -7, and -8, which is protected from protease digestion because of its insertion of a membrane. We also identified a putative heparin binding site (HBS) located in the VVA2 C terminus (166-194 residues), which was conserved among 10 kinds of snake venom cardiotoxins. VVA2 or the reHBS fragment was shown to interact with sulfated glycoaminoglycans by affinity column chromatography. The finding of a higher number of glycoaminoglycans in the membrane of cardiac myocytes suggested that they could be the specific membrane target for VVA2. Taken together, these findings indicate that VVA2 contains two functional domains, NTF and CTF. The NTF domain is responsible for VVA2 oligomerization and the CTF domain for membrane binding and insertion. Our results support a model whereby the formation of VVA2 oligomeric pre-pore complexes precedes their membrane insertion. PMID:14645370

  9. Advanced flight deck/crew station simulator functional requirements

    NASA Technical Reports Server (NTRS)

    Wall, R. L.; Tate, J. L.; Moss, M. J.

    1980-01-01

    This report documents a study of flight deck/crew system research facility requirements for investigating issues involved with developing systems, and procedures for interfacing transport aircraft with air traffic control systems planned for 1985 to 2000. Crew system needs of NASA, the U.S. Air Force, and industry were investigated and reported. A matrix of these is included, as are recommended functional requirements and design criteria for simulation facilities in which to conduct this research. Methods of exploiting the commonality and similarity in facilities are identified, and plans for exploiting this in order to reduce implementation costs and allow efficient transfer of experiments from one facility to another are presented.

  10. Structural Elements in the Transmembrane and Cytoplasmic Domains of the Metal Transporter SLC30A10 Are Required for Its Manganese Efflux Activity.

    PubMed

    Zogzas, Charles E; Aschner, Michael; Mukhopadhyay, Somshuvra

    2016-07-29

    Homozygous mutations in SLC30A10 lead to the development of familial manganese-induced parkinsonism. We previously demonstrated that SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its trafficking and efflux activity. Interestingly, other transporters in the SLC30 family mediate zinc efflux. Determining the mechanisms that allow SLC30A10 to transport manganese, which are unclear, is essential to understand its role in parkinsonism. Here, we generated a predicted structure of SLC30A10, based on the structure of the bacterial zinc transporter YiiP, and performed functional studies. In YiiP, side chains of residues Asp-45 and Asp-49 in the second and His-153 and Asp-157 in the fifth transmembrane segments coordinate zinc and are required for transport. In SLC30A10, the corresponding residues are Asn-43 and Asp-47 in the second and His-244 and Asp-248 in the fifth transmembrane segments. Surprisingly, although alanine substitution of Asp-248 abolished manganese efflux, that of Asn-43 and Asp-47 did not. Instead, side chains of charged or polar residues adjacent to Asp-248 in the first (Glu-25) or fourth (Asn-127) transmembrane segments were required. Further analyses revealed that residues His-333 and His-350 in the cytoplasmic C-terminal domain were required for full activity. However, the C-terminal domain failed to transfer manganese transport capability to a related zinc transporter. Overall, our results indicate that residues in the transmembrane and C-terminal domains together confer optimal manganese transport capability to SLC30A10 and suggest that the mechanism of ion coordination in the transmembrane domain of SLC30A10 may be substantially different from that in YiiP/other SLC30 proteins. PMID:27307044

  11. pRB-Dependent, J Domain-Independent Function of Simian Virus 40 Large T Antigen in Override of p53 Growth Suppression

    PubMed Central

    Gjoerup, Ole; Chao, Herta; DeCaprio, James A.; Roberts, Thomas M.

    2000-01-01

    Simian virus 40 (SV40) large T antigen (LT) can immortalize and transform many cell types. These activities are attributed in large part to the binding and functional inactivation by LT of two major tumor suppressors: p53 and the retinoblastoma protein, pRB. Most effects of LT on pRB have been shown to additionally require an intact J domain, which mediates binding to Hsc70. We show here that the J domain is not required for p53 override in full-length LT. Although LT binds p53, it was shown previously that overcoming a p53-induced cell cycle arrest requires binding to pRB family members (R. S. Quartin et al., J. Virol. 68:1334–1341). We demonstrate that an LT mutant defective for pRB family member binding (K1) can be complemented for efficient override of p53 arrest by a construct encoding the first 135 amino acids of LT with a J domain-inactivating mutation, H42Q. Hence, complementation does not require the J domain, and pRB binding by LT is important for more than dissociating pRB-E2F complexes, which is J dependent. In accordance with this notion, LT alleviates pRB small-pocket-mediated transcriptional repression independently of the J domain. The LT K1 mutant can also be complemented for p53 override by small t antigen (st) in a manner independent of its J domain. Our observations underscore the importance of multiple SV40 functions, two in LT and one in st, that act cooperatively to counteract p53 growth suppression. PMID:10623749

  12. The crystal structure of human IRE1 luminal domain reveals a conserved dimerization interface required for activation of the unfolded protein response

    SciTech Connect

    Zhou, Jiahai; Liu, Chuan Yin; Back, Sung Hoon; Clark, Robert L.; Peisach, Daniel; Xu, Zhaohui; Kaufman, Randal J.

    2010-03-08

    The unfolded protein response (UPR) is an evolutionarily conserved mechanism by which all eukaryotic cells adapt to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring kinase 1 (IRE1) and PKR-related ER kinase (PERK) are two type I transmembrane ER-localized protein kinase receptors that signal the UPR through a process that involves homodimerization and autophosphorylation. To elucidate the molecular basis of the ER transmembrane signaling event, we determined the x-ray crystal structure of the luminal domain of human IRE1{alpha}. The monomer of the luminal domain comprises a unique fold of a triangular assembly of {beta}-sheet clusters. Structural analysis identified an extensive dimerization interface stabilized by hydrogen bonds and hydrophobic interactions. Dimerization creates an MHC-like groove at the interface. However, because this groove is too narrow for peptide binding and the purified luminal domain forms high-affinity dimers in vitro, peptide binding to this groove is not required for dimerization. Consistent with our structural observations, mutations that disrupt the dimerization interface produced IRE1{alpha} molecules that failed to either dimerize or activate the UPR upon ER stress. In addition, mutations in a structurally homologous region within PERK also prevented dimerization. Our structural, biochemical, and functional studies in vivo altogether demonstrate that IRE1 and PERK have conserved a common molecular interface necessary and sufficient for dimerization and UPR signaling.

  13. Simulation domain size requirements for elastic response of 3D polycrystalline materials

    NASA Astrophysics Data System (ADS)

    Ozturk, Tugce; Stein, Clayton; Pokharel, Reeju; Hefferan, Christopher; Tucker, Harris; Jha, Sushant; John, Reji; Lebensohn, Ricardo A.; Kenesei, Peter; Suter, Robert M.; Rollett, Anthony D.

    2016-01-01

    A fast Fourier transform (FFT) based spectral algorithm is used to compute the full field mechanical response of polycrystalline microstructures. The field distributions in a specific region are used to determine the sensitivity of the method to the number of surrounding grains through quantification of the divergence of the field values from the largest simulation domain, as successively smaller surrounding volumes are included in the simulation. The analysis considers a mapped 3D structure where the location of interest is taken to be a particular pair of surface grains that enclose a small fatigue crack, and synthetically created statistically representative microstructures to further investigate the effect of anisotropy, loading condition, loading direction, and texture. The synthetic structures are generated via DREAM3D and the measured material is a cyclically loaded, Ni-based, low solvus high refractory (LSHR) superalloy that was characterized via 3D high energy x-ray diffraction microscopy (HEDM). Point-wise comparison of distributions in the grain pairs shows that, in order to obtain a Pearson correlation coefficient larger than 99%, the domain must extend to at least the third nearest neighbor. For an elastic FFT calculation, the stress-strain distributions are not sensitive to the shape of the domain. The main result is that convergence can be specified in terms of the number of grains surrounding a region of interest.

  14. Dimerization of the DYT6 dystonia protein, THAP1, requires residues within the coiled-coil domain.

    PubMed

    Sengel, Cem; Gavarini, Sophie; Sharma, Nutan; Ozelius, Laurie J; Bragg, D Cristopher

    2011-09-01

    Thanatos-associated [THAP] domain-containing apoptosis-associated protein 1 (THAP1) is a DNA-binding protein that has been recently associated with DYT6 dystonia, a hereditary movement disorder involving sustained, involuntary muscle contractions. A large number of dystonia-related mutations have been identified in THAP1 in diverse patient populations worldwide. Previous reports have suggested that THAP1 oligomerizes with itself via a C-terminal coiled-coil domain, raising the possibility that DYT6 mutations in this region might affect this interaction. In this study, we examined the ability of wild-type THAP1 to bind itself and the effects on this interaction of the following disease mutations: C54Y, F81L, ΔF132, T142A, I149T, Q154fs180X, and A166T. The results confirmed that wild-type THAP1 associated with itself and most of the DYT6 mutants tested, except for the Q154fs180X variant, which loses most of the coiled-coil domain because of a frameshift at position 154. However, deletion of C-terminal residues after position 166 produced a truncated variant of THAP1 that was able to bind the wild-type protein. The interaction of THAP1 with itself therefore required residues within a 13-amino acid region (aa 154-166) of the coiled-coil domain. Further inspection of this sequence revealed elements highly consistent with previous descriptions of leucine zippers, which serve as dimerization domains in other transcription factor families. Based on this similarity, a structural model was generated to predict how hydrophobic residues in this region may mediate dimerization. These observations offer additional insight into the role of the coiled-coil domain in THAP1, which may facilitate future analyses of DYT6 mutations in this region. PMID:21752024

  15. Quality functions for requirements engineering in system development methods.

    PubMed

    Johansson, M; Timpka, T

    1996-01-01

    Based on a grounded theory framework, this paper analyses the quality characteristics for methods to be used for requirements engineering in the development of medical decision support systems (MDSS). The results from a Quality Function Deployment (QFD) used to rank functions connected to user value and a focus group study were presented to a validation focus group. The focus group studies take advantage of a group process to collect data for further analyses. The results describe factors considered by the participants as important in the development of methods for requirements engineering in health care. Based on the findings, the content which, according to the user a MDSS method should support is established. PMID:8947891

  16. Functional Requirements for Information Resource Provenance on the Web

    SciTech Connect

    McCusker, James P.; Lebo, Timothy; Graves, Alvaro; Difranzo, Dominic; Pinheiro da Silva, Paulo; McGuinness, Deborah L.

