Science.gov

Sample records for distinct functional domains

  1. 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

  2. Functional diversity of Robo receptor immunoglobulin domains promotes distinct axon guidance decisions.

    PubMed

    Evans, Timothy A; Bashaw, Greg J

    2010-03-23

    Recognition molecules of the immunoglobulin (Ig) superfamily control axon guidance in the developing nervous system. Ig-like domains are among the most widely represented protein domains in the human genome, and the number of Ig superfamily proteins is strongly correlated with cellular complexity. In Drosophila, three Roundabout (Robo) Ig superfamily receptors respond to their common Slit ligand to regulate axon guidance at the midline: Robo and Robo2 mediate midline repulsion, Robo2 and Robo3 control longitudinal pathway selection, and Robo2 can promote midline crossing. How these closely related receptors mediate distinct guidance functions is not understood. We report that the differential functions of Robo2 and Robo3 are specified by their ectodomains and do not reflect differences in cytoplasmic signaling. Functional modularity of Robo2's ectodomain facilitates multiple guidance decisions: Ig1 and Ig3 of Robo2 confer lateral positioning activity, whereas Ig2 confers promidline crossing activity. Robo2's distinct functions are not dependent on greater Slit affinity but are instead due in part to differences in multimerization and receptor-ligand stoichiometry conferred by Robo2's Ig domains. Together, our findings suggest that diverse responses to the Slit guidance cue are imparted by intrinsic structural differences encoded in the extracellular Ig domains of the Robo receptors.

  3. Replication-coupled chromatin assembly of newly synthesized histones: distinct functions for the histone tail domains.

    PubMed

    Ejlassi-Lassallette, Aïda; Thiriet, Christophe

    2012-02-01

    The maintenance of the genome during replication requires the assembly of nucleosomes with newly synthesized histones. Achieving the deposition of newly synthesized histones in chromatin implies their transport from the cytoplasm to the nucleus at the replication sites. Several lines of evidence have revealed critical functions of the histone tail domains in these conserved cellular processes. In this review, we discuss the role of the amino termini of the nucleosome building blocks, H2A/H2B and H3/H4, in different model systems. The experimental data showed that H2A/H2B tails and H3/H4 tails display distinct functions in nuclear import and chromatin assembly. Furthermore, we describe recent studies exploiting the unique properties of the slime mold, Physarum polycephalum , that have advanced understanding of the function of the highly conserved replication-dependent diacetylation of H4.

  4. The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components*

    PubMed Central

    Soldi, Monica; Bonaldi, Tiziana

    2013-01-01

    Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational modifications, histone variants, and DNA methylation. Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. Here we describe a modified protocol for chromatin immunoprecipitation combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH, both enriched in silent domains. Chromatin proteomics expands the arsenal of tools for deciphering how all the distinct protein components act together to enforce a given region-specific chromatin status. PMID:23319141

  5. Plasmodium alveolins possess distinct but structurally and functionally related multi-repeat domains.

    PubMed

    Al-Khattaf, Fatimah S; Tremp, Annie Z; Dessens, Johannes T

    2015-02-01

    The invasive and motile life stages of malaria parasites (merozoite, ookinete and sporozoite) possess a distinctive cortical structure termed the pellicle. The pellicle is characterised by a double-layered 'inner membrane complex' (IMC) located underneath the plasma membrane, which is supported by a cytoskeletal structure termed the subpellicular network (SPN). The SPN consists of intermediate filaments, whose major constituents include a family of proteins called alveolins. Here, we re-appraise the alveolins in the genus Plasmodium with respect to their repertoire, structure and interrelatedness. Amongst 13 family members identified, we distinguish two domain types that, albeit distinct at the primary structure level, are structurally related and contain tandem repeats with a consensus 12-amino acid periodicity. Analysis in Plasmodium berghei of the most divergent alveolin, PbIMC1d, reveals a zoite-specific expression in ookinetes and a subcellular localisation in the pellicle, consistent with its predicted role as a SPN component. Knockout of PbIMC1d gives rise to a wild-type phenotype with respect to ookinete morphogenesis, tensile strength, gliding motility and infectivity, presenting the first example of apparent functional redundancy amongst alveolin family members.

  6. Distinct protein domains and expression patterns confer divergent axon guidance functions for Drosophila Robo receptors.

    PubMed

    Spitzweck, Bettina; Brankatschk, Marko; Dickson, Barry J

    2010-02-05

    The orthogonal array of axon pathways in the Drosophila CNS is constructed in part under the control of three Robo family axon guidance receptors: Robo1, Robo2 and Robo3. Each of these receptors is responsible for a distinct set of guidance decisions. To determine the molecular basis for these functional specializations, we used homologous recombination to create a series of 9 "robo swap" alleles: expressing each of the three Robo receptors from each of the three robo loci. We demonstrate that the lateral positioning of longitudinal axon pathways relies primarily on differences in gene regulation, not distinct combinations of Robo proteins as previously thought. In contrast, specific features of the Robo1 and Robo2 proteins contribute to their distinct functions in commissure formation. These specializations allow Robo1 to prevent crossing and Robo2 to promote crossing. These data demonstrate how diversification of expression and structure within a single family of guidance receptors can shape complex patterns of neuronal wiring.

  7. Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses

    PubMed Central

    Nguyen, Quynh-Anh; Horn, Meryl E; Nicoll, Roger A

    2016-01-01

    Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that interact trans-synaptically with neurexins to mediate synapse development and function. NLGN2 is only at inhibitory synapses while NLGN3 is at both excitatory and inhibitory synapses. We found that NLGN3 function at inhibitory synapses in rat CA1 depends on the presence of NLGN2 and identified a domain in the extracellular region that accounted for this functional difference between NLGN2 and 3 specifically at inhibitory synapses. We further show that the presence of a cytoplasmic tail (c-tail) is indispensible, and identified two domains in the c-tail that are necessary for NLGN function at inhibitory synapses. These domains point to a gephyrin-dependent mechanism that is disrupted by an autism-associated mutation at R705 and a gephyrin-independent mechanism reliant on a putative phosphorylation site at S714. Our work highlights unique and separate roles for the extracellular and intracellular regions in specifying and carrying out NLGN function respectively. DOI: http://dx.doi.org/10.7554/eLife.19236.001 PMID:27805570

  8. Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses.

    PubMed

    Nguyen, Quynh-Anh; Horn, Meryl E; Nicoll, Roger A

    2016-11-02

    Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that interact trans-synaptically with neurexins to mediate synapse development and function. NLGN2 is only at inhibitory synapses while NLGN3 is at both excitatory and inhibitory synapses. We found that NLGN3 function at inhibitory synapses in rat CA1 depends on the presence of NLGN2 and identified a domain in the extracellular region that accounted for this functional difference between NLGN2 and 3 specifically at inhibitory synapses. We further show that the presence of a cytoplasmic tail (c-tail) is indispensible, and identified two domains in the c-tail that are necessary for NLGN function at inhibitory synapses. These domains point to a gephyrin-dependent mechanism that is disrupted by an autism-associated mutation at R705 and a gephyrin-independent mechanism reliant on a putative phosphorylation site at S714. Our work highlights unique and separate roles for the extracellular and intracellular regions in specifying and carrying out NLGN function respectively.

  9. Distinct and redundant roles of the non-muscle myosin II isoforms and functional domains.

    PubMed

    Wang, Aibing; Ma, Xuefei; Conti, Mary Anne; Adelstein, Robert S

    2011-10-01

    We propose that the in vivo functions of NM II (non-muscle myosin II) can be divided between those that depend on the N-terminal globular motor domain and those less dependent on motor activity but more dependent on the C-terminal domain. The former, being more dependent on the kinetic properties of NM II to translocate actin filaments, are less amenable to substitution by different NM II isoforms, whereas the in vivo functions of the latter, which involve the structural properties of NM II to cross-link actin filaments, are more amenable to substitution. In light of this hypothesis, we examine the ability of NM II-A, as well as a motor-compromised form of NM II-B, to replace NM II-B and rescue neuroepithelial cell-cell adhesion defects and hydrocephalus in the brain of NM II-B-depleted mice. We also examine the ability of NM II-B as well as chimaeric forms of NM II (II-A head and II-B tail and vice versa) to substitute for NM II-A in cell-cell adhesions in II-A-ablated mice. However, we also show that certain functions, such as neuronal cell migration in the developing brain and vascularization of the mouse embryo and placenta, specifically require NM II-B and II-A respectively.

  10. Two functionally distinct domains generated by in vivo cleavage of Nup145p: a novel biogenesis pathway for nucleoporins.

    PubMed Central

    Teixeira, M T; Siniossoglou, S; Podtelejnikov, S; Bénichou, J C; Mann, M; Dujon, B; Hurt, E; Fabre, E

    1997-01-01

    Nup145p is an essential yeast nucleoporin involved in nuclear export of polyadenylated RNAs. We demonstrate here that Nup145p is cleaved in vivo to yield two functionally distinct domains: a carboxy-terminal domain (C-Nup145p) which is located at the nuclear pore complex (NPC) and assembles into the Nup84p complex, and a GLFG-containing amino-terminal domain (N-Nup145p) which is not part of this complex. Whereas the essential C-Nup145p accomplishes the functions required for efficient mRNA export and normal NPC distribution, N-Nup145p, which is homologous to the GLFG-containing nucleoporins Nup100p and Nup116p, is not necessary for cell growth. However, the N-Nup145p becomes essential in a nup188 mutant background. Strikingly, generation of a free N-domain is a prerequisite for complementation of this peculiar synthetic lethal mutant. These data suggest that N- and C-domains of Nup145p perform independent functions, and that the in vivo cleavage observed is of functional importance. PMID:9305650

  11. Paget disease of bone-associated UBA domain mutations of SQSTM1 exert distinct effects on protein structure and function

    PubMed Central

    Goode, Alice; Long, Jed E.; Shaw, Barry; Ralston, Stuart H.; Visconti, Micaela Rios; Gianfrancesco, Fernando; Esposito, Teresa; Gennari, Luigi; Merlotti, Daniela; Rendina, Domenico; Rea, Sarah L.; Sultana, Melanie; Searle, Mark S.; Layfield, Robert

    2014-01-01

    SQSTM1 mutations are common in patients with Paget disease of bone (PDB), with most affecting the C-terminal ubiquitin-associated (UBA) domain of the SQSTM1 protein. We performed structural and functional analyses of two UBA domain mutations, an I424S mutation relatively common in UK PDB patients, and an A427D mutation associated with a severe phenotype in Southern Italian patients. Both impaired SQSTM1's ubiquitin-binding function in pull-down assays and resulted in activation of basal NF-κB signalling, compared to wild-type, in reporter assays. We found evidence for a relationship between the ability of different UBA domain mutants to activate NF-κB signalling in vitro and number of affected sites in vivo in 1152 PDB patients from the UK and Italy, with A427D-SQSTM1 producing the greatest level of activation (relative to wild-type) of all PDB mutants tested to date. NMR and isothermal titration calorimetry studies were able to demonstrate that I424S is associated with global structural changes in the UBA domain, resulting in 10-fold weaker UBA dimer stability than wild-type and reduced ubiquitin-binding affinity of the UBA monomer. Our observations provide insights into the role of SQSTM1-mediated NF-κB signalling in PDB aetiology, and demonstrate that different mutations in close proximity within loop 2/helix 3 of the SQSTM1 UBA domain exert distinct effects on protein structure and stability, including indirect effects at the UBA/ubiquitin-binding interface. PMID:24642144

  12. 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

  13. Tropomyosin variants describe distinct functional subcellular domains in differentiated vascular smooth muscle cells.