    2012-06-19

    We provide a means to formally explain the relationship between HTTP URLs and the representations returned when they are requested. According to existing World Wide Web architecture, the URL serves as an identier for a semiotic referent while the document returned via HTTP serves as a representation of the same referent. This begins with two sides of a semiotic triangle; the third side is the relationship between the URL and the representation received. We complete this description by extending the library science resource model Functional Requirements for Bibliographic Resources (FRBR) with cryptographic message and content digests to create a Functional Requirements for Information Resources (FRIR). We show how applying the FRIR model to HTTP GET and POST transactions disambiguates the many relationships between a given URL and all representations received from its request, provides fine-grained explanations that are complementary to existing explanations of web resources, and integrates easily into the emerging W3C provenance standard.

  17. Functional Analysis of the Leading Malaria Vaccine Candidate AMA-1 Reveals an Essential Role for the Cytoplasmic Domain in the Invasion Process

    PubMed Central

    Treeck, Moritz; Zacherl, Sonja; Herrmann, Susann; Cabrera, Ana; Kono, Maya; Struck, Nicole S.; Engelberg, Klemens; Haase, Silvia; Frischknecht, Friedrich; Miura, Kota; Spielmann, Tobias; Gilberger, Tim W.

    2009-01-01

    A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies. PMID:19283086

  18. Microtubule-Actin Cross-Linking Factor 1: Domains, Interaction Partners, and Tissue-Specific Functions.

    PubMed

    Goryunov, Dmitry; Liem, Ronald K H

    2016-01-01

    The cytoskeleton of most eukaryotic cells is composed of three principal filamentous components: actin filaments, microtubules (MTs), and intermediate filaments. It is a highly dynamic system that plays crucial roles in a wide range of cellular processes, including migration, adhesion, cytokinesis, morphogenesis, intracellular traffic and signaling, and structural flexibility. Among the large number of cytoskeleton-associated proteins characterized to date, microtubule-actin cross-linking factor 1 (MACF1) is arguably the most versatile integrator and modulator of cytoskeleton-related processes. MACF1 belongs to the plakin family of proteins, and within it, to the spectraplakin subfamily. These proteins are characterized by the ability to bridge MT and actin cytoskeletal networks in a dynamic fashion, which underlies their involvement in the regulation of cell migration, axonal extension, and vesicular traffic. Studying MACF1 functions has provided insights not only into the regulation of the cytoskeleton but also into molecular mechanisms of both normal cellular physiology and cellular pathology. Multiple MACF1 isoforms exist, composed of a large variety of alternatively spliced domains. Each of these domains mediates a specific set of interactions and functions. These functions are manifested in tissue and cell-specific phenotypes observed in conditional MACF1 knockout mice. The conditional models described to date reveal critical roles of MACF1 in mammalian skin, nervous system, heart muscle, and intestinal epithelia. Complete elimination of MACF1 is early embryonic lethal, indicating an essential role for MACF1 in early development. Further studies of MACF1 domains and their interactions will likely reveal multiple new roles of this protein in various tissues. PMID:26778566

  19. The InterPro database, an integrated documentation resource for protein families, domains and functional sites.

    PubMed

    Apweiler, R; Attwood, T K; Bairoch, A; Bateman, A; Birney, E; Biswas, M; Bucher, P; Cerutti, L; Corpet, F; Croning, M D; Durbin, R; Falquet, L; Fleischmann, W; Gouzy, J; Hermjakob, H; Hulo, N; Jonassen, I; Kahn, D; Kanapin, A; Karavidopoulou, Y; Lopez, R; Marx, B; Mulder, N J; Oinn, T M; Pagni, M; Servant, F; Sigrist, C J; Zdobnov, E M

    2001-01-01

    Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1,000,000 hits from 462,500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk. PMID:11125043

  20. The InterPro database, an integrated documentation resource for protein families, domains and functional sites

    PubMed Central

    Apweiler, R.; Attwood, T. K.; Bairoch, A.; Bateman, A.; Birney, E.; Biswas, M.; Bucher, P.; Cerutti, L.; Corpet, F.; Croning, M. D. R.; Durbin, R.; Falquet, L.; Fleischmann, W.; Gouzy, J.; Hermjakob, H.; Hulo, N.; Jonassen, I.; Kahn, D.; Kanapin, A.; Karavidopoulou, Y.; Lopez, R.; Marx, B.; Mulder, N. J.; Oinn, T. M.; Pagni, M.; Servant, F.; Sigrist, C. J. A.; Zdobnov, E. M.

    2001-01-01

    Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1 000 000 hits from 462 500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk. PMID:11125043

  1. Seventh Graders' Academic Achievement, Creativity, and Ability to Construct a Cross-Domain Concept Map--A Brain Function Perspective

    ERIC Educational Resources Information Center

    Yeh, Yu-Chu

    2004-01-01

    This study proposes an interactive model of "cross-domain" concept mapping with an emphasis on brain functions, and it further investigates the relationships between academic achievement, creative thinking, and cross-domain concept mapping. Sixty-nine seventh graders participated in this study which employed two 50-minute instructional sessions.…

  2. Assessment of the Social Functions and Vitality of the Yi Language from the Perspective of Its Domains of Use

    ERIC Educational Resources Information Center

    Suhua, Hu

    2008-01-01

    In general, the vitality and social functions of a language are assessed in connection with such indices as the language's intergenerational transmission, the absolute number of speakers and proportion of speakers in the population, its present domains of use, its development within diverse domains of use, and the availability of its educational…

  3. Functional dissection of the Pax6 paired domain: Roles in neural tube patterning and peripheral nervous system development.

    PubMed

    Huettl, Rosa-Eva; Eckstein, Simone; Stahl, Tessa; Petricca, Stefania; Ninkovic, Jovica; Götz, Magdalena; Huber, Andrea B

    2016-05-01

    During development of the CNS, stem and progenitor cell proliferation, cell fate designation, and patterning decisions are tightly regulated by interdependent networks of key transcriptional regulators. In a genetic approach we analyzed divergent functionality of the PAI and RED sub-domains of the Pax6 Paired domain (PD) during progenitor zone formation, motor and interneuron development, and peripheral connectivity at distinct levels within the neural tube: within the hindbrain, mutation of the PAI sub-domain severely affected patterning of the p3 and pMN domains and establishment of the corresponding motor neurons. Exit point designation of hypoglossal axons was disturbed in embryos harboring either mutations in the PD sub-domains or containing a functional Pax6 Null allele. At brachial spinal levels, we propose a selective involvement of the PAI sub-domain during patterning of ventral p2 and pMN domains, critically disturbing generation of specific motor neuron subtypes and increasing V2 interneuron numbers. Our findings present a novel aspect of how Pax6 not only utilizes its modular structure to perform distinct functions via its paired and homeodomain. Individual sub-domains can exert distinct functions, generating a new level of complexity for transcriptional regulation by one single transcription factor not only in dorso-ventral, but also rostro-caudal neural tube patterning. PMID:26187199

  4. Structural Conservation and Functional Diversity of the Poxvirus Immune Evasion (PIE) Domain Superfamily

    PubMed Central

    Nelson, Christopher A.; Epperson, Megan L.; Singh, Sukrit; Elliott, Jabari I.; Fremont, Daved H.

    2015-01-01

    Poxviruses encode a broad array of proteins that serve to undermine host immune defenses. Structural analysis of four of these seemingly unrelated proteins revealed the recurrent use of a conserved beta-sandwich fold that has not been observed in any eukaryotic or prokaryotic protein. Herein we propose to call this unique structural scaffolding the PIE (Poxvirus Immune Evasion) domain. PIE domain containing proteins are abundant in chordopoxvirinae, with our analysis identifying 20 likely PIE subfamilies among 33 representative genomes spanning 7 genera. For example, cowpox strain Brighton Red appears to encode 10 different PIEs: vCCI, A41, C8, M2, T4 (CPVX203), and the SECRET proteins CrmB, CrmD, SCP-1, SCP-2, and SCP-3. Characterized PIE proteins all appear to be nonessential for virus replication, and all contain signal peptides for targeting to the secretory pathway. The PIE subfamilies differ primarily in the number, size, and location of structural embellishments to the beta-sandwich core that confer unique functional specificities. Reported ligands include chemokines, GM-CSF, IL-2, MHC class I, and glycosaminoglycans. We expect that the list of ligands and receptors engaged by the PIE domain will grow as we come to better understand how this versatile structural architecture can be tailored to manipulate host responses to infection. PMID:26343707

  5. The nuclear factor SPBP contains different functional domains and stimulates the activity of various transcriptional activators.

    PubMed

    Rekdal, C; Sjøttem, E; Johansen, T

    2000-12-22

    SPBP (stromelysin-1 platelet-derived growth factor-responsive element binding protein) was originally cloned from a cDNA expression library by virtue of its ability to bind to a platelet-derived growth factor-responsive element in the human stromelysin-1 promoter. A 937-amino acid-long protein was deduced from a 3995-nucleotide murine cDNA sequence. By analyses of both human and murine cDNAs, we now show that SPBP is twice as large as originally found. The human SPBP gene contains six exons and is located on chromosome 22q13.1-13.3. Two isoforms differing in their C termini are expressed due to alternative splicing. PCR analyses of multitissue cDNA panels showed that SPBP is expressed in most tissues except for ovary and prostate. Functional mapping revealed that SPBP is a nuclear, multidomain protein containing an N-terminal region with transactivating ability, a novel type of DNA-binding domain containing an AT hook motif, and a bipartite nuclear localization signal as well as a C-terminal zinc finger domain. This type of zinc finger domain is also found in the trithorax family of chromatin-based transcriptional regulator proteins. Using cotransfection experiments, we find that SPBP enhances the transcriptional activity of various transcription factors such as c-Jun, Ets1, Sp1, and Pax6. Hence, SPBP seems to act as a transcriptional coactivator. PMID:10995766

  6. Time domain investigation of transceiver functions using a known reference target.

    PubMed

    Feuillade, C; Meredith, R W; Chotiros, N P; Clay, C S

    2002-12-01

    During August 1998, a bottom scattering tank experiment was performed at the Applied Research Laboratory, University of Texas to measure wideband acoustic reverberation from multiple objects (e.g., cobbles and pebbles) placed on a sediment simulation of the sea floor. In preparation for processing and analyzing the experimental data, time domain scattering measurements made with stainless steel and glass balls suspended in the water column were used to calibrate the sonar transceiver system by deconvolving the theoretical impulse response for steel and glass spheres, obtained via the Faran elastic sphere scattering model, from the scattered time signals. It is the analysis of these calibration measurements which forms the subject of this paper. Results show the critical importance of accurate input-output system calibrations for time domain sound scattering research, and successfully demonstrate a time domain method for accurately calibrating the complete sonar transceiver function, i.e., both the amplitude and time dependence, using a known reference target. The work has implications for boundary and volume scattering applications. PMID:12508990

  7. Special functions arising in the study of semi-linear equations in circular domains

    NASA Astrophysics Data System (ADS)

    Varlamov, Vladimir

    2007-05-01

    Rayleigh functions are defined by the formulawhere are zeros of the Bessel function J[nu](x) and n=1,2,3,..., is the number of the zero. These functions appear in the classical problems of vibrating circular membranes, heat conduction in cylinders and diffraction through circular apertures. In the present paper it is shown that a new family of special functions, convolutions of Rayleigh functions with respect to the Bessel index,arises in constructing solutions of semi-linear evolution equations in circular domains (see also [V. Varlamov, Convolution of Rayleigh functions with respect to the Bessel index, J. Math. Anal. Appl. 306 (2005) 413-424]). As an example of its application a forced Cahn-Hilliard equation is considered in a unit disc with homogeneous boundary and initial conditions. Construction of its global-in-time solutions involves the use of R1(m) and R2(m). A general representation of Rl(m) is deduced and on the basis of that a particular result for R2(m) is obtained convenient for computing its asymptotics as m-->[infinity]. The latter issue is important for establishing a function space to which a solution of the corresponding problem belongs.

  8. Functional characterization of spectrin-actin-binding domains in 4.1 family of proteins.

    PubMed

    Gimm, J Aura; An, Xiuli; Nunomura, Wataru; Mohandas, Narla

    2002-06-11

    Protein 4.1R is the prototypical member of a protein family that includes 4.1G, 4.1B, and 4.1N. 4.1R plays a crucial role in maintaining membrane mechanical integrity by binding cooperatively to spectrin and actin through its spectrin-actin-binding (SAB) domain. While the binary interaction between 4.1R and spectrin has been well characterized, the actin binding site in 4.1R remains unidentified. Moreover, little is known about the interaction of 4.1R homologues with spectrin and actin. In the present study, we showed that the 8 aa motif (LKKNFMES) within the 10 kDa spectrin-actin-binding domain of 4.1R plays a critical role in binding of 4.1R to actin. Recombinant 4.1R SAB domain peptides with mutations in this motif showed a marked decrease in their ability to form ternary complexes with spectrin and actin. Binary protein-protein interaction studies revealed that this decrease resulted from the inability of mutant SAB peptides to bind to actin filaments while affinity for spectrin was unchanged. We also documented that the 14 C-terminal residues of the 21 amino acid cassette encoded by exon 16 in conjunction with residues 27-43 encoded by exon 17 constituted a fully functional minimal spectrin-binding motif. Finally, we showed that 4.1N SAB domain was unable to form a ternary complex with spectrin and actin, while 4.1G and 4.1B SAB domains were able to form such a complex but less efficiently than 4.1R SAB. This was due to a decrease in the ability of 4.1G and 4.1B SAB domain to interact with actin but not with spectrin. These data enabled us to propose a model for the 4.1R-spectrin-actin ternary complex which may serve as a general paradigm for regulation of spectrin-based cytoskeleton interaction in various cell types. PMID:12044158

  9. Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa.

    PubMed

    Bai, Erdeni; Rosell, Federico I; Lige, Bao; Mauk, Marcia R; Lelj-Garolla, Barbara; Moore, Geoffrey R; Mauk, A Grant

    2006-12-15

    The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (K(A)) of 10(+/-7)x10(6), 5.7(+/-3)x10(6), 2.0(+/-2)x10(6) and 2.0(+/-3)x10(4) M(-1) for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(+/-2)x10(6), 3.2(+/-2)x10(4), 1.76(+/-1)x10(5) and 1.5(+/-2)x10(3) M(-1) respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 degrees C). The stability of metal ion binding to the sensory site follows the Irving-Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents. PMID:16928194

  10. Dissection of Rad9 BRCT Domain Function In The Mitotic Checkpoint Response To Telomere Uncapping

    PubMed Central

    Nnakwe, Chinonye C.; Altaf, Mohammed; Côté, Jacques; Kron, Stephen J.

    2009-01-01

    In Saccharomyces cerevisiae, destabilizing telomeres, via inactivation of telomeric repeat binding factor Cdc13, induces a cell cycle checkpoint that arrests cells at the metaphase to anaphase transition—much like the response to an unrepaired DNA double-strand break (DSB). Throughout the cell cycle, the multi-domain adaptor protein Rad9 is required for activation of checkpoint effector kinase Rad53 in response to DSBs and is similarly necessary for checkpoint signaling in response to telomere uncapping. Rad53 activation in G1 and S phase depends on Rad9 association with modified chromatin adjacent to DSBs, which is mediated by Tudor domains binding histone H3 di-methylated at K79 and BRCT domains to histone H2A phosphorylated at S129. Nonetheless, Rad9 Tudor or BRCT mutants can initiate a checkpoint response to DNA damage in nocodazole-treated cells. Mutations affecting di-methylation of H3 K79, or its recognition by Rad9 enhance 5' strand resection upon telomere uncapping, and potentially implicate Rad9 chromatin binding in the checkpoint response to telomere uncapping. Indeed, we report that Rad9 binds to sub-telomeric chromatin, upon telomere uncapping, up to 10 kb from the telomere. Rad9 binding occurred within 30 min after inactivating Cdc13, preceding Rad53 phosphorylation. In turn, Rad9 Tudor and BRCT domain mutations blocked chromatin binding and led to attenuated checkpoint signaling as evidenced by decreased Rad53 phosphorylation and impaired cell cycle arrest. Our work identifies a role for Rad9 chromatin association, during mitosis, in the DNA damage checkpoint response to telomere uncapping, suggesting that chromatin binding may be an initiating event for checkpoints throughout the cell cycle. PMID:19880356

  11. Time Domain Identification of an Optimal Control Pilot Model with Emphasis on the Objective Function

    NASA Technical Reports Server (NTRS)

    Schmidt, D. K.

    1982-01-01

    A method for the identification of the pilot's control compensation using time domain techniques is proposed. From this information we hope to infer a quadratic cost function, supported by the data, that represents a reasonable expression for the pilot's control objective in the task being performed, or an inferred piloting strategy. The objectives for this method are: (1) obtain a better understanding of the fundamental piloting techniques in complex tasks, such as landing approach; (2) the development of a metric measurable in simulations and flight test that correlate with subjective pilot opinion; and (3) to further validate pilot models and pilot vehicle analysis methods.

  12. Inner products of Bethe states as partial domain wall partition functions

    NASA Astrophysics Data System (ADS)

    Kostov, Ivan; Matsuo, Yutaka

    2012-10-01

    We study the inner product of Bethe states in the inhomogeneous periodic XXX spin-1/2 chain of length L, which is given by the Slavnov determinant formula. We show that the inner product of an on-shell M -magnon state with a generic M -magnon state is given by the same expression as the inner product of a 2 M -magnon state with a vacuum descendent. The second inner product is proportional to the partition function of the six-vertex model on a rectangular L × 2 M grid, with partial domain-wall boundary conditions.