    PubMed

    Gallant, Cynthia; Appel, Sarah; Graceffa, Philip; Leavis, Paul; Lin, Jim Jung-Ching; Gunning, Peter W; Schevzov, Galina; Chaponnier, Christine; DeGnore, Jon; Lehman, William; Morgan, Kathleen G

    2011-06-01

    Tropomyosin (Tm) is known to be an important gatekeeper of actin function. Tm isoforms are encoded by four genes, and each gene produces several variants by alternative splicing, which have been proposed to play roles in motility, proliferation, and apoptosis. Smooth muscle studies have focused on gizzard smooth muscle, where a heterodimer of Tm from the α-gene (Tmsm-α) and from the β-gene (Tmsm-β) is associated with contractile filaments. In this study we examined Tm in differentiated mammalian vascular smooth muscle (dVSM). Liquid chromatography-tandem mass spectrometry (LC MS/MS) analysis and Western blot screening with variant-specific antibodies revealed that at least five different Tm proteins are expressed in this tissue: Tm6 (Tmsm-α) and Tm2 from the α-gene, Tm1 (Tmsm-β) from the β-gene, Tm5NM1 from the γ-gene, and Tm4 from the δ-gene. Tm6 is by far most abundant in dVSM followed by Tm1, Tm2, Tm5NM1, and Tm4. Coimmunoprecipitation and coimmunofluorescence studies demonstrate that Tm1 and Tm6 coassociate with different actin isoforms and display different intracellular localizations. Using an antibody specific for cytoplasmic γ-actin, we report here the presence of a γ-actin cortical cytoskeleton in dVSM cells. Tm1 colocalizes with cortical cytoplasmic γ-actin and coprecipitates with γ-actin. Tm6, on the other hand, is located on contractile bundles. These data indicate that Tm1 and Tm6 do not form a classical heterodimer in dVSM but rather describe different functional cellular compartments.

  14. Modulation of elasticity in functionally distinct domains of the tropomyosin coiled-coil.

    PubMed

    Lakkaraju, Sirish Kaushik; Hwang, Wonmuk

    2009-03-01

    Alpha-helical coiled-coils are common protein structural motifs. Whereas vast information is available regarding their structure, folding, and stability, far less is known about their elastic properties, even though they play mechanical roles in many cases such as tropomyosin in muscle contraction or neck stalks of kinesin or myosin motor proteins. Using computer simulations, we characterized elastic properties of coiled-coils, either globally or locally. Global bending stiffness of standard leucine zipper coiled-coils was calculated using normal mode analysis. Mutations in hydrophobic residues involved in the knob-into-hole interface between the two alpha-helices affect elasticity significantly, whereas charged side chains forming inter-helical salt bridges do not. This suggests that coiled-coils with less regular heptad periodicity may have regional variations in flexibility. We show this by the flexibility map of tropomyosin, which was constructed by a local fluctuation analysis. Overall, flexibility varies by more than twofold and increases towards the C-terminal region of the molecule. Describing the coiled-coil as a twisted tape, it is generally more flexible in the splay bending than in the bending of the broad face. Actin binding sites in alpha zones show local rigidity minima. Broken core regions due to acidic residues at the hydrophobic face such as the Asp137 and the Glu218 are found to be the most labile with moduli for splay and broad face bending as 70 nm and 116 nm respectively. Such variation in flexibility could be relevant to the tropomyosin function, especially for moving across the non-uniform surface of F-actin to regulate myosin binding.

  15. Modulation of elasticity in functionally distinct domains of the tropomyosin coiled-coil

    PubMed Central

    Lakkaraju, Sirish Kaushik; Hwang, Wonmuk

    2009-01-01

    Alpha-helical coiled-coils are common protein structural motifs. Whereas vast information is available regarding their structure, folding, and stability, far less is known about their elastic properties, even though they play mechanical roles in many cases such as tropomyosin in muscle contraction or neck stalks of kinesin or myosin motor proteins. Using computer simulations, we characterized elastic properties of coiled-coils, either globally or locally. Global bending stiffness of standard leucine zipper coiled-coils was calculated using normal mode analysis. Mutations in hydrophobic residues involved in the knob-into-hole interface between the two α-helices affect elasticity significantly, whereas charged side chains forming inter-helical salt bridges do not. This suggests that coiled-coils with less regular heptad periodicity may have regional variations in flexibility. We show this by the flexibility map of tropomyosin, which was constructed by a local fluctuation analysis. Overall, flexibility varies by more than twofold and increases towards the C-terminal region of the molecule. Describing the coiled-coil as a twisted tape, it is generally more flexible in the splay bending than in the bending of the broad face. Actin binding sites in α zones show local rigidity minima. Broken core regions due to acidic residues at the hydrophobic face such as the Asp137 and the Glu218 are found to be the most labile with moduli for splay and broad face bending as 70 nm and 116 nm respectively. Such variation in flexibility could be relevant to the tropomyosin function, especially for moving across the non-uniform surface of F-actin to regulate myosin binding. PMID:19830262

  16. FENS-1 and DFCP1 are FYVE domain-containing proteins with distinct functions in the endosomal and Golgi compartments.

    PubMed

    Ridley, S H; Ktistakis, N; Davidson, K; Anderson, K E; Manifava, M; Ellson, C D; Lipp, P; Bootman, M; Coadwell, J; Nazarian, A; Erdjument-Bromage, H; Tempst, P; Cooper, M A; Thuring, J W; Lim, Z Y; Holmes, A B; Stephens, L R; Hawkins, P T

    2001-11-01

    FENS-1 and DFCP1 are recently discovered proteins containing one or two FYVE-domains respectively. We show that the FYVE domains in these proteins can bind PtdIns3P in vitro with high specificity over other phosphoinositides. Exogenously expressed FENS-1 localises to early endosomes: this localisation requires an intact FYVE domain and is sensitive to wortmannin inhibition. The isolated FYVE domain of FENS-1 also localises to endosomes. These results are consistent with current models of FYVE-domain function in this cellular compartment. By contrast, exogenously expressed DFCP1 displays a predominantly Golgi, endoplasmic reticulum (ER) and vesicular distribution with little or no overlap with FENS-1 or other endosomal markers. Overexpression of DFCP1 was found to cause dispersal of the Golgi compartment defined by giantin and gpp130-staining. Disruption of the FYVE domains of DFCP1 causes a shift to more condensed and compact Golgi structures and overexpression of this mutant was found to confer significant protection to the Golgi against brefeldin-induced dispersal. These properties of DFCP1 are surprising, and suggest FYVE domain-localisation and function may not be exclusively endosomal. Movies available on-line

  17. Structural and Functional Modularity of the Orange Carotenoid Protein: Distinct Roles for the N- and C-Terminal Domains in Cyanobacterial Photoprotection[C][W

    PubMed Central

    Leverenz, Ryan L.; Jallet, Denis; Li, Ming-De; Mathies, Richard A.; Kirilovsky, Diana; Kerfeld, Cheryl A.

    2014-01-01

    The orange carotenoid protein (OCP) serves as a sensor of light intensity and an effector of phycobilisome (PB)–associated photoprotection in cyanobacteria. Structurally, the OCP is composed of two distinct domains spanned by a single carotenoid chromophore. Functionally, in response to high light, the OCP converts from a dark-stable orange form, OCPO, to an active red form, OCPR. The C-terminal domain of the OCP has been implicated in the dynamic response to light intensity and plays a role in switching off the OCP’s photoprotective response through its interaction with the fluorescence recovery protein. The function of the N-terminal domain, which is uniquely found in cyanobacteria, is unclear. To investigate its function, we isolated the N-terminal domain in vitro using limited proteolysis of native OCP. The N-terminal domain retains the carotenoid chromophore; this red carotenoid protein (RCP) has constitutive PB fluorescence quenching activity comparable in magnitude to that of active, full-length OCPR. A comparison of the spectroscopic properties of the RCP with OCPR indicates that critical protein–chromophore interactions within the C-terminal domain are weakened in the OCPR form. These results suggest that the C-terminal domain dynamically regulates the photoprotective activity of an otherwise constitutively active carotenoid binding N-terminal domain. PMID:24399299

  18. Novel exons in the tbx5 gene locus generate protein isoforms with distinct expression domains and function.

    PubMed

    Yamak, Abir; Georges, Romain O; Sheikh-Hassani, Massomeh; Morin, Martin; Komati, Hiba; Nemer, Mona

    2015-03-13

    TBX5 is the gene mutated in Holt-Oram syndrome, an autosomal dominant disorder with complex heart and limb deformities. Its protein product is a member of the T-box family of transcription factors and an evolutionarily conserved dosage-sensitive regulator of heart and limb development. Understanding TBX5 regulation is therefore of paramount importance. Here we uncover the existence of novel exons and provide evidence that TBX5 activity may be extensively regulated through alternative splicing to produce protein isoforms with differing N- and C-terminal domains. These isoforms are also present in human heart, indicative of an evolutionarily conserved regulatory mechanism. The newly identified isoforms have different transcriptional properties and can antagonize TBX5a target gene activation. Droplet Digital PCR as well as immunohistochemistry with isoform-specific antibodies reveal differential as well as overlapping expression domains. In particular, we find that the predominant isoform in skeletal myoblasts is Tbx5c, and we show that it is dramatically up-regulated in differentiating myotubes and is essential for myotube formation. Mechanistically, TBX5c antagonizes TBX5a activation of pro-proliferative signals such as IGF-1, FGF-10, and BMP4. The results provide new insight into Tbx5 regulation and function that will further our understanding of its role in health and disease. The finding of new exons in the Tbx5 locus may also be relevant to mutational screening especially in the 30% of Holt-Oram syndrome patients with no mutations in the known TBX5a exons.

  19. Genes encoding proteins with peritrophin A-type chitin-binding domains in Tribolium castaneum are grouped into three distinct families based on phylogeny, expression and function.

    PubMed

    Jasrapuria, Sinu; Arakane, Yasuyuki; Osman, Gamal; Kramer, Karl J; Beeman, Richard W; Muthukrishnan, Subbaratnam

    2010-03-01

    different orders suggests that ChtBD2s are ancient protein domains whose affinity for chitin in extracellular matrices has been exploited many times for a range of biological functions. The differences in the expression profiles of PMPs and CPAPs indicate that even though they share the peritrophin A motif for chitin binding, these three families of proteins have quite distinct biological functions.

  20. Distinct functional domains within the acidic cluster of tegument protein pp28 required for trafficking and cytoplasmic envelopment of human cytomegalovirus.

    PubMed

    Seo, Jun-Young; Jeon, Hyejin; Hong, Sookyung; Britt, William J

    2016-10-01

    Human cytomegalovirus UL99-encoded tegument protein pp28 contains a 16 aa acidic cluster that is required for pp28 trafficking to the assembly compartment (AC) and the virus assembly. However, functional signals within the acidic cluster of pp28 remain undefined. Here, we demonstrated that an acidic cluster rather than specific sorting signals was required for trafficking to the AC. Recombinant viruses with chimeric pp28 proteins expressing non-native acidic clusters exhibited delayed viral growth kinetics and decreased production of infectious virus, indicating that the native acidic cluster of pp28 was essential for wild-type virus assembly. These results suggested that the acidic cluster of pp28 has distinct functional domains required for trafficking and for efficient virus assembly. The first half (aa 44-50) of the acidic cluster was sufficient for pp28 trafficking, whereas the native acidic cluster consisting of aa 51-59 was required for the assembly of wild-type levels of infectious virus.

  1. Distinct functional and conformational states of the human lymphoid tyrosine phosphatase catalytic domain can be targeted by choice of the inhibitor chemotype

    NASA Astrophysics Data System (ADS)

    Vidović, Dušica; Xie, Yuli; Rinderspacher, Alison; Deng, Shi-Xian; Landry, Donald W.; Chung, Caty; Smith, Deborah H.; Tautz, Lutz; Schürer, Stephan C.

    2011-09-01

    The lymphoid tyrosine phosphatase (LYP), encoded by the PTPN22 gene, has recently been identified as a promising drug target for human autoimmunity diseases. Like the majority of protein-tyrosine phosphatases LYP can adopt two functionally distinct forms determined by the conformation of the WPD-loop. The WPD-loop plays an important role in the catalytic dephosphorylation by protein-tyrosine phosphatases. Here we investigate the binding modes of two chemotypes of small molecule LYP inhibitors with respect to both protein conformations using computational modeling. To evaluate binding in the active form, we built a LYP protein structure model of high quality. Our results suggest that the two different compound classes investigated, bind to different conformations of the LYP phosphatase domain. Binding to the closed form is facilitated by an interaction with Asp195 in the WPD-loop, presumably stabilizing the active conformation. The analysis presented here is relevant for the design of inhibitors that specifically target either the closed or the open conformation of LYP in order to achieve better selectivity over phosphatases with similar binding sites.

  2. 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…

  3. Functional characterization of a StyS sensor kinase reveals distinct domains associated with intracellular and extracellular sensing of styrene in P. putida CA-3.

    PubMed

    O'Leary, Niall D; Mooney, Aisling; O'Mahony, Mark; Dobson, Alan Dw

    2014-01-01

    Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor and/or bio-reporter strains. In this study we functionally characterize the multi-domain StyS sensor kinase associated with sensing of the aromatic pollutant styrene by Pseudomonas putida CA-3. Deletion analysis of discrete domains was performed and the ability of the truncated StyS sensor proteins to activate a cognate reporter system in an E. coli host assessed. The essential histidine kinase and PAS input domains were identified for StyS dependent activation of the reporter system. However, co-expression of an ABC-transporter protein StyE, previously linked to styrene transport in P. putida CA-3, enabled activation of the reporter system with a StyS construct containing a non-essential PAS input domain, suggesting a novel role for intracellular detection and/or activation. Site directed mutagenesis and amino acid deletions were employed to further characterize the PAS sensing domains of both input regions. The potential implications of these findings in the use of multi-domain sensor kinases in rational design strategies and the potential link between transport and intracellular sensing are discussed.

  4. Functional characterization of a StyS sensor kinase reveals distinct domains associated with intracellular and extracellular sensing of styrene in P. putida CA-3

    PubMed Central

    O'Leary, Niall D; Mooney, Aisling; O'Mahony, Mark; Dobson, Alan DW

    2014-01-01

    Bacterial two-component systems (TCSs) are of vital importance in the translation of rapidly changing environmental conditions into appropriate cellular regulatory responses enabling adaptation, growth, and survival. The diverse range of environmental signals that TCSs can process, coupled with discrete modular domains within TCS proteins, offers considerable potential for the rational design of bio-sensor and/or bio-reporter strains. In this study we functionally characterize the multi-domain StyS sensor kinase associated with sensing of the aromatic pollutant styrene by Pseudomonas putida CA-3. Deletion analysis of discrete domains was performed and the ability of the truncated StyS sensor proteins to activate a cognate reporter system in an E. coli host assessed. The essential histidine kinase and PAS input domains were identified for StyS dependent activation of the reporter system. However, co-expression of an ABC-transporter protein StyE, previously linked to styrene transport in P. putida CA-3, enabled activation of the reporter system with a StyS construct containing a non-essential PAS input domain, suggesting a novel role for intracellular detection and/or activation. Site directed mutagenesis and amino acid deletions were employed to further characterize the PAS sensing domains of both input regions. The potential implications of these findings in the use of multi-domain sensor kinases in rational design strategies and the potential link between transport and intracellular sensing are discussed. PMID:24637704

  5. Distinct function of COAR and B3 domains of maize VP1 in induction of ectopic gene expression and plant developmental phenotypes in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize VP1 enhancement of ABA sensitivity in roots is B3 independent. ABA and VP1 mediated suppression of auxin induced lateral root development is B3 independent. Arabidopsis transgenic system to delineate B3 dependent and COAR domain dependent regulatory functions of VP1. Analyses of ectopic ABA re...

  6. Functional domains in tetraspanin proteins.

    PubMed

    Stipp, Christopher S; Kolesnikova, Tatiana V; Hemler, Martin E

    2003-02-01

    Exciting new findings have emerged about the structure, function and biochemistry of tetraspanin proteins. Five distinct tetraspanin regions have now been delineated linking structural features to specific functions. Within the large extracellular loop of tetraspanins, there is a variable region that mediates specific interactions with other proteins, as well as a more highly conserved region that has been suggested to mediate homodimerization. Within the transmembrane region, the four tetraspanin transmembrane domains are probable sites of both intra- and inter-molecular interactions that are crucial during biosynthesis and assembly of the network of tetraspanin-linked membrane proteins known as the 'tetraspanin web'. In the intracellular juxtamembrane region, palmitoylation of cysteine residues also contributes to tetraspanin web assembly, and the C-terminal cytoplasmic tail region could provide specific functional links to cytoskeletal or signaling proteins.

  7. Molecular mechanisms of protein-cholesterol interactions in plasma membranes: Functional distinction between topological (tilted) and consensus (CARC/CRAC) domains.

    PubMed

    Fantini, Jacques; Di Scala, Coralie; Baier, Carlos J; Barrantes, Francisco J

    2016-09-01

    The molecular mechanisms that control the multiple possible modes of protein association with membrane cholesterol are remarkably convergent. These mechanisms, which include hydrogen bonding, CH-π stacking and dispersion forces, are used by a wide variety of extracellular proteins (e.g. microbial or amyloid) and membrane receptors. Virus fusion peptides penetrate the membrane of host cells with a tilted orientation that is compatible with a transient interaction with cholesterol; this tilted orientation is also characteristic of the process of insertion of amyloid proteins that subsequently form oligomeric pores in the plasma membrane of brain cells. Membrane receptors that are associated with cholesterol generally display linear consensus binding motifs (CARC and CRAC) characterized by a triad of basic (Lys/Arg), aromatic (Tyr/phe) and aliphatic (Leu/Val) amino acid residues. In some cases, the presence of both CARC and CRAC within the same membrane-spanning domain allows the simultaneous binding of two cholesterol molecules, one in each membrane leaflet. In this review the molecular basis and the functional significance of the different modes of protein-cholesterol interactions in plasma membranes are discussed.

  8. Genes encoding proteins with peritrophin A-type chitin-binding domains in Tribolium castaneum are grouped into three distinct families based on phylogeny, expression and function

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study is focused on the characterization and expression of genes in the red flour beetle, Tribolium castaneum, encoding proteins that possess six-cysteine-containing chitin-binding domains (CBDs) related to the peritrophin A domain (ChtBD2). An exhaustive bioinformatics search of the genome of...

  9. Distinct Functional Domains Contribute to Degradation of the Low Density Lipoprotein Receptor (LDLR) by the E3 Ubiquitin Ligase Inducible Degrader of the LDLR (IDOL)

    PubMed Central

    Sorrentino, Vincenzo; Scheer, Lilith; Santos, Ana; Reits, Eric; Bleijlevens, Boris; Zelcer, Noam

    2011-01-01

    We recently identified the liver X receptor-regulated E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) as a modulator of lipoprotein metabolism. Acting as an E3 ubiquitin ligase, IDOL triggers ubiquitination and subsequent degradation of the low density lipoprotein receptor (LDLR). We demonstrate here that this outcome requires the conserved FERM and RING domains present in IDOL. The RING domain promotes ubiquitination in vitro and Lys-63-specific ubiquitination of the LDLR in vivo in response to IDOL or liver X receptor activation. We further identify RING residues that differentially influence ubiquitination of the LDLR or stability of IDOL. The FERM domain interacts with the LDLR and in living cells co-localizes with the receptor at the plasma membrane. Homology modeling revealed a phosphotyrosine-binding element embedded in the FERM domain. Mutating residues within this region or residues in the LDLR preceding the NPVY endocytosis motif abrogate LDLR degradation by IDOL. Collectively, our results indicate that both the FERM and RING domains are required for promoting lysosomal degradation of the LDLR by IDOL. Our findings may facilitate development of structure-based IDOL inhibitors aimed at increasing LDLR abundance in therapeutic strategies to treat cardiovascular disease. PMID:21734303

  10. Two distinct variants of erythrocyte spectrin beta IV domain.

    PubMed

    Pothier, B; Alloisio, N; Morlé, L; Maréchal, J; Barthélemy, H; Ducluzeau, M T; Dorier, A; Delaunay, J

    1989-11-01

    We report two distinct variants affecting the beta IV domain of erythrocyte spectrin, designated spectrin Saint-Chamond and spectrin Tlemcen. They were discovered in a French family and an Algerian individual, respectively. They appeared clinically and morphologically asymptomatic in the heterozygous state. In two-dimensional maps of spectrin partial digests, both mutants were manifested by cathodic shifts (with no change of the molecular weights) of the peptides that cover the N-terminal region of spectrin beta IV domain. The relevance of the abnormal peptides to the beta IV domain was established by quantitative analysis and by Western blotting using anti-beta IV domain-specific antibodies. These two variants are thus far the most distal variants of spectrin to be defined on an unequivocal structural basis.