  13. Adapting ORAP to wind plants : industry value and functional requirements.

    SciTech Connect

    Not Available

    2010-08-01

    Strategic Power Systems (SPS) was contracted by Sandia National Laboratories to assess the feasibility of adapting their ORAP (Operational Reliability Analysis Program) tool for deployment to the wind industry. ORAP for Wind is proposed for use as the primary data source for the CREW (Continuous Reliability Enhancement for Wind) database which will be maintained by Sandia to enable reliability analysis of US wind fleet operations. The report primarily addresses the functional requirements of the wind-based system. The SPS ORAP reliability monitoring system has been used successfully for over twenty years to collect RAM (Reliability, Availability, Maintainability) and operations data for benchmarking and analysis of gas and steam turbine performance. This report documents the requirements to adapt the ORAP system for the wind industry. It specifies which existing ORAP design features should be retained, as well as key new requirements for wind. The latter includes alignment with existing and emerging wind industry standards (IEEE 762, ISO 3977 and IEC 61400). There is also a comprehensive list of thirty critical-to-quality (CTQ) functional requirements which must be considered and addressed to establish the optimum design for wind.

  14. Type I Interferon Signaling Is Decoupled from Specific Receptor Orientation through Lenient Requirements of the Transmembrane Domain.

    PubMed

    Sharma, Nanaocha; Longjam, Geeta; Schreiber, Gideon

    2016-02-12

    Type I interferons serve as the first line of defense against pathogen invasion. Binding of IFNs to its receptors, IFNAR1 and IFNAR2, is leading to activation of the IFN response. To determine whether structural perturbations observed during binding are propagated to the cytoplasmic domain, multiple mutations were introduced into the transmembrane helix and its surroundings. Insertion of one to five alanine residues near either the N or C terminus of the transmembrane domain (TMD) likely promotes a rotation of 100° and a translation of 1.5 Å per added residue. Surprisingly, the added alanines had little effect on the binding affinity of IFN to the cell surface receptors, STAT phosphorylation, or gene induction. Similarly, substitution of the juxtamembrane residues of the TMD with alanines, or replacement of the TMD of IFNAR1 with that of IFNAR2, did not affect IFN binding or activity. Finally, only the addition of 10 serine residues (but not 2 or 4) between the extracellular domain of IFNAR1 and the TMD had some effect on signaling. Bioinformatic analysis shows a correlation between high sequence conservation of TMDs of cytokine receptors and the ability to transmit structural signals. Sequence conservation near the TMD of IFNAR1 is low, suggesting limited functional importance for this region. Our results suggest that IFN binding to the extracellular domains of IFNAR1 and IFNAR2 promotes proximity between the intracellular domains and that differential signaling is a function of duration of activation and affinity of binding rather than specific conformational changes transmitted from the outside to the inside of the cell. PMID:26679999

  15. Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression

    SciTech Connect

    Wek, R.C.; Ramirez, M.; Jackson, B.M.; Hinnebusch, A.G. )

    1990-06-01

    GCN4 is a transcriptional activator of amino acid-biosynthetic genes in the yeast {ital Saccharomyces cerevisiae}. GCN2, a translational activator of {ital GCN4} expression, contains a domain homologous to the catalytic subunit of eukaryotic protein kinases. Substitution of a highly conserved lysine residue in the kinase domain abolished GCN2 regulatory function in vivo and its ability to autophosphorylate in vitro, indicating that GCN2 acts as a protein kinase in stimulating {ital GCN4} expression. Elevated {ital GCN2} gene dosage led to depression of {ital GCN4} under nonstarvation conditions; however, the authors found that {ital GCN2} mRNA and protein levels did not increase in wild-type cells in response to amino acid starvation. Therefore, it appears that GCN2 protein kinase function is stimulated postranslationally in amino acid-starved cells. Three dominant-constitutive {ital GCN2} point mutations were isolated that led to derepressed {ital GCN4} expression under nonstarvation conditions. Two of the {ital GCN2}(Con) mutations mapped in the kinase domain itself. The third mapped just downstream from a carboxyl-terminal segment homologous to histidyl-tRNA synthetase (HisRS), which the authors suggest might function to detect uncharged tRNA in amino acid-starved cells and activate the adjacent protein kinase moiety.

  16. Viral suppression function of intracellular antibody against C-terminal domain of rabies virus phosphoprotein.

    PubMed

    Liu, Yang; Sun, Lina; Yu, Pengcheng; Li, Aqian; Li, Chuan; Tang, Qing; Li, Dexin; Liang, Mifang

    2015-10-01

    Rabies virus (RV) causes a fatal disease in both human and animals. The disease can be prevented by post-exposure prophylaxis in individuals exposed to RV. However, the neutralization effect is limited after the virus enters into the host cells. So, it is important to identify new targets for rabies therapy. In this study, a human antibody RV1A2 specific to RV phosphoprotein (RV-P) was generated from a human naïve immune antibody library. The antibody recognized all forms of the phosphoproteins including the full length (P1) and short length of the P proteins (P2, P3, P4, and P5). The epitope mapping and the molecular docking of antigen-antibody complex showed that the antibody targets at a conserved epitope of 'VLGWV' ranging from amino acid (aa) 262 to 266 at C-terminal domain of the P protein, which locates at a hydrophobic pocket region in the C-terminal of the RV-P. The aa W265 within the epitope is on the flat surface of the domain, suggesting that it may be a critical amino acid for the functions of the P protein. Our results further showed that intracellular antibody RV1A2 which targets at the C-terminal domain of the P protein could effectively inhibit RV propagation 2-4 days post infection. These results suggest that the conserved C-terminal domain may be used as a new target for drug discovery, which highlights an intracellular inhibition of RV propagation and provides a potential novel way to treat RV infection. PMID:26188200

  17. Pegylation of fibronectin and its functional domains: Effect on stability and biological activity

    NASA Astrophysics Data System (ADS)

    Zhang, Chen

    Delayed wound healing in many chronic wounds has been linked to the lack of extracellular matrix (ECM) support and the degradation of fibronectin (FN) by an abnormally high protease level. The ECM provides physical and chemical cues that direct tissue growth and development while FN is a key ECM protein that attracts and binds different molecules and cells. The goal of my study is creating an ECM analogue based on a composite of polyethylene glycol (PEG) hydrogels and FN binding domains and stabilizing FN against proteolytic degradation by conjugating it to PEG. The work presented here shows a two-prong approach by which the problem of ECM degradation and deficiency chronic wound healing can be addressed. The first approach for addressing ECM deficiency is through a scaffold design methodology. The novelty of the scaffold approach is that it uses the cell-binding domains of FN instead of the often-used RGD peptide. I demonstrate that a PEG hydrogel with the cell-binding domain produces a more robust biological response in cells than a PEG hydrogel with the RGD peptide. I also demonstrate that varying different functional domains of FN can be used to controllably stimulate multiple biological responses. The second approach demonstrates a method by which FN, a key ECM protein, is stabilized against proteolytic degradation without perturbing its activity. These studies of creating PEG-FN conjugates are the first of their kind. Collectively, the data that I present in this thesis will lead to novel therapeutic methods for treating chronic wounds.

  18. Functional connectivity between tRNA binding domains in glutaminyl-tRNA synthetase.

    PubMed

    Sherman, J M; Thomann, H U; Söll, D

    1996-03-15

    The structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) in complex with tRNAGln and ATP has identified a number a sequence-specific protein-tRNA interactions. The contribution to glutamine identity has previously been determined for the nucleotides in tRNAGln. Here, we report the mutational analysis of residues in all three tRNA recognition domains of GlnRS, thus completing a survey of the major sequence-specific contacts between GlnRS and tRNAGln. Specifically, we analyzed the GlnRS determinants involved in recognition of the anticodon which is essential for glutamine identity and in the communication of anticodon recognition to the acceptor binding domain in GlnRS. A combined in vivo and in vitro approach has demonstrated that Arg341, which makes a single sequence-specific hydrogen bond with U35 in the anticodon of tRNAGln, is involved in initial RNA recognition and is an important positive determinant for this base in both cognate and non- cognate tRNA contexts. However, Arg341, as well as Arg402, which interacts with G36 in the anticodon, are negative determinants for non-cognate nucleotides at their respective positions. Analysis of acceptor-anticodon binding double mutants and of a mutation of Glu323 in the loop-strand-helix connectivity subdomain in GlnRS has further implicated this domain in the functional communication of anticodon recognition. The better than expected activity (anticooperativity) of these double mutants has led us to propose an "anticodon-independent" mechanism, in which the removal of certain synthetase interactions with the anticodon eliminates structural constraints, thus allowing the relaxed specificity mutants in the acceptor binding domain ot make more productive interactions. PMID:8601833

  19. Functional Domain Analysis of the Remorin Protein LjSYMREM1 in Lotus japonicus

    PubMed Central

    Madsen, Esben B.; Ye, Juanying; Popp, Claudia; Antolín-Llovera, Meritxell; Grossmann, Christina; Jensen, Ole N.; Schüßler, Arthur; Parniske, Martin; Ott, Thomas

    2012-01-01

    In legumes rhizobial infection during root nodule symbiosis (RNS) is controlled by a conserved set of receptor proteins and downstream components. MtSYMREM1, a protein of the Remorin family in Medicago truncatula, was shown to interact with at least three receptor-like kinases (RLKs) that are essential for RNS. Remorins are comprised of a conserved C-terminal domain and a variable N-terminal region that defines the six different Remorin groups. While both N- and C-terminal regions of Remorins belonging to the same phylogenetic group are similar to each other throughout the plant kingdom, the N-terminal domains of legume-specific group 2 Remorins show exceptional high degrees of sequence divergence suggesting evolutionary specialization of this protein within this clade. We therefore identified and characterized the MtSYMREM1 ortholog from Lotus japonicus (LjSYMREM1), a model legume that forms determinate root nodules. Here, we resolved its spatio-temporal regulation and showed that over-expression of LjSYMREM1 increases nodulation on transgenic roots. Using a structure-function approach we show that protein interactions including Remorin oligomerization are mainly mediated and stabilized by the Remorin C-terminal region with its coiled-coil domain while the RLK kinase domains transiently interact in vivo and phosphorylate a residue in the N-terminal region of the LjSYMREM1 protein in vitro. These data provide novel insights into the mechanism of this putative molecular scaffold protein and underline its importance during rhizobial infection. PMID:22292047

  20. Co-operative DNA binding by GAGA transcription factor requires the conserved BTB/POZ domain and reorganizes promoter topology.