  11. The C-Terminal Region of Lymphocytic Choriomeningitis Virus Nucleoprotein Contains Distinct and Segregable Functional Domains Involved in NP-Z Interaction and Counteraction of the Type I Interferon Response▿

    PubMed Central

    Ortiz-Riaño, Emilio; Cheng, Benson Yee Hin; de la Torre, Juan Carlos; Martínez-Sobrido, Luis

    2011-01-01

    Several arenaviruses cause hemorrhagic fever (HF) disease in humans that is associated with high morbidity and significant mortality. Arenavirus nucleoprotein (NP), the most abundant viral protein in infected cells and virions, encapsidates the viral genome RNA, and this NP-RNA complex, together with the viral L polymerase, forms the viral ribonucleoprotein (vRNP) that directs viral RNA replication and gene transcription. Formation of infectious arenavirus progeny requires packaging of vRNPs into budding particles, a process in which arenavirus matrix-like protein (Z) plays a central role. In the present study, we have characterized the NP-Z interaction for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). The LCMV NP domain that interacted with Z overlapped with a previously documented C-terminal domain that counteracts the host type I interferon (IFN) response. However, we found that single amino acid mutations that affect the anti-IFN function of LCMV NP did not disrupt the NP-Z interaction, suggesting that within the C-terminal region of NP different amino acid residues critically contribute to these two distinct and segregable NP functions. A similar NP-Z interaction was confirmed for the HF arenavirus Lassa virus (LASV). Notably, LCMV NP interacted similarly with both LCMV Z and LASV Z, while LASV NP interacted only with LASV Z. Our results also suggest the presence of a conserved protein domain within NP but with specific amino acid residues playing key roles in determining the specificity of NP-Z interaction that may influence the viability of reassortant arenaviruses. In addition, this NP-Z interaction represents a potential target for the development of antiviral drugs to combat human-pathogenic arenaviruses. PMID:21976642

  12. SH3 Domains Differentially Stimulate Distinct Dynamin I Assembly Modes and G Domain Activity

    PubMed Central

    Krishnan, Sai; Collett, Michael; Robinson, Phillip J.

    2015-01-01

    Dynamin I is a highly regulated GTPase enzyme enriched in nerve terminals which mediates vesicle fission during synaptic vesicle endocytosis. One regulatory mechanism involves its interactions with proteins containing Src homology 3 (SH3) domains. At least 30 SH3 domain-containing proteins bind dynamin at its proline-rich domain (PRD). Those that stimulate dynamin activity act by promoting its oligomerisation. We undertook a systematic parallel screening of 13 glutathione-S-transferase (GST)-tagged endocytosis-related SH3 domains on dynamin binding, GTPase activity and oligomerisation. No correlation was found between dynamin binding and their potency to stimulate GTPase activity. There was limited correlation between the extent of their ability to stimulate dynamin activity and the level of oligomerisation, indicating an as yet uncharacterised allosteric coupling of the PRD and G domain. We examined the two variants, dynamin Iab and Ibb, which differ in the alternately splice middle domain α2 helix. They responded differently to the panel of SH3s, with the extent of stimulation between the splice variants varying greatly between the SH3s. This study reveals that SH3 binding can act as a heterotropic allosteric regulator of the G domain via the middle domain α2 helix, suggesting an involvement of this helix in communicating the PRD-mediated allostery. This indicates that SH3 binding both stabilises multiple conformations of the tetrameric building block of dynamin, and promotes assembly of dynamin-SH3 complexes with distinct rates of GTP hydrolysis. PMID:26659814

  13. 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.

  14. Structural and Functional Analysis of Multi-Interface Domains

    PubMed Central

    Zhao, Liang; Hoi, Steven C. H.; Wong, Limsoon; Hamp, Tobias; Li, Jinyan

    2012-01-01

    A multi-interface domain is a domain that can shape multiple and distinctive binding sites to contact with many other domains, forming a hub in domain-domain interaction networks. The functions played by the multiple interfaces are usually different, but there is no strict bijection between the functions and interfaces as some subsets of the interfaces play the same function. This work applies graph theory and algorithms to discover fingerprints for the multiple interfaces of a domain and to establish associations between the interfaces and functions, based on a huge set of multi-interface proteins from PDB. We found that about 40% of proteins have the multi-interface property, however the involved multi-interface domains account for only a tiny fraction (1.8%) of the total number of domains. The interfaces of these domains are distinguishable in terms of their fingerprints, indicating the functional specificity of the multiple interfaces in a domain. Furthermore, we observed that both cooperative and distinctive structural patterns, which will be useful for protein engineering, exist in the multiple interfaces of a domain. PMID:23272073

  15. Apo raver1 structure reveals distinct RRM domain orientations

    SciTech Connect

    Rangarajan, Erumbi S.; Lee, Jun Hyuck; Izard, Tina

    2012-09-17

    Raver1 is a multifunctional protein that modulates both alternative splicing and focal adhesion assembly by binding to the nucleoplasmic splicing repressor polypyrimidine tract protein (PTB) or to the cytoskeletal proteins vinculin and {alpha}-actinin. The amino-terminal region of raver1 has three RNA recognition motif (RRM1, RRM2, and RRM3) domains, and RRM1 interacts with the vinculin tail (Vt) domain and vinculin mRNA. We previously determined the crystal structure of the raver1 RRM1-3 domains in complex with Vt at 2.75 {angstrom} resolution. Here, we report crystal structure of the unbound raver1 RRM1-3 domains at 2 {angstrom} resolution. The apo structure reveals that a bound sulfate ion disrupts an electrostatic interaction between the RRM1 and RRM2 domains, triggering a large relative domain movement of over 30{sup o}. Superposition with other RNA-bound RRM structures places the sulfate ion near the superposed RNA phosphate group suggesting that this is the raver1 RNA binding site. While several single and some tandem RRM domain structures have been described, to the best of our knowledge, this is the second report of a three-tandem RRM domain structure.

  16. Three Distinct Domains Contribute to Nuclear Transport of Murine Foxp3

    PubMed Central

    Hancock, Wayne W.; Özkaynak, Engin

    2009-01-01

    Foxp3, a 47-kDa transcription factor, is necessary for the function of CD4+CD25+ regulatory T cells (Tregs), with an essential role in the control of self-reactive T cells and in preventing autoimmunity. Activation of Tregs by TCR engagement results in upregulation of Foxp3 expression, followed by its rapid nuclear transport and binding to chromatin. Here, we identify three distinct Foxp3 domains that contribute to nuclear transport. The first domain (Domain 1) comprises the C-terminal 12 amino acids. The second domain (Domain 2) is located immediately N-terminal to the forkhead domain (FHD), recently reported to be a binding site for the runt-related transcription factor 1/acute myeloid leukemia 1 (Runx1/AML1). The third domain (Domain 3) is located within the N-terminal first 51 amino acids. Unlike the known nuclear localization signals (NLSs), none of these three regions are rich in basic residues and do not bear any similarity to known monopartite or bipartite NLSs that have one or more clusters of basic amino acids. The basic arginine-lysine-lysine-arginine (RKKR) sequence, located 12-aa from the C-terminal end of Foxp3 was previously reported to be a nuclear localization signal (NLS) for several proteins, including for a GFP-Foxp3 hybrid. Evidence is provided here that in the full-length native Foxp3 RKKR does not function as an NLS. The data reported in this study indicates that Foxp3 achieves nuclear transport by binding to other nuclear factors and co-transporting with them to the nucleus. PMID:19924293

  17. Morphologically and Functionally Distinct Lipid Droplet Subpopulations

    PubMed Central

    Zhang, Shuyan; Wang, Yang; Cui, Liujuan; Deng, Yaqin; Xu, Shimeng; Yu, Jinhai; Cichello, Simon; Serrero, Ginette; Ying, Yunshu; Liu, Pingsheng

    2016-01-01

    Lipid droplet (LD), a multi-functional organelle, is often found to associate with other cellular membranous structures and vary in size in a given cell, which may be related to their functional diversity. Here we established a method to separate LD subpopulations from isolated CHO K2 LDs into three different size categories. The subpopulation with smallest LDs was nearly free of ER and other membranous structures while those with larger LDs contained intact ER. These distinct subpopulations of LDs differed in their protein composition and ability to recruit proteins. This method was also applicable to LDs obtained from other sources, such as Huh7 cells, mouse liver and brown adipose tissue, et al. We developed an in vitro assay requiring only isolated LDs, Coenzyme A, and ATP to drive lipid synthesis. The LD subpopulation nearly depleted of ER was able to incorporate fatty acids into triacylglycerol and phospholipids. Together, our data demonstrate that LDs in a given cell are heterogeneous in size and function, and suggest that LDs are one of cellular lipid synthetic organelles. PMID:27386790

  18. Cooperative unfolding of distinctive mechanoreceptor domains transduces force into signals

    PubMed Central

    Ju, Lining; Chen, Yunfeng; Xue, Lingzhou; Du, Xiaoping; Zhu, Cheng

    2016-01-01

    How cells sense their mechanical environment and transduce forces into biochemical signals is a crucial yet unresolved question in mechanobiology. Platelets use receptor glycoprotein Ib (GPIb), specifically its α subunit (GPIbα), to signal as they tether and translocate on von Willebrand factor (VWF) of injured arterial surfaces against blood flow. Force elicits catch bonds to slow VWF–GPIbα dissociation and unfolds the GPIbα leucine-rich repeat domain (LRRD) and juxtamembrane mechanosensitive domain (MSD). How these mechanical processes trigger biochemical signals remains unknown. Here we analyze these extracellular events and the resulting intracellular Ca2+ on a single platelet in real time, revealing that LRRD unfolding intensifies Ca2+ signal whereas MSD unfolding affects the type of Ca2+ signal. Therefore, LRRD and MSD are analog and digital force transducers, respectively. The >30 nm macroglycopeptide separating the two domains transmits force on the VWF–GPIbα bond (whose lifetime is prolonged by LRRD unfolding) to the MSD to enhance its unfolding, resulting in unfolding cooperativity at an optimal force. These elements may provide design principles for a generic mechanosensory protein machine. DOI: http://dx.doi.org/10.7554/eLife.15447.001 PMID:27434669

  19. Domain-specific knowledge systems in the brain the animate-inanimate distinction.

    PubMed

    Caramazza, A; Shelton, J R

    1998-01-01

    We claim that the animate and inanimate conceptual categories represent evolutionarily adapted domain-specific knowledge systems that are subserved by distinct neural mechanisms, thereby allowing for their selective impairment in conditions of brain damage. On this view, (some of) the category-specific deficits that have recently been reported in the cognitive neuropsychological literature - for example, the selective damage or sparing of knowledge about animals - are truly categorical effects. Here, we articulate and defend this thesis against the dominant, reductionist theory of category-specific deficits, which holds that the categorical nature of the deficits is the result of selective damage to noncategorically organized visual or functional semantic subsystems. On the latter view, the sensory/functional dimension provides the fundamental organizing principle of the semantic system. Since, according to the latter theory, sensory and functional properties are differentially important in determining the meaning of the members of different semantic categories, selective damage to the visual or the functional semantic subsystem will result in a category-like deficit. A review of the literature and the results of a new case of category-specific deficit will show that the domain-specific knowledge framework provides a better account of category-specific deficits than the sensory/functional dichotomy theory.