    PubMed Central

    Katsani, K R; Hajibagheri, M A; Verrijzer, C P

    1999-01-01

    The POZ domain is a conserved protein-protein interaction motif present in a variety of transcription factors involved in development, chromatin remodelling and human cancers. Here, we study the role of the POZ domain of the GAGA transcription factor in promoter recognition. Natural target promoters for GAGA typically contain multiple GAGA-binding elements. Our results show that the POZ domain mediates strong co-operative binding to multiple sites but inhibits binding to single sites. Protein cross-linking and gel filtration chromatography experiments established that the POZ domain is required for GAGA oligomerization into higher order complexes. Thus, GAGA oligomerization increases binding specificity by selecting only promoters with multiple sites. Electron microscopy revealed that GAGA binds to multiple sites as a large oligomer and induces bending of the promoter DNA. Our results indicate a novel mode of DNA binding by GAGA, in which a large GAGA complex binds multiple GAGA elements that are spread out over a region of a few hundred base pairs. We suggest a model in which the promoter DNA is wrapped around a GAGA multimer in a conformation that may exclude normal nucleosome formation. PMID:9927429

  1. A unique spumavirus Gag N-terminal domain with functional properties of orthoretroviral matrix and capsid.

    PubMed

    Goldstone, David C; Flower, Thomas G; Ball, Neil J; Sanz-Ramos, Marta; Yap, Melvyn W; Ogrodowicz, Roksana W; Stanke, Nicole; Reh, Juliane; Lindemann, Dirk; Stoye, Jonathan P; Taylor, Ian A

    2013-05-01

    The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of gag genes where structurally unrelated molecules have become functionally equivalent. PMID:23675305

  2. Nuclear lamins are not required for lamina-associated domain organization in mouse embryonic stem cells

    PubMed Central

    Amendola, Mario; van Steensel, Bas

    2015-01-01

    In mammals, the nuclear lamina interacts with hundreds of large genomic regions, termed lamina-associated domains (LADs) that are generally in a transcriptionally repressed state. Lamins form the major structural component of the lamina and have been reported to bind DNA and chromatin. Here, we systematically evaluate whether lamins are necessary for the LAD organization in murine embryonic stem cells. Surprisingly, removal of essentially all lamins does not have any detectable effect on the genome-wide interaction pattern of chromatin with emerin, a marker of the inner nuclear membrane. This suggests that other components of the lamina mediate these interactions. PMID:25784758

  3. Domain Structure of the Redβ Single-Strand Annealing Protein: the C-terminal Domain is Required for Fine-Tuning DNA-binding Properties, Interaction with the Exonuclease Partner, and Recombination in vivo.

    PubMed

    Smith, Christopher E; Bell, Charles E

    2016-02-13

    Redβ is a component of the Red recombination system of bacteriophage λ that promotes a single strand annealing (SSA) reaction to generate end-to-end concatemers of the phage genome for packaging. Redβ interacts with λ exonuclease (λexo), the other component of the Red system, to form a "synaptosome" complex that somehow integrates the end resection and annealing steps of the reaction. Previous work using limited proteolysis and chemical modification revealed that Redβ consists of an N-terminal DNA binding domain, residues 1-177, and a flexible C-terminal "tail", residues 178-261. Here, we quantitatively compare the binding of the full-length protein (Redβ(FL)) and the N-terminal domain (Redβ(177)) to different lengths of ssDNA substrate and annealed duplex product. We find that in general, Redβ(FL) binds more tightly to annealed duplex product than to ssDNA substrate, while Redβ(177) binds more tightly to ssDNA. In addition, the C-terminal region of Redβ corresponding to residues 182-261 was purified and found to fold into an α-helical domain that is required for the interaction with λexo to form the synaptosome complex. Deletion analysis of Redβ revealed that removal of just eleven residues from the C-terminus disrupts the interaction with λexo as well as ssDNA and dsDNA recombination in vivo. By contrast, the determinants for self-oligomerization of Redβ appear to reside solely within the N-terminal domain. The subtle but significant differences in the relative binding of Redβ(FL) and Redβ(177) to ssDNA substrate and annealed duplex product may be important for Redβ to function as a SSA protein in vivo. PMID:26780547

  4. Multifunctional G-Rich and RRM-Containing Domains of TbRGG2 Perform Separate yet Essential Functions in Trypanosome RNA Editing

    PubMed Central

    Foda, Bardees M.; Downey, Kurtis M.; Fisk, John C.

    2012-01-01

    Efficient editing of Trypanosoma brucei mitochondrial RNAs involves the actions of multiple accessory factors. T. brucei RGG2 (TbRGG2) is an essential protein crucial for initiation and 3′-to-5′ progression of editing. TbRGG2 comprises an N-terminal G-rich region containing GWG and RG repeats and a C-terminal RNA recognition motif (RRM)-containing domain. Here, we perform in vitro and in vivo separation-of-function studies to interrogate the mechanism of TbRGG2 action in RNA editing. TbRGG2 preferentially binds preedited mRNA in vitro with high affinity attributable to its G-rich region. RNA-annealing and -melting activities are separable, carried out primarily by the G-rich and RRM domains, respectively. In vivo, the G-rich domain partially complements TbRGG2 knockdown, but the RRM domain is also required. Notably, TbRGG2's RNA-melting activity is dispensable for RNA editing in vivo. Interactions between TbRGG2 and MRB1 complex proteins are mediated by both G-rich and RRM-containing domains, depending on the binding partner. Overall, our results are consistent with a model in which the high-affinity RNA binding and RNA-annealing activities of the G-rich domain are essential for RNA editing in vivo. The RRM domain may have key functions involving interactions with the MRB1 complex and/or regulation of the activities of the G-rich domain. PMID:22798390

  5. Multifunctional G-rich and RRM-containing domains of TbRGG2 perform separate yet essential functions in trypanosome RNA editing.

    PubMed

    Foda, Bardees M; Downey, Kurtis M; Fisk, John C; Read, Laurie K

    2012-09-01

    Efficient editing of Trypanosoma brucei mitochondrial RNAs involves the actions of multiple accessory factors. T. brucei RGG2 (TbRGG2) is an essential protein crucial for initiation and 3'-to-5' progression of editing. TbRGG2 comprises an N-terminal G-rich region containing GWG and RG repeats and a C-terminal RNA recognition motif (RRM)-containing domain. Here, we perform in vitro and in vivo separation-of-function studies to interrogate the mechanism of TbRGG2 action in RNA editing. TbRGG2 preferentially binds preedited mRNA in vitro with high affinity attributable to its G-rich region. RNA-annealing and -melting activities are separable, carried out primarily by the G-rich and RRM domains, respectively. In vivo, the G-rich domain partially complements TbRGG2 knockdown, but the RRM domain is also required. Notably, TbRGG2's RNA-melting activity is dispensable for RNA editing in vivo. Interactions between TbRGG2 and MRB1 complex proteins are mediated by both G-rich and RRM-containing domains, depending on the binding partner. Overall, our results are consistent with a model in which the high-affinity RNA binding and RNA-annealing activities of the G-rich domain are essential for RNA editing in vivo. The RRM domain may have key functions involving interactions with the MRB1 complex and/or regulation of the activities of the G-rich domain. PMID:22798390

  6. Development of the aboral domain in Nematostella requires β-catenin and the opposing activities of Six3/6 and Frizzled5/8

    PubMed Central

    Leclère, Lucas; Bause, Markus; Sinigaglia, Chiara; Steger, Julia; Rentzsch, Fabian

    2016-01-01

    ABSTRACT The development of the oral pole in cnidarians and the posterior pole in bilaterians is regulated by canonical Wnt signaling, whereas a set of transcription factors, including Six3/6 and FoxQ2, controls aboral development in cnidarians and anterior identity in bilaterians. However, it is poorly understood how these two patterning systems are initially set up in order to generate correct patterning along the primary body axis. Investigating the early steps of aboral pole formation in the sea anemone Nematostella vectensis, we found that, at blastula stage, oral genes are expressed before aboral genes and that Nvβ-catenin regulates both oral and aboral development. In the oral hemisphere, Nvβ-catenin specifies all subdomains except the oral-most, NvSnailA-expressing domain, which is expanded upon Nvβ-catenin knockdown. In addition, Nvβ-catenin establishes the aboral patterning system by promoting the expression of NvSix3/6 at the aboral pole and suppressing the Wnt receptor NvFrizzled5/8 at the oral pole. NvFrizzled5/8 expression thereby gets restricted to the aboral domain. At gastrula stage, NvSix3/6 and NvFrizzled5/8 are both expressed in the aboral domain, but they have opposing activities, with NvSix3/6 maintaining and NvFrizzled5/8 restricting the size of the aboral domain. At planula stage, NvFrizzled5/8 is required for patterning within the aboral domain and for regulating the size of the apical organ by modulation of a previously characterized FGF feedback loop. Our findings suggest conserved roles for Six3/6 and Frizzled5/8 in aboral/anterior development and reveal key functions for Nvβ-catenin in the patterning of the entire oral-aboral axis of Nematostella. PMID:26989171

  7. Development of the aboral domain in Nematostella requires β-catenin and the opposing activities of Six3/6 and Frizzled5/8.