  20. Natural Microbial Assemblages Reflect Distinct Organismal and Functional Partitioning

    NASA Astrophysics Data System (ADS)

    Wilmes, P.; Andersson, A.; Kalnejais, L. H.; Verberkmoes, N. C.; Lefsrud, M. G.; Wexler, M.; Singer, S. W.; Shah, M.; Bond, P. L.; Thelen, M. P.; Hettich, R. L.; Banfield, J. F.

    2007-12-01

    The ability to link microbial community structure to function has long been a primary focus of environmental microbiology. With the advent of community genomic and proteomic techniques, along with advances in microscopic imaging techniques, it is now possible to gain insights into the organismal and functional makeup of microbial communities. Biofilms growing within highly acidic solutions inside the Richmond Mine (Iron Mountain, Redding, California) exhibit distinct macro- and microscopic morphologies. They are composed of microorganisms belonging to the three domains of life, including archaea, bacteria and eukarya. The proportion of each organismal type depends on sampling location and developmental stage. For example, mature biofilms floating on top of acid mine drainage (AMD) pools exhibit layers consisting of a densely packed bottom layer of the chemoautolithotroph Leptospirillum group II, a less dense top layer composed mainly of archaea, and fungal filaments spanning across the entire biofilm. The expression of cytochrome 579 (the most highly abundant protein in the biofilm, believed to be central to iron oxidation and encoded by Leptospirillum group II) is localized at the interface of the biofilm with the AMD solution, highlighting that biofilm architecture is reflected at the functional gene expression level. Distinct functional partitioning is also apparent in a biological wastewater treatment system that selects for distinct polyphosphate accumulating organisms. Community genomic data from " Candidatus Accumulibacter phosphatis" dominated activated sludge has enabled high mass-accuracy shotgun proteomics for identification of key metabolic pathways. Comprehensive genome-wide alignment of orthologous proteins suggests distinct partitioning of protein variants involved in both core-metabolism and specific metabolic pathways among the dominant population and closely related species. In addition, strain- resolved proteogenomic analysis of the AMD biofilms

  1. Transcriptional activation by the acidic domain of Vmw65 requires the integrity of the domain and involves additional determinants distinct from those necessary for TFIIB binding.

    PubMed

    Walker, S; Greaves, R; O'Hare, P

    1993-09-01

    In this work we have examined the requirements for activity of the acidic domain of Vmw65 (VP16) by deletion and site-directed mutagenesis of the region in the context of GAL4 fusion proteins. The results indicate that the present interpretation of what actually constitutes the activation domain is not correct. We demonstrate, using a promoter with one target site which is efficiently activated by the wild-type (wt) fusion protein, that amino acids distal to residue 453 are critical for activity. Truncation of the domain or substitution of residues in the distal region almost completely abrogate activity. However, inactivating mutations within the distal region are complemented by using a promoter containing multiple target sites. Moreover, duplication of the proximal region, but not the distal region, restores the ability to activate a promoter with a single target site. These results indicate some distinct qualitative difference between the proximal and distal regions. We have also examined the binding of nuclear proteins to the wt domain and to a variant with the distal region inactivated by mutation. The lack of activity of this variant is not explained by a lack of binding of TFIIB, a protein previously reported to be the likely target of the acidic domain. Therefore some additional function is involved in transcriptional activation by the acid domain, and determinants distinct from those involved in TFIIB binding are required for this function. Analysis of the total protein profiles binding to the wt and mutant domains has demonstrated the selective binding to the wt domain of a 135-kDa polypeptide, which is therefore a candidate component involved in this additional function. This is the first report to provide evidence for the proposal of a multiplicity of interactions within the acidic domain, by uncoupling requirements for one function from those for another.

  2. DEP domains: structurally similar but functionally different.

    PubMed

    Consonni, Sarah V; Maurice, Madelon M; Bos, Johannes L

    2014-05-01

    The Dishevelled, EGL-10 and pleckstrin (DEP) domain is a globular protein domain that is present in about ten human protein families with well-defined structural features. A picture is emerging that DEP domains mainly function in the spatial and temporal control of diverse signal transduction events by recruiting proteins to the plasma membrane. DEP domains can interact with various partners at the membrane, including phospholipids and membrane receptors, and their binding is subject to regulation.

  3. Epigenetic and genetic deregulation in cancer target distinct signaling pathway domains

    PubMed Central

    Gao, Yang; Teschendorff, Andrew E.

    2017-01-01

    Cancer is characterized by both genetic and epigenetic alterations. While cancer driver mutations and copy-number alterations have been studied at a systems-level, relatively little is known about the systems-level patterns exhibited by their epigenetic counterparts. Here we perform a pan-cancer wide systems-level analysis, mapping candidate cancer-driver DNA methylation (DNAm) alterations onto a human interactome. We demonstrate that functional DNAm alterations in cancer tend to map to nodes of lower connectivity and inter-connectivity, compared to the corresponding alterations at the genomic level. We find that epigenetic alterations are relatively over-represented in extracellular and transmembrane signaling domains, whereas cancer genes undergoing amplification or deletion tend to be enriched within the intracellular domain. A pan-cancer wide meta-analysis identifies WNT and chemokine signaling, as two key pathways where epigenetic deregulation preferentially targets extracellular components. We further pinpoint specific chemokine ligands/receptors whose epigenetic deregulation associates with key epigenetic enzymes, representing potential targets for epigenetic therapy. Our results suggest that epigenetic deregulation in cancer not only targets tissue-specific transcription factors, but also modulates signaling within the extra-cellular domain, providing novel system-level insight into the potential distinctive role of genetic and epigenetic alterations in cancer. PMID:27899617

  4. Structural and functional diversity of Topologically Associating Domains.

    PubMed

    Dekker, Job; Heard, Edith

    2015-10-07

    Recent studies have shown that chromosomes in a range of organisms are compartmentalized in different types of chromatin domains. In mammals, chromosomes form compartments that are composed of smaller Topologically Associating Domains (TADs). TADs are thought to represent functional domains of gene regulation but much is still unknown about the mechanisms of their formation and how they exert their regulatory effect on embedded genes. Further, similar domains have been detected in other organisms, including flies, worms, fungi and bacteria. Although in all these cases these domains appear similar as detected by 3C-based methods, their biology appears to be quite distinct with differences in the protein complexes involved in their formation and differences in their internal organization. Here we outline our current understanding of such domains in different organisms and their roles in gene regulation.

  5. Functions and Functional Domains of the GTPase Cdc42p

    PubMed Central

    Kozminski, Keith G.; Chen, Ann J.; Rodal, Avital A.; Drubin, David G.

    2000-01-01

    Cdc42p, a Rho family GTPase of the Ras superfamily, is a key regulator of cell polarity and morphogenesis in eukaryotes. Using 37 site-directed cdc42 mutants, we explored the functions and interactions of Cdc42p in the budding yeast Saccharomyces cerevisiae. Cytological and genetic analyses of these cdc42 mutants revealed novel and diverse phenotypes, showing that Cdc42p possesses at least two distinct essential functions and acts as a nodal point of cell polarity regulation in vivo. In addition, mapping the functional data for each cdc42 mutation onto a structural model of the protein revealed as functionally important a surface of Cdc42p that is distinct from the canonical protein-interacting domains (switch I, switch II, and the C terminus) identified previously in members of the Ras superfamily. This region overlaps with a region (α5-helix) recently predicted by structural models to be a specificity determinant for Cdc42p-protein interactions. PMID:10637312

  6. Structure and function of KH domains.

    PubMed

    Valverde, Roberto; Edwards, Laura; Regan, Lynne

    2008-06-01

    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.

  7. 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.

  8. Distinct functional domains in nesprin-1{alpha} and nesprin-2{beta} bind directly to emerin and both interactions are disrupted in X-linked Emery-Dreifuss muscular dystrophy

    SciTech Connect

    Wheeler, Matthew A.; Davies, John D.; Zhang Qiuping; Emerson, Lindsay J.; Hunt, James; Shanahan, Catherine M.; Ellis, Juliet A. . E-mail: juliet.ellis@kcl.ac.uk

    2007-08-01

    Emerin and specific isoforms of nesprin-1 and -2 are nuclear membrane proteins which are binding partners in multi-protein complexes spanning the nuclear envelope. We report here the characterisation of the residues both in emerin and in nesprin-1{alpha} and -2{beta} which are involved in their interaction and show that emerin requires nesprin-1 or -2 to retain it at the nuclear membrane. Using several protein-protein interaction methods, we show that residues 368 to 627 of nesprin-1{alpha} and residues 126 to 219 of nesprin-2{beta}, which show high homology to one another, both mediate binding to emerin residues 140-176. This region has previously been implicated in binding to F-actin, {beta}-catenin and lamin A/C suggesting that it is critical for emerin function. Confirmation that these protein domains interact in vivo was shown using GFP-dominant negative assays. Exogenous expression of either of these nesprin fragments in mouse myoblast C2C12 cells displaced endogenous emerin from the nuclear envelope and reduced the targeting of newly synthesised emerin. Furthermore, we are the first to report that emerin mutations which give rise to X-linked Emery-Dreifuss muscular dystrophy, disrupt binding to both nesprin-1{alpha} and -2{beta} isoforms, further indicating a role of nesprins in the pathology of Emery-Dreifuss muscular dystrophy.

  9. 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.