    PubMed

    Leclère, Lucas; Bause, Markus; Sinigaglia, Chiara; Steger, Julia; Rentzsch, Fabian

    2016-05-15

    The development of the oral pole in cnidarians and the posterior pole in bilaterians is regulated by canonical Wnt signaling, whereas a set of transcription factors, including Six3/6 and FoxQ2, controls aboral development in cnidarians and anterior identity in bilaterians. However, it is poorly understood how these two patterning systems are initially set up in order to generate correct patterning along the primary body axis. Investigating the early steps of aboral pole formation in the sea anemone Nematostella vectensis, we found that, at blastula stage, oral genes are expressed before aboral genes and that Nvβ-catenin regulates both oral and aboral development. In the oral hemisphere, Nvβ-catenin specifies all subdomains except the oral-most, NvSnailA-expressing domain, which is expanded upon Nvβ-catenin knockdown. In addition, Nvβ-catenin establishes the aboral patterning system by promoting the expression of NvSix3/6 at the aboral pole and suppressing the Wnt receptor NvFrizzled5/8 at the oral pole. NvFrizzled5/8 expression thereby gets restricted to the aboral domain. At gastrula stage, NvSix3/6 and NvFrizzled5/8 are both expressed in the aboral domain, but they have opposing activities, with NvSix3/6 maintaining and NvFrizzled5/8 restricting the size of the aboral domain. At planula stage, NvFrizzled5/8 is required for patterning within the aboral domain and for regulating the size of the apical organ by modulation of a previously characterized FGF feedback loop. Our findings suggest conserved roles for Six3/6 and Frizzled5/8 in aboral/anterior development and reveal key functions for Nvβ-catenin in the patterning of the entire oral-aboral axis of Nematostella. PMID:26989171

  8. Functional Domains of Autoimmune Regulator (AIRE) Modulate INS-VNTR Transcription in Human Thymic Epithelial Cells.

    PubMed

    Sparks, Avis E; Chen, Chiachen; Breslin, Mary B; Lan, Michael S

    2016-05-20

    INS-VNTR (insulin-variable number of tandem repeats) and AIRE (autoimmune regulator) have been associated with the modulation of insulin gene expression in thymus, which is essential to induce either insulin tolerance or the development of insulin autoimmunity and type 1 diabetes. We sought to analyze whether each functional domain of AIRE is critical for the activation of INS-VNTR in human thymic epithelial cells. Twelve missense or nonsense mutations in AIRE and two chimeric AIRE constructs were generated. A luciferase reporter assay and a pulldown assay using biotinylated INS-class I VNTR probe were performed to examine the transactivation and binding activities of WT, mutant, and chimeric AIREs on the INS-VNTR promoter. Confocal microscopy analysis was performed for WT or mutant AIRE cellular localization. We found that all of the AIRE mutations resulted in loss of transcriptional activation of INS-VNTR except mutant P252L. Using WT/mutant AIRE heterozygous forms to modulate the INS-VNTR target revealed five mutations (R257X, G228W, C311fsX376, L397fsX478, and R433fsX502) that functioned in a dominant negative fashion. The LXXLL-3 motif is identified for the first time to be essential for DNA binding to INS-VNTR, whereas the intact PHD1, PHD2, LXXLL-3, and LXXLL-4 motifs were important for successful transcriptional activation. AIRE nuclear localization in the human thymic epithelial cell line was disrupted by mutations in the homogenously staining region domain and the R257X mutation in the PHD1 domain. This study supports the notion that AIRE mutation could specifically affect human insulin gene expression in thymic epithelial cells through INS-VNTR and subsequently induce either insulin tolerance or autoimmunity. PMID:27048654

  9. Syntaxin-1 N-peptide and Habc-domain perform distinct essential functions in synaptic vesicle fusion

    PubMed Central

    Zhou, Peng; Pang, Zhiping P; Yang, Xiaofei; Zhang, Yingsha; Rosenmund, Christian; Bacaj, Taulant; Südhof, Thomas C

    2013-01-01

    Among SNARE proteins mediating synaptic vesicle fusion, syntaxin-1 uniquely includes an N-terminal peptide (‘N-peptide') that binds to Munc18-1, and a large, conserved Habc-domain that also binds to Munc18-1. Previous in vitro studies suggested that the syntaxin-1 N-peptide is functionally important, whereas the syntaxin-1 Habc-domain is not, but limited information is available about the in vivo functions of these syntaxin-1 domains. Using rescue experiments in cultured syntaxin-deficient neurons, we now show that the N-peptide and the Habc-domain of syntaxin-1 perform distinct and independent roles in synaptic vesicle fusion. Specifically, we found that the N-peptide is essential for vesicle fusion as such, whereas the Habc-domain regulates this fusion, in part by forming the closed syntaxin-1 conformation. Moreover, we observed that deletion of the Habc-domain but not deletion of the N-peptide caused a loss of Munc18-1 which results in a decrease in the readily releasable pool of vesicles at a synapse, suggesting that Munc18 binding to the Habc-domain stabilizes Munc18-1. Thus, the N-terminal syntaxin-1 domains mediate different functions in synaptic vesicle fusion, probably via formation of distinct Munc18/SNARE-protein complexes. PMID:23188083

  10. Cognitive Functioning in Space Exploration Missions: A Human Requirement

    NASA Technical Reports Server (NTRS)

    Fiedler, Edan; Woolford, Barbara

    2005-01-01

    Solving cognitive issues in the exploration missions will require implementing results from both Human Behavior and Performance, and Space Human Factors Engineering. Operational and research cognitive requirements need to reflect a coordinated management approach with appropriate oversight and guidance from NASA headquarters. First, this paper will discuss one proposed management method that would combine the resources of Space Medicine and Space Human Factors Engineering at JSC, other NASA agencies, the National Space Biomedical Research Institute, Wyle Labs, and other academic or industrial partners. The proposed management is based on a Human Centered Design that advocates full acceptance of the human as a system equal to other systems. Like other systems, the human is a system with many subsystems, each of which has strengths and limitations. Second, this paper will suggest ways to inform exploration policy about what is needed for optimal cognitive functioning of the astronaut crew, as well as requirements to ensure necessary assessment and intervention strategies for the human system if human limitations are reached. Assessment strategies will include clinical evaluation and fitness-to-perform evaluations. Clinical intervention tools and procedures will be available to the astronaut and space flight physician. Cognitive performance will be supported through systematic function allocation, task design, training, and scheduling. Human factors requirements and guidelines will lead to well-designed information displays and retrieval systems that reduce crew time and errors. Means of capturing process, design, and operational requirements to ensure crew performance will be discussed. Third, this paper will describe the current plan of action, and future challenges to be resolved before a lunar or Mars expedition. The presentation will include a proposed management plan for research, involvement of various organizations, and a timetable of deliverables.

  11. C. elegans patched-3 is an essential gene implicated in osmoregulation and requiring an intact permease transporter domain

    PubMed Central

    Soloviev, Alexander; Gallagher, Joseph; Marnef, Aline; Kuwabara, Patricia E.

    2011-01-01

    The nematode Caenorhabditis elegans has retained a rudimentary Hedgehog (Hh) signalling pathway; Hh and Smoothened (Smo) homologs are absent, but two highly related Patched gene homologs, ptc-1 and ptc-3, and 24 ptc-related (ptr) genes are present. We previously showed that ptc-1 is essential for germ line cytokinesis. Here, we report that ptc-3 is also an essential gene; the absence of ptc-3 results in a late embryonic lethality due to an apparent defect in osmoregulation. Rescue of a ptc-3 mutant with a ptc-3::gfp translational reporter reveals that ptc-3 is dynamically expressed in multiple tissues across development. Consistent with this pattern of expression, ptc-3(RNAi) reveals an additional postembryonic requirement for ptc-3 activity. Tissue-specific promoter studies indicate that hypodermal expression of ptc-3 is required for normal development. Missense changes in key residues of the sterol sensing domain (SSD) and the permease transporter domain GxxxD/E motif reveal that the transporter domain is essential for PTC-3 activity, whereas an intact SSD is dispensable. Taken together, our studies indicate that PTC proteins have retained essential roles in C. elegans that are independent of Smoothened (Smo). These observations reveal novel, and perhaps ancestral, roles for PTC proteins. PMID:21215260

  12. Lymphatic function is required prenatally for lung inflation at birth.

    PubMed

    Jakus, Zoltán; Gleghorn, Jason P; Enis, David R; Sen, Aslihan; Chia, Stephanie; Liu, Xi; Rawnsley, David R; Yang, Yiqing; Hess, Paul R; Zou, Zhiying; Yang, Jisheng; Guttentag, Susan H; Nelson, Celeste M; Kahn, Mark L

    2014-05-01

    Mammals must inflate their lungs and breathe within minutes of birth to survive. A key regulator of neonatal lung inflation is pulmonary surfactant, a lipoprotein complex which increases lung compliance by reducing alveolar surface tension (Morgan, 1971). Whether other developmental processes also alter lung mechanics in preparation for birth is unknown. We identify prenatal lymphatic function as an unexpected requirement for neonatal lung inflation and respiration. Mice lacking lymphatic vessels, due either to loss of the lymphangiogenic factor CCBE1 or VEGFR3 function, appear cyanotic and die shortly after birth due to failure of lung inflation. Failure of lung inflation is not due to reduced surfactant levels or altered development of the lung but is associated with an elevated wet/dry ratio consistent with edema. Embryonic studies reveal active lymphatic function in the late gestation lung, and significantly reduced total lung compliance in late gestation embryos that lack lymphatics. These findings reveal that lymphatic vascular function plays a previously unrecognized mechanical role in the developing lung that prepares it for inflation at birth. They explain respiratory failure in infants with congenital pulmonary lymphangiectasia, and suggest that inadequate late gestation lymphatic function may also contribute to respiratory failure in premature infants. PMID:24733830

  13. A singularly perturbed nonlinear traction problem in a periodically perforated domain: a functional analytic approach

    NASA Astrophysics Data System (ADS)

    Dalla Riva, M.; Musolino, P.