  10. Dystrophin colocalizes with beta-spectrin in distinct subsarcolemmal domains in mammalian skeletal muscle

    PubMed Central

    1992-01-01

    Duchenne's muscular dystrophy (DMD) is caused by the absence or drastic decrease of the structural protein, dystrophin, and is characterized by sarcolemmal lesions in skeletal muscle due to the stress of contraction. Dystrophin has been localized to the sarcolemma, but its organization there is not known. We report immunofluorescence studies which show that dystrophin is concentrated, along with the major muscle isoform of beta-spectrin, in three distinct domains at the sarcolemma: in elements overlying both I bands and M lines, and in occasional strands running along the longitudinal axis of the myofiber. Vinculin, which has previously been found at the sarcolemma overlying the I bands and in longitudinal strands, was present in the same three structures as spectrin and dystrophin. Controls demonstrated that the labeling was intracellular. Comparison to labeling of the lipid bilayer and of the extracellular matrix showed that the labeling for spectrin and dystrophin is associated with the intact sarcolemma and is not a result of processing artifacts. Dystrophin is not required for this lattice- like organization, as similar domains containing spectrin but not dystrophin are present in muscle from the mdx mouse and from humans with Duchenne's muscular dystrophy. We discuss the possibility that dystrophin and spectrin, along with vinculin, may function to link the contractile apparatus to the sarcolemma of normal skeletal muscle. PMID:1577872

  11. Functional divergence in the Arabidopsis LOB-domain gene family

    PubMed Central

    Mangeon, Amanda; Lin, Wan-ching; Springer, Patricia S.

    2012-01-01

    The Arabidopsis LOB-domain (LBD) gene family is composed by 43 members divided in two classes based on amino acid conservation within the LOB-domain. The LOB domain is known to be responsible for DNA binding and protein-protein interactions. There is very little functional information available for most genes in the LBD family and many lbd single mutants do not exhibit conspicuous phenotypes. One plausible explanation for the limited loss-of-function phenotypes observed in this family is that LBD genes exhibit significant functional redundancy. Here we discuss an example of one phylogenetic subgroup of the LBD family, in which genes that are closely related based on phylogeny exhibit distinctly different expression patterns and do not have overlapping functions. We discuss the challenges of using phylogenetic analyses to predict redundancy in gene families. PMID:23073009

  12. TIM-3 Regulates Distinct Functions in Macrophages

    PubMed Central

    Ocaña-Guzman, Ranferi; Torre-Bouscoulet, Luis; Sada-Ovalle, Isabel

    2016-01-01

    The transmembrane protein TIM-3 is a type I protein expressed by sub-types of lymphoid cells, such as lymphocytes Th1, Th17, Tc1, NK, as well as in myeloid cells. Scientific evidence indicates that this molecule acts as a negative regulator of T lymphocyte activation and that its expression is modified in viral infections or autoimmune diseases. In addition to evidence from lymphoid cells, the function of TIM-3 has been investigated in myeloid cells, such as monocytes, macrophages, and dendritic cells (DC), where studies have demonstrated that it can regulate cytokine production, cell activation, and the capture of apoptotic bodies. Despite these advances, the function of TIM-3 in myeloid cells and the molecular mechanisms that this protein regulates are not yet fully understood. This review examines the most recent evidence concerning the function of TIM-3 when expressed in myeloid cells, primarily macrophages, and the potential impact of that function on the field of basic immunology. PMID:27379093

  13. Temporal stability and representational distinctiveness: Key functions of orthographic working memory

    PubMed Central

    Costa, Vanessa; Fischer-Baum, Simon; Capasso, Rita; Miceli, Gabriele; Rapp, Brenda

    2012-01-01

    A primary goal of working memory research has been to understand the mechanisms that permit working memory systems to effectively maintain the identity and order of the elements held in memory for sufficient time as to allow for their selection and transfer to subsequent processing stages. Based on the performance of two individuals with acquired dysgraphia affecting orthographic WM (the graphemic buffer) we present evidence of two distinct and dissociable functions of orthographic WM. One function is responsible for maintaining the temporal stability of letters held in orthographic WM, while the other is responsible for maintaining their representational distinctiveness. The failure to maintain temporal stability and representational distinctiveness give rise, respectively, to decay and interference effects that manifest themselves in distinctive error patterns, including distinct serial position effects. The findings we report have implications beyond our understanding of orthographic WM, as the need to maintain temporal stability and representational distinctiveness in WM is common across cognitive domains. PMID:22248210

  14. J domain independent functions of J proteins.

    PubMed

    Ajit Tamadaddi, Chetana; Sahi, Chandan

    2016-07-01

    Heat shock proteins of 40 kDa (Hsp40s), also called J proteins, are obligate partners of Hsp70s. Via their highly conserved and functionally critical J domain, J proteins interact and modulate the activity of their Hsp70 partners. Mutations in the critical residues in the J domain often result in the null phenotype for the J protein in question. However, as more J proteins have been characterized, it is becoming increasingly clear that a significant number of J proteins do not "completely" rely on their J domains to carry out their cellular functions, as previously thought. In some cases, regions outside the highly conserved J domain have become more important making the J domain dispensable for some, if not for all functions of a J protein. This has profound effects on the evolution of such J proteins. Here we present selected examples of J proteins that perform J domain independent functions and discuss this in the context of evolution of J proteins with dispensable J domains and J-like proteins in eukaryotes.

  15. The structure of a conserved piezo channel domain reveals a topologically distinct β sandwich fold.

    PubMed

    Kamajaya, Aron; Kaiser, Jens T; Lee, Jonas; Reid, Michelle; Rees, Douglas C

    2014-10-07

    Piezo has recently been identified as a family of eukaryotic mechanosensitive channels composed of subunits containing over 2,000 amino acids, without recognizable sequence similarity to other channels. Here, we present the crystal structure of a large, conserved extramembrane domain located just before the last predicted transmembrane helix of C. elegans PIEZO, which adopts a topologically distinct β sandwich fold. The structure was also determined of a point mutation located on a conserved surface at the position equivalent to the human PIEZO1 mutation found in dehydrated hereditary stomatocytosis patients (M2225R). While the point mutation does not change the overall domain structure, it does alter the surface electrostatic potential that may perturb interactions with a yet-to-be-identified ligand or protein. The lack of structural similarity between this domain and any previously characterized fold, including those of eukaryotic and bacterial channels, highlights the distinctive nature of the Piezo family of eukaryotic mechanosensitive channels.

  16. The activation domain of a basic helix-loop-helix protein is masked by repressor interaction with domains distinct from that required for transcription regulation.

    PubMed Central

    Jayaraman, P S; Hirst, K; Goding, C R

    1994-01-01

    While there are many examples of protein-protein interactions modulating the DNA-binding activity of transcription factors, little is known of the molecular mechanisms underlying the regulation of the transcription activation function. Using a two-hybrid system we show here that transcription repression of the basic domain/helix-loop-helix factor PHO4 is mediated by complex formation with the PHO80 repressor. In contrast to other systems, such as inhibition of GAL4 by GAL80 or of p53 by MDM2, where repression is mediated by direct interaction at regions overlapping the transcription activation domain, interaction with PHO80 involves two regions of PHO4 distinct from those involved in transcription activation or DNA-binding and dimerization. The possibility that repression of PHO4 by PHO80 may represent a general mechanism of transcription control, including regulation of the cell-type-specific transcription activation domain of c-Jun, is discussed. Images PMID:8187772

  17. The C2 Domains of Human Synaptotagmin 1 Have Distinct Mechanical Properties

    PubMed Central

    Fuson, Kerry L.; Ma, Liang; Sutton, R. Bryan; Oberhauser, Andres F.

    2009-01-01

    Synaptotagmin 1 (Syt1) is the Ca+2 receptor for fast, synchronous vesicle fusion in neurons. Because membrane fusion is an inherently mechanical, force-driven event, Syt1 must be able to adapt to the energetics of the fusion apparatus. Syt1 contains two C2 domains (C2A and C2B) that are homologous in sequence and three-dimensional in structure; yet, a number of observations have suggested that they have distinct biochemical and biological properties. In this study, we analyzed the mechanical stability of the C2A and C2B domains of human Syt1 using single-molecule atomic force microscopy. We found that stretching the C2AB domains of Syt1 resulted in two distinct unfolding force peaks. The larger force peak of ∼100 pN was identified as C2B and the second peak of ∼50 pN as C2A. Furthermore, a significant fraction of C2A domains unfolded through a low force intermediate that was not observed in C2B. We conclude that these domains have different mechanical properties. We hypothesize that a relatively small stretching force may be sufficient to deform the effector-binding regions of the C2A domain and modulate the affinity for soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), phospholipids, and Ca+2. PMID:19186144

  18. Two distinct functions for PI3-kinases in macropinocytosis

    PubMed Central

    Hoeller, Oliver; Bolourani, Parvin; Clark, Jonathan; Stephens, Len R.; Hawkins, Phillip T.; Weiner, Orion D.; Weeks, Gerald; Kay, Robert R.

    2013-01-01

    Summary Class-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P3, with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins. PMID:23843627

  19. An introduction to recognizing functional domains.

    PubMed

    Stormo, Gary D

    2006-10-01

    This unit provides an overview of issues involved in domain recognition in protein and DNA sequences. It opens with a discussion of the two primary methods of domain representation, namely consensus sequences and alignment matrices (e.g., the log-odds matrix). The unit continues with a brief overview of some of the resources available for identifying functional domains in nucleotide sequences (e.g., TRANSFAC). In addition, it reviews databases such as Pfam, InterPro and Blocks, which are available for protein analysis.

  20. Protein function prediction using domain families

    PubMed Central

    2013-01-01

    Here we assessed the use of domain families for predicting the functions of whole proteins. These 'functional families' (FunFams) were derived using a protocol that combines sequence clustering with supervised cluster evaluation, relying on available high-quality Gene Ontology (GO) annotation data in the latter step. In essence, the protocol groups domain sequences belonging to the same superfamily into families based on the GO annotations of their parent proteins. An initial test based on enzyme sequences confirmed that the FunFams resemble enzyme (domain) families much better than do families produced by sequence clustering alone. For the CAFA 2011 experiment, we further associated the FunFams with GO terms probabilistically. All target proteins were first submitted to domain superfamily assignment, followed by FunFam assignment and, eventually, function assignment. The latter included an integration step for multi-domain target proteins. The CAFA results put our domain-based approach among the top ten of 31 competing groups and 56 prediction methods, confirming that it outperforms simple pairwise whole-protein sequence comparisons. PMID:23514456

  1. Association of astrocytes with neurons and astrocytes derived from distinct progenitor domains in the subpallium

    PubMed Central

    Torigoe, Makio; Yamauchi, Kenta; Zhu, Yan; Kobayashi, Hiroaki; Murakami, Fujio

    2015-01-01

    Astrocytes play pivotal roles in metabolism and homeostasis as well as in neural development and function in a manner thought to depend on their region-specific diversity. In the mouse spinal cord, astrocytes and neurons, which are derived from a common progenitor domain (PD) and controlled by common PD-specific transcription factors, migrate radially and share their final positions. However, whether astrocytes can only interact with neurons from common PDs in the brain remains unknown. Here, we focused on subpallium-derived cells, because the subpallium generates neurons that show a diverse mode of migration. We tracked their fate by in utero electroporation of plasmids that allow for chromosomal integration of transgenes or of a Cre recombinase expression vector to reporter mice. We also used an Nkx2.1Cre mouse line to fate map the cells originating from the medial ganglionic eminence and preoptic area. We find that although neurons and astrocytes are labeled in various regions, only neurons are labeled in the neocortex, hippocampus and olfactory bulb. Furthermore, we find astrocytes derived from an Nkx 2.1-negative PD are associated with neurons from the Nkx2.1+ PD. Thus, forebrain astrocytes can associate with neurons as well as astrocytes derived from a distinct PD. PMID:26193445

  2. Distinct cytoplasmic domains in Plexin-A4 mediate diverse responses to semaphorin 3A in developing mammalian neurons.

    PubMed

    Mlechkovich, Guy; Peng, Sheng-Shiang; Shacham, Vered; Martinez, Edward; Gokhman, Irena; Minis, Adi; Tran, Tracy S; Yaron, Avraham

    2014-03-11

    Guidance receptor signaling is crucial for neural circuit formation and elicits diverse cellular events in specific neurons. We found that signaling from the guidance cue semaphorin 3A diverged through distinct cytoplasmic domains in its receptor Plexin-A4 to promote disparate cellular behavior in different neuronal cell types. Plexin-A4 has three main cytoplasmic domains--C1, Hinge/RBD, and C2--and interacts with family members of the Rho guanine nucleotide exchange factor FARP proteins. We show that growth cone collapse occurred in Plexin-A4-deficient dorsal root ganglion sensory neurons reconstituted with Plexin-A4 containing either the Hinge/RBD or C2 domain, whereas both of the Hinge/RBD and C1 domains were required for dendritic arborization in cortical neurons. Although knockdown studies indicated that both the collapse and arborization responses involved FARP2, mutations in the cytoplasmic region of Plexin-A4 that reduced its interaction with FARP2 strongly inhibited semaphorin 3A-induced dendritic branching but not growth cone collapse, suggesting that different degrees of interaction are required for the two responses or that developing axons have an indirect path to FARP2 activation. Thus, our study provided insights into the multifunctionality of guidance receptors, in particular showing that the semaphorin 3A signal diverges through specific functions of the modular domains of Plexin-A4.

  3. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures.

    PubMed

    Marchler-Bauer, Aron; Bo, Yu; Han, Lianyi; He, Jane; Lanczycki, Christopher J; Lu, Shennan; Chitsaz, Farideh; Derbyshire, Myra K; Geer, Renata C; Gonzales, Noreen R; Gwadz, Marc; Hurwitz, David I; Lu, Fu; Marchler, Gabriele H; Song, James S; Thanki, Narmada; Wang, Zhouxi; Yamashita, Roxanne A; Zhang, Dachuan; Zheng, Chanjuan; Geer, Lewis Y; Bryant, Stephen H

    2017-01-04

    NCBI's Conserved Domain Database (CDD) aims at annotating biomolecular sequences with the location of evolutionarily conserved protein domain footprints, and functional sites inferred from such footprints. An archive of pre-computed domain annotation is maintained for proteins tracked by NCBI's Entrez database, and live search services are offered as well. CDD curation staff supplements a comprehensive collection of protein domain and protein family models, which have been imported from external providers, with representations of selected domain families that are curated in-house and organized into hierarchical classifications of functionally distinct families and sub-families. CDD also supports comparative analyses of protein families via conserved domain architectures, and a recent curation effort focuses on providing functional characterizations of distinct subfamily architectures using SPARCLE: Subfamily Protein Architecture Labeling Engine. CDD can be accessed at https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.

  4. Distinctive Pattern of Behavioral Functioning in Angelman Syndrome.

    ERIC Educational Resources Information Center

    Summers, Jane A.; Feldman, Maurice A.

    1999-01-01

    A study compared 27 participants with Angelman syndrome to clinical and community participants (n=948) with developmental disabilities of mixed etiology to determine whether Angelman syndrome is associated with a distinctive patterns of behavioral functioning. Those with Angelman syndrome had significantly lower scores on measures of irritability…

  5. Individual domains of Tensin2 exhibit distinct subcellular localisations and migratory effects.

    PubMed

    Hafizi, Sassan; Sernstad, Emma; Swinny, Jerome D; Gomez, Maria F; Dahlbäck, Björn

    2010-01-01

    Tensins are large intracellular proteins believed to link the extracellular matrix to the cytoskeleton via integrins. Tensins are multidomain proteins consisting of homologous C1, PTPase, C2, SH2 and PTB domains. Full-length Tensin proteins can undergo cleavage inside cells, thus yielding domains in isolation that may have discrete subcellular localisations and downstream effects. We expressed different isoforms of Tensin2 and their individual domains as recombinant green fluorescent protein (GFP)-fusion constructs in DU145 human prostate cancer cells. Under fluorescence confocal microscopy, the isolated domains of Tensin2 all displayed discrete distributions throughout the cytoplasm and the nucleus. In particular, partial constructs containing the C1 domain localised preferentially to the nucleus, including the isolated C1 domain and the PTPase domain. In contrast, all three full-length isoforms of Tensin2 were present exclusively in discrete punctate bodies throughout the cytoplasm. This punctate staining showed colocalisation with the tumour suppressor protein DLC-1 as well as with actin (phalloidin). Furthermore, DU145 cells transiently expressing partial Tensin2 constructs containing the PTB domain showed an increased haptotactic migration. In addition, stimulation of renal carcinoma cells stably expressing Tensin2 by the survival factor Gas6 caused phosphorylation of its receptor Axl, but no effect on Tensin2, which was already maximally phosphorylated at time 0. In conclusion, our results indicate that differential proteolytic cleavage of Tensin2 can liberate domains with discrete localisations and functions, which has implications for the role of Tensins in cancer cell survival and motility.

  6. Isolation and characterization of distinct domains of sarcolemma and T-tubules from rat skeletal muscle.

    PubMed Central

    Muñoz, P; Rosemblatt, M; Testar, X; Palacín, M; Zorzano, A

    1995-01-01

    1. Several cell-surface domains of sarcolemma and T-tubule from skeletal-muscle fibre were isolated and characterized. 2. A protocol of subcellular fractionation was set up that involved the sequential low- and high-speed homogenization of rat skeletal muscle followed by KCl washing, Ca2+ loading and sucrose-density-gradient centrifugation. This protocol led to the separation of cell-surface membranes from membranes enriched in sarcoplasmic reticulum and intracellular GLUT4-containing vesicles. 3. Agglutination of cell-surface membranes using wheat-germ agglutinin allowed the isolation of three distinct cell-surface membrane domains: sarcolemmal fraction 1 (SM1), sarcolemmal fraction 2 (SM2) and a T-tubule fraction enriched in protein tt28 and the alpha 2-component of dihydropyridine receptor. 4. Fractions SM1 and SM2 represented distinct sarcolemmal subcompartments based on different compositions of biochemical markers: SM2 was characterized by high levels of beta 1-integrin and dystrophin, and SM1 was enriched in beta 1-integrin but lacked dystrophin. 5. The caveolae-associated molecule caveolin was very abundant in SM1, SM2 and T-tubules, suggesting the presence of caveolae or caveolin-rich domains in these cell-surface membrane domains. In contrast, clathrin heavy chain was abundant in SM1 and T-tubules, but only trace levels were detected in SM2. 6. Immunoadsorption of T-tubule vesicles with antibodies against protein tt28 and against GLUT4 revealed the presence of GLUT4 in T-tubules under basal conditions and it also allowed the identification of two distinct pools of T-tubules showing different contents of tt28 and dihydropyridine receptors. 7. Our data on distribution of clathrin and dystrophin reveal the existence of subcompartments in sarcolemma from muscle fibre, featuring selective mutually exclusive components. T-tubules contain caveolin and clathrin suggesting that they contain caveolin- and clathrin-rich domains. Furthermore, evidence for the

  7. Microdissection of Shoot Meristem Functional Domains

    PubMed Central

    Zhang, Xiaolan; Ohtsu, Kazuhiro; Zhou, Ruilian; Sarkar, Ananda; Hargreaves, Sarah; Elshire, Robert J.; Eudy, Douglas; Pawlowska, Teresa; Ware, Doreen; Janick-Buckner, Diane; Buckner, Brent; Timmermans, Marja C. P.; Schnable, Patrick S.; Nettleton, Dan; Scanlon, Michael J.

    2009-01-01

    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 that function during leaf development. Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf (P0/P1) or in the SAM proper verified the precision of the microdissections. Genes involved in cell division/growth, cell wall biosynthesis, chromatin remodeling, RNA binding, and translation are especially upregulated in initiating leaves, whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper. In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1. P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation. Reverse genetic analyses of asceapen1 (asc1), a maize D4-cyclin gene upregulated in the P0/P1, revealed novel leaf phenotypes, less genetic redundancy, and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis. These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize. PMID:19424435

  8. Microdissection of shoot meristem functional domains.

    PubMed

    Brooks, Lionel; Strable, Josh; Zhang, Xiaolan; Ohtsu, Kazuhiro; Zhou, Ruilian; Sarkar, Ananda; Hargreaves, Sarah; Elshire, Robert J; Eudy, Douglas; Pawlowska, Teresa; Ware, Doreen; Janick-Buckner, Diane; Buckner, Brent; Timmermans, Marja C P; Schnable, Patrick S; Nettleton, Dan; Scanlon, Michael J

    2009-05-01

    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 that function during leaf development. Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf (P0/P1) or in the SAM proper verified the precision of the microdissections. Genes involved in cell division/growth, cell wall biosynthesis, chromatin remodeling, RNA binding, and translation are especially upregulated in initiating leaves, whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper. In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1. P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation. Reverse genetic analyses of asceapen1 (asc1), a maize D4-cyclin gene upregulated in the P0/P1, revealed novel leaf phenotypes, less genetic redundancy, and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis. These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize.