    2014-01-01

    We consider a periodically perforated domain obtained by making in R^n a periodic set of holes, each of them of size proportional to \\epsilon. Then we introduce a nonlinear boundary value problem for the Lam\\'e equations in such a periodically perforated domain. The unknown of the problem is a vector valued function u which represents the displacement attained in the equilibrium configuration by the points of a periodic linearly elastic matrix with a hole of size \\epsilon contained in each periodic cell. We assume that the traction exerted by the matrix on the boundary of each hole depends (nonlinearly) on the displacement attained by the points of the boundary of the hole. Then our aim is to describe what happens to the displacement vector function u when \\epsilon tends to 0. Under suitable assumptions we prove the existence of a family of solutions {u(\\epsilon,.) : \\epsilon \\in ]0,\\epsilon'[} with a prescribed limiting behaviour when \\epsilon approaches 0. Moreover, the family {u(\\epsilon,.) : \\epsilon \\in ]0,\\epsilon'[} is in a sense locally unique and can be continued real analytically for negative values of \\epsilon.

  14. Evidence-Based Structural Model of the Staphylococcal Repressor Protein: Separation of Functions into Different Domains

    PubMed Central

    Nyíri, Kinga; Kőhegyi, Bianka; Micsonai, András; Kardos, József; Vertessy, Beata G.

    2015-01-01

    Horizontal transfer of mobile genetic elements within Staphylococci is of high biomedical significance as such elements are frequently responsible for virulence and toxic effects. Staphylococcus-encoded repressor proteins regulate the replication of these mobile genetic elements that are located within the so-called pathogenicity islands. Here, we report structural and functional characterization of one such repressor protein, namely the Stl protein encoded by the pathogenicity island SaPIbov1. We create a 3D structural model and based on this prediction, we investigate the different functionalities of truncated and point mutant constructs. Results suggest that a helix-turn-helix motif governs the interaction of the Stl protein with its cognate DNA site: point mutations within this motif drastically decrease DNA-binding ability, whereas the interaction with the Stl-binding partner protein dUTPase is unperturbed by these point mutations. The 3D model also suggested the potential independent folding of a carboxy-terminal domain. This suggestion was fully verified by independent experiments revealing that the carboxy-terminal domain does not bind to DNA but is still capable of binding to and inhibiting dUTPase. A general model is proposed, which suggests that among the several structurally different repressor superfamilies Stl-like Staphylococcal repressor proteins belong to the helix-turn-helix transcription factor group and the HTH motif is suggested to reside within N-terminal segment. PMID:26414067

  15. Analysis of functional domains of rat mitochondrial Fis1, the mitochondrial fission-stimulating protein

    SciTech Connect

    Jofuku, Akihiro; Ishihara, Naotada; Mihara, Katsuyoshi . E-mail: mihara@cell.med.kyushu-u.ac.jp

    2005-07-29

    In yeast, mitochondrial-fission is regulated by the cytosolic dynamin-like GTPase (Dnm1p) in conjunction with a peripheral protein, Mdv1p, and a C-tail-anchored outer membrane protein, Fis1p. In mammals, a dynamin-related protein (Drp1) and Fis1 are involved in the mitochondrial-fission reaction as Dnm1 and Fis1 orthologues, respectively. The involvement of other component(s), such as the Mdv1 homologue, and the mechanisms regulating mitochondrial-fission remain unclear. Here, we identified rat Fis1 (rFis1) and analyzed its structure-function relationship. Blue-native-polyacrylamide gel electrophoresis revealed that rFis1 formed a {approx}200-kDa complex in the outer mitochondrial membrane. Its expression in HeLa cells promoted extensive mitochondrial fragmentation, and gene knock-down by RNAi induced extension of the mitochondrial networks. Taking advantage of these properties, we analyzed functional domains of rFis1. These experiments revealed that the N-terminal and C-terminal segments are both essential for oligomeric rFis1 interaction, and the middle TPR-like domains regulate proper oligomer assembly. Any mutations that disturb the proper oligomeric assembly compromise mitochondrial division-stimulating activity of rFis1.

  16. InterPro: an integrated documentation resource for protein families, domains and functional sites.

    PubMed

    Mulder, Nicola J; Apweiler, Rolf; Attwood, Terri K; Bairoch, Amos; Bateman, Alex; Binns, David; Biswas, Margaret; Bradley, Paul; Bork, Peer; Bucher, Phillip; Copley, Richard; Courcelle, Emmanuel; Durbin, Richard; Falquet, Laurent; Fleischmann, Wolfgang; Gouzy, Jerome; Griffith-Jones, Sam; Haft, Daniel; Hermjakob, Henning; Hulo, Nicolas; Kahn, Daniel; Kanapin, Alexander; Krestyaninova, Maria; Lopez, Rodrigo; Letunic, Ivica; Orchard, Sandra; Pagni, Marco; Peyruc, David; Ponting, Chris P; Servant, Florence; Sigrist, Christian J A

    2002-09-01

    The exponential increase in the submission of nucleotide sequences to the nucleotide sequence database by genome sequencing centres has resulted in a need for rapid, automatic methods for classification of the resulting protein sequences. There are several signature and sequence cluster-based methods for protein classification, each resource having distinct areas of optimum application owing to the differences in the underlying analysis methods. In recognition of this, InterPro was developed as an integrated documentation resource for protein families, domains and functional sites, to rationalise the complementary efforts of the individual protein signature database projects. The member databases - PRINTS, PROSITE, Pfam, ProDom, SMART and TIGRFAMs - form the InterPro core. Related signatures from each member database are unified into single InterPro entries. Each InterPro entry includes a unique accession number, functional descriptions and literature references, and links are made back to the relevant member database(s). Release 4.0 of InterPro (November 2001) contains 4,691 entries, representing 3,532 families, 1,068 domains, 74 repeats and 15 sites of post-translational modification (PTMs) encoded by different regular expressions, profiles, fingerprints and hidden Markov models (HMMs). Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (2,141,621 InterPro hits from 586,124 SWISS-PROT and TrEMBL protein sequences). The database is freely accessible for text- and sequence-based searches. PMID:12230031

  17. Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors

    SciTech Connect

    Nie,Y.; Hobbs, J.; Vigues, S.; Olson, W.; Conn, G.; Munger, S.

    2006-01-01

    Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain a large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.

  18. Thrombospondin-1 domain-containing peptide properdistatin improves vascular function in human melanoma xenografts.

    PubMed

    Gaustad, Jon-Vidar; Simonsen, Trude G; Andersen, Lise Mari K; Rofstad, Einar K

    2015-03-01

    Properdistatin is a novel peptide derived from the thrombospondin-1 domain of the plasma protein properdin. The purpose of this study was to investigate the effect of properdistatin treatment on the morphology and function of tumor vasculature. A-07 human melanoma xenografts grown in dorsal window chambers were used as preclinical model. Tumors were treated with 80 mg/kg/day properdistatin or vehicle for 4 days. Morphologic parameters of tumor vascular networks were assessed from high-resolution transillumination images, and tumor blood supply time and plasma velocities were assessed from first-pass imaging movies recorded after a bolus of 155 kDa tetramethylrhodamine isothiocyanate-labeled dextran had been administered intravenously. Properdistatin-treated tumors showed reduced density of small-diameter vessels, reduced blood supply time, and increased plasma velocities. In conclusion, properdistatin treatment inhibited angiogenesis and improved vascular function in A-07 tumors. PMID:24555949

  19. The Caenorhabditis elegans Iodotyrosine Deiodinase Ortholog SUP-18 Functions through a Conserved Channel SC-Box to Regulate the Muscle Two-Pore Domain Potassium Channel SUP-9

    PubMed Central

    de la Cruz, Ignacio Perez; Ma, Long; Horvitz, H. Robert

    2014-01-01

    Loss-of-function mutations in the Caenorhabditis elegans gene sup-18 suppress the defects in muscle contraction conferred by a gain-of-function mutation in SUP-10, a presumptive regulatory subunit of the SUP-9 two-pore domain K+ channel associated with muscle membranes. We cloned sup-18 and found that it encodes the C. elegans ortholog of mammalian iodotyrosine deiodinase (IYD), an NADH oxidase/flavin reductase that functions in iodine recycling and is important for the biosynthesis of thyroid hormones that regulate metabolism. The FMN-binding site of mammalian IYD is conserved in SUP-18, which appears to require catalytic activity to function. Genetic analyses suggest that SUP-10 can function with SUP-18 to activate SUP-9 through a pathway that is independent of the presumptive SUP-9 regulatory subunit UNC-93. We identified a novel evolutionarily conserved serine-cysteine-rich region in the C-terminal cytoplasmic domain of SUP-9 required for its specific activation by SUP-10 and SUP-18 but not by UNC-93. Since two-pore domain K+ channels regulate the resting membrane potentials of numerous cell types, we suggest that the SUP-18 IYD regulates the activity of the SUP-9 channel using NADH as a coenzyme and thus couples the metabolic state of muscle cells to muscle membrane excitability. PMID:24586202

  20. Dimerization of the DYT6 dystonia protein, THAP1, requires residues within the coiled-coil domain

    PubMed Central

    Sengel, Cem; Gavarini, Sophie; Sharma, Nutan; Ozelius, Laurie J.; Bragg, D. Cristopher

    2011-01-01

    THAP1 is a DNA binding protein that has been recently associated with DYT6 dystonia, a hereditary movement disorder involving sustained, involuntary muscle contractions. A large number of dystonia-related mutations have been identified in THAP1 in diverse patient populations worldwide. Previous reports have suggested that THAP1 oligomerizes with itself via a C-terminal coiled-coil domain, raising the possibility that DYT6 mutations in this region might affect this interaction. In this study we examined the ability of wild-type THAP1 to bind itself and the effects on this interaction of the following disease mutations: C54Y, F81L, ΔF132, T142A, I149T, Q154fs180X, and A166T. The results confirmed that wild-type THAP1 associated with itself and most of the DYT6 mutants tested, except for the Q154fs180X variant, which loses most of the coiled-coil domain due to a frameshift at position 154. However, deletion of C-terminal residues after position 166 produced a truncated variant of THAP1 that was able to bind the wild-type protein. The interaction of THAP1 with itself therefore required residues within a 13-amino acid region (aa 154–166) of the coiled-coil domain. Further inspection of this sequence revealed elements highly consistent with previous descriptions of leucine zippers, which serve as dimerization domains in other transcription factor families. Based on this similarity, a structural model was generated to predict how hydrophobic residues in this region may mediate dimerization. These observations offer additional insight into the role of the coiled-coil domain in THAP1, which may facilitate future analyses of DYT6 mutations in this region. PMID:21752024

  1. The C-terminal Kinase and ERK-binding Domains of Drosophila S6KII (RSK) Are Required for Phosphorylation of the Protein and Modulation of Circadian Behavior*

    PubMed Central

    Tangredi, Michelle M.; Ng, Fanny S.; Jackson, F. Rob

    2012-01-01

    A detailed structure/function analysis of Drosophila p90 ribosomal S6 kinase (S6KII) or its mammalian homolog RSK has not been performed in the context of neuronal plasticity or behavior. We previously reported that S6KII is required for normal circadian periodicity. Here we report a site-directed mutagenesis of S6KII and analysis of mutants, in vivo, that identifies functional domains and phosphorylation sites critical for the regulation of circadian period. We demonstrate, for the first time, a role for the S6KII C-terminal kinase that is independent of its known role in activation of the N-terminal kinase. Both S6KII C-terminal kinase activity and its ERK-binding domain are required for wild-type circadian period and normal phosphorylation status of the protein. In contrast, the N-terminal kinase of S6KII is dispensable for modulation of circadian period and normal phosphorylation of the protein. We also show that particular sites of S6KII phosphorylation, Ser-515 and Thr-732, are essential for normal circadian behavior. Surprisingly, the phosphorylation of S6KII residues, in vivo, does not follow a strict sequential pattern, as implied by certain cell-based studies of mammalian RSK protein. PMID:22447936

  2. Rigidifying Acyl Carrier Protein Domain in Iterative Type I PKS CalE8 Does Not Affect Its Function

    PubMed Central

    Lim, Jackwee; Sun, Huihua; Fan, Jing-Song; Hameed, Iman Fahim; Lescar, Julien; Liang, Zhao-Xun; Yang, Daiwen

    2012-01-01

    Acyl carrier protein (ACP) domains shuttle acyl intermediates among the catalytic domains of multidomain type I fatty acid synthase and polyketide synthase (PKS) systems. It is believed that the unique function of ACPs is associated with their dynamic property, but it remains to be fully elucidated what type of protein dynamics is critical for the shuttling domain. Using NMR techniques, we found that the ACP domain of iterative type I PKS CalE8 from Micromonospora echinospora is highly dynamic on the millisecond-second timescale. Introduction of an interhelical disulfide linkage in the ACP domain suppresses the dynamics on the millisecond-second timescale and reduces the mobility on the picosecond-nanosecond timescale. We demonstrate that the full-length PKS is fully functional upon rigidification of the ACP domain, suggesting that although the flexibility of the disordered terminal linkers may be important for the function of the ACP domain, the internal dynamics of the helical regions is not critical for that function. PMID:23009853

  3. Frequency-domain Green's functions for radar waves in heterogeneous 2.5D media

    USGS Publications Warehouse

    Ellefsen, K.J.; Croize, D.; Mazzella, A.T.; McKenna, J.R.

    2009-01-01

    Green's functions for radar waves propagating in heterogeneous 2.5D media might be calculated in the frequency domain using a hybrid method. The model is defined in the Cartesian coordinate system, and its electromagnetic properties might vary in the x- and z-directions, but not in the y-direction. Wave propagation in the x- and z-directions is simulated with the finite-difference method, and wave propagation in the y-direction is simulated with an analytic function. The absorbing boundaries on the finite-difference grid are perfectly matched layers that have been modified to make them compatible with the hybrid method. The accuracy of these numerical Greens functions is assessed by comparing them with independently calculated Green's functions. For a homogeneous model, the magnitude errors range from -4.16% through 0.44%, and the phase errors range from -0.06% through 4.86%. For a layered model, the magnitude errors range from -2.60% through 2.06%, and the phase errors range from -0.49% through 2.73%. These numerical Green's functions might be used for forward modeling and full waveform inversion. ?? 2009 Society of Exploration Geophysicists. All rights reserved.

  4. Software with partial functions: Automating correctness proofs via nonstrict explicit domains

    SciTech Connect

    Yakhnis, A.; Yakhnis, V.; Winter, V.

    1996-07-30

    As our society becomes technologically more complex, computers are being used in greater and greater numbers of high consequence systems. Giving a machine control over the lives of humans can be disturbing, especially if the software that is run on such a machine has bugs. Formal reasoning is one of the most powerful techniques available to demonstrate the correctness of a piece of software. When reasoning about software and its development, one frequently encounters expressions that contain partial functions. As might be expected, the presence of partial functions introduces an additional dimension of difficulty to the reasoning framework. This difficulty produces an especially strong impact in the case of high consequence systems. An ability to use formal methods for constructing software is essential if we want to obtain greater confidence in such systems through formal reasoning. This is only reasonable under automation of software development and verification. However, the ubiquitous presence of partial functions prevents a uniform application to software of any tools not specifically accounting for partial functions. In this paper we will describe a framework for reasoning about software, based on the nonstrict explicit domain approach, that is applicable to a large class of software/hardware systems. In this framework the Hoare triples containing partial functions can be reasoned about automatically in a well-defined and uniform manner.

  5. Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1: FUNCTIONAL INTERACTIONS BETWEEN THE KUNITZ-TYPE INHIBITOR DOMAIN-1 AND THE NEIGHBORING POLYCYSTIC KIDNEY DISEASE-LIKE DOMAIN.

    PubMed

    Hong, Zebin; De Meulemeester, Laura; Jacobi, Annemarie; Pedersen, Jan Skov; Morth, J Preben; Andreasen, Peter A; Jensen, Jan K

    2016-07-01

    Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knock-out mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain). PMID:27189939

  6. Hepatitis C Virus RNA Replication and Virus Particle Assembly Require Specific Dimerization of the NS4A Protein Transmembrane Domain

    PubMed Central

    Kohlway, Andrew; Pirakitikulr, Nathan; Barrera, Francisco N.; Potapova, Olga; Engelman, Donald M.

    2014-01-01

    Hepatitis C virus (HCV) NS4A is a single-pass transmembrane (TM) protein essential for viral replication and particle assembly. The sequence of the NS4A TM domain is highly conserved, suggesting that it may be important for protein-protein interactions. To test this hypothesis, we measured the potential dimerization of the NS4A TM domain in a well-characterized two-hybrid TM protein interaction system. The NS4A TM domain exhibited a strong homotypic interaction that was comparable in affinity to glycophorin A, a well-studied human blood group antigen that forms TM homodimers. Several mutations predicted to cluster on a common surface of the NS4A TM helix caused significant reductions in dimerization, suggesting that these residues form an interface for NS4A dimerization. Mutations in the NS4A TM domain were further examined in the JFH-1 genotype 2a replicon system; importantly, all mutations that destabilized NS4A dimers also caused defects in RNA replication and/or virus assembly. Computational modeling of NS4A TM interactions suggests a right-handed dimeric interaction of helices with an interface that is consistent with the mutational effects. Furthermore, defects in NS4A oligomerization and virus particle assembly of two mutants were rescued by NS4A A15S, a TM mutation recently identified through forward genetics as a cell culture-adaptive mutation. Together, these data provide the first example of a functionally important TM dimer interface within an HCV nonstructural protein and reveal a fundamental role of the NS4A TM domain in coordinat