  9. Two distinct forms of functional lateralization in the human brain

    PubMed Central

    Gotts, Stephen J.; Jo, Hang Joon; Wallace, Gregory L.; Saad, Ziad S.; Cox, Robert W.; Martin, Alex

    2013-01-01

    The hemispheric lateralization of certain faculties in the human brain has long been held to be beneficial for functioning. However, quantitative relationships between the degree of lateralization in particular brain regions and the level of functioning have yet to be established. Here we demonstrate that two distinct forms of functional lateralization are present in the left vs. the right cerebral hemisphere, with the left hemisphere showing a preference to interact more exclusively with itself, particularly for cortical regions involved in language and fine motor coordination. In contrast, right-hemisphere cortical regions involved in visuospatial and attentional processing interact in a more integrative fashion with both hemispheres. The degree of lateralization present in these distinct systems selectively predicted behavioral measures of verbal and visuospatial ability, providing direct evidence that lateralization is associated with enhanced cognitive ability. PMID:23959883

  10. Different Functions of Phylogenetically Distinct Bacterial Complex I Isozymes

    PubMed Central

    Spero, Melanie A.; Brickner, Joshua R.; Mollet, Jordan T.; Pisithkul, Tippapha; Amador-Noguez, Daniel

    2016-01-01

    ABSTRACT NADH:quinone oxidoreductase (complex I) is a bioenergetic enzyme that transfers electrons from NADH to quinone, conserving the energy of this reaction by contributing to the proton motive force. While the importance of NADH oxidation to mitochondrial aerobic respiration is well documented, the contribution of complex I to bacterial electron transport chains has been tested in only a few species. Here, we analyze the function of two phylogenetically distinct complex I isozymes in Rhodobacter sphaeroides, an alphaproteobacterium that contains well-characterized electron transport chains. We found that R. sphaeroides complex I activity is important for aerobic respiration and required for anaerobic dimethyl sulfoxide (DMSO) respiration (in the absence of light), photoautotrophic growth, and photoheterotrophic growth (in the absence of an external electron acceptor). Our data also provide insight into the functions of the phylogenetically distinct R. sphaeroides complex I enzymes (complex IA and complex IE) in maintaining a cellular redox state during photoheterotrophic growth. We propose that the function of each isozyme during photoheterotrophic growth is either NADH synthesis (complex IA) or NADH oxidation (complex IE). The canonical alphaproteobacterial complex I isozyme (complex IA) was also shown to be important for routing electrons to nitrogenase-mediated H2 production, while the horizontally acquired enzyme (complex IE) was dispensable in this process. Unlike the singular role of complex I in mitochondria, we predict that the phylogenetically distinct complex I enzymes found across bacterial species have evolved to enhance the functions of their respective electron transport chains. IMPORTANCE Cells use a proton motive force (PMF), NADH, and ATP to support numerous processes. In mitochondria, complex I uses NADH oxidation to generate a PMF, which can drive ATP synthesis. This study analyzed the function of complex I in bacteria, which contain more

  11. Concomitant prediction of function and fold at the domain level with GO-based profiles

    PubMed Central

    2013-01-01

    Predicting the function of newly sequenced proteins is crucial due to the pace at which these raw sequences are being obtained. Almost all resources for predicting protein function assign functional terms to whole chains, and do not distinguish which particular domain is responsible for the allocated function. This is not a limitation of the methodologies themselves but it is due to the fact that in the databases of functional annotations these methods use for transferring functional terms to new proteins, these annotations are done on a whole-chain basis. Nevertheless, domains are the basic evolutionary and often functional units of proteins. In many cases, the domains of a protein chain have distinct molecular functions, independent from each other. For that reason resources with functional annotations at the domain level, as well as methodologies for predicting function for individual domains adapted to these resources are required. We present a methodology for predicting the molecular function of individual domains, based on a previously developed database of functional annotations at the domain level. The approach, which we show outperforms a standard method based on sequence searches in assigning function, concomitantly predicts the structural fold of the domains and can give hints on the functionally important residues associated to the predicted function. PMID:23514233

  12. Discoidin domain receptor functions in physiological and pathological conditions

    PubMed Central

    Leitinger, Birgit

    2014-01-01

    The discoidin domain receptors, DDR1 and DDR2, are non-integrin collagen receptors that are members of the receptor tyrosine kinase family. Both DDRs bind a number of different collagen types and play important roles in embryo development. Dysregulated DDR function is associated with progression of various human diseases, including fibrosis, arthritis and cancer. By interacting with key components of the extracellular matrix and displaying distinct activation kinetics, the DDRs form a unique subfamily of receptor tyrosine kinases. DDR-facilitated cellular functions include cell migration, cell survival, proliferation and differentiation, as well as remodelling of extracellular matrices. This review summarises the current knowledge of DDR-ligand interactions, DDR-initiated signal pathways and the molecular mechanisms that regulate receptor function. Also discussed are the roles of DDRs in development and disease progression. PMID:24725424

  13. Human fibronectin contains distinct adhesion- and motility-promoting domains for metastatic melanoma cells

    PubMed Central

    1986-01-01

    The active migration of tumor cells through extracellular matrices has been proposed to play a role in certain aspects of metastasis. Metastatic tumor cells migrate in vitro in response to substratum-bound adhesive glycoproteins such as fibronectin. The present studies use affinity-purified proteolytic fragments of fibronectin to determine the nature of adhesion- and/or motility-promoting domains within the protein. Two distinct fragments were identified with cell adhesion- promoting activities. By a number of criteria, the adhesive activity promoted by these two fragments was distinct. One fragment, a 75-kD tryptic fragment purified by monoclonal antibody chromatography, promoted the adhesion, spreading, and haptotactic motility of melanoma cells. Experiments using a synthetic cell attachment peptide in solution indicated that at least part of the attachment activity exhibited by the 75-kD fragment is mediated by the sequence arg-gly-asp- ser. It was not possible to demonstrate migration-stimulating activity using a small (11.5 kD) peptic fragment containing this sequence (Pierschbacher, M.D., E. G. Hayman, and E. Ruoslahti, 1981, Cell, 26:259-267) suggesting that another cell-binding activity within the 75 kD fragment distinct from arg-gly-asp-ser might be required for motility. The second fragment that stimulated melanoma adhesion was a 33-kD tryptic/catheptic carboxyl-terminal heparin-binding fragment, which is localized to the A chain of fibronectin. This fragment promotes adhesion and spreading but not the motility of these cells. Melanoma adhesion to this heparin-binding fragment was sensitive to the effects of cycloheximide, which contrasted adhesion to the haptotaxis- promoting fragment. Importantly, these studies illustrate that haptotaxis in response to fibronectin is not due to simple adhesion gradients of this protein. The results are discussed in light of a model for multiple distinct cell surface constituents mediating cell adhesion and motility on

  14. A distinct switch in interactions of the histone H4 tail domain upon salt-dependent folding of nucleosome arrays.

    PubMed

    Pepenella, Sharon; Murphy, Kevin J; Hayes, Jeffrey J

    2014-09-26

    The core histone tail domains mediate inter-nucleosomal interactions that direct folding and condensation of nucleosome arrays into higher-order chromatin structures. The histone H4 tail domain facilitates inter-array interactions by contacting both the H2A/H2B acidic patch and DNA of neighboring nucleosomes. Likewise, H4 tail-H2A contacts stabilize array folding. However, whether the H4 tail domains stabilize array folding via inter-nucleosomal interactions with the DNA of neighboring nucleosomes remains unclear. We utilized defined oligonucleosome arrays containing a single specialized nucleosome with a photo-inducible cross-linker in the N terminus of the H4 tail to characterize these interactions. We observed that the H4 tail participates exclusively in intra-array interactions with DNA in unfolded arrays. These interactions are diminished during array folding, yet no inter-nucleosome, intra-array H4 tail-DNA contacts are observed in condensed chromatin. However, we document contacts between the N terminus of the H4 tail and H2A. Installation of acetylation mimics known to disrupt H4-H2A surface interactions did not increase observance of H4-DNA inter-nucleosomal interactions. These results suggest the multiple functions of the H4 tail require targeted distinct interactions within condensed chromatin.

  15. Mycoplasma hyopneumoniae and Mycoplasma flocculare differential domains from orthologous surface proteins induce distinct cellular immune responses in mice.

    PubMed

    Leal, Fernanda Munhoz Dos Anjos; Virginio, Veridiana Gomes; Martello, Carolina Lumertz; Paes, Jéssica Andrade; Borges, Thiago J; Jaeger, Natália; Bonorino, Cristina; Ferreira, Henrique Bunselmeyer

    2016-07-15

    Mycoplasma hyopneumoniae and Mycoplasma flocculare are two genetically close species found in the swine respiratory tract. Despite their similarities, while M. hyopneumoniae is the causative agent of porcine enzootic pneumonia, M. flocculare is a commensal bacterium. Genomic and transcriptional comparative analyses so far failed to explain the difference in pathogenicity between these two species. We then hypothesized that such difference might be, at least in part, explained by amino acid sequence and immunological or functional differences between ortholog surface proteins. In line with that, it was verified that approximately 85% of the ortholog surface proteins from M. hyopneumoniae 7448 and M. flocculare present one or more differential domains. To experimentally assess possible immunological implications of this kind of difference, the extracellular differential domains from one pair of orthologous surface proteins (MHP7448_0612, from M. hyopneumoniae, and MF_00357, from M. flocculare) were expressed in E. coli and used to immunize mice. The recombinant polypeptides (rMHP61267-169 and rMF35767-196, respectively) induced distinct cellular immune responses. While, rMHP61267-169 induced both Th1 and Th2 responses, rMF35767-196 induced just an early pro-inflammatory response. These results indicate that immunological properties determined by differential domains in orthologous surface protein might play a role in pathogenicity, contributing to elicit specific and differential immune responses against each species.

  16. The two distinctive metal ion binding domains of the wheat metallothionein Ec-1.

    PubMed

    Peroza, Estevão A; Kaabi, Ali Al; Meyer-Klaucke, Wolfram; Wellenreuther, Gerd; Freisinger, Eva

    2009-03-01

    Metallothioneins are small cysteine-rich proteins believed to play a role, among others, in the homeostasis of essential metal ions such as Zn(II) and Cu(I). Recently, we could show that wheat E(c)-1 is coordinating its six Zn(II) ions in form of metal-thiolate clusters analogously to the vertebrate metallothioneins. Specifically, two Zn(II) ions are bound in the N-terminal and four in the C-terminal domain. In the following, we will present evidence for the relative independence of the two domains from each other with respect to their metal ion binding abilities, and uncover three intriguing peculiarities of the protein. Firstly, one Zn(II) ion of the N-terminal domain is relative resistant to complete replacement with Cd(II) indicating the presence of a Zn(II)-binding site with increased stability. Secondly, the C-terminal domain is able to coordinate an additional fifth metal ion, though with reduced affinity, which went undetected so far. Finally, reconstitution of apoE(c)-1 with an excess of Zn(II) shows a certain amount of sub-stoichiometrically metal-loaded species. The possible relevance of these finding for the proposed biological functions of wheat E(c)-1 will be discussed. In addition, extended X-ray absorption fine structure (EXAFS) measurements on both, the full-length and the truncated protein, provide final evidence for His participation in metal ion binding.

  17. Hox genes define distinct progenitor sub-domains within the second heart field

    PubMed Central

    Bertrand, Nicolas; Roux, Marine; Ryckebüsch, Lucile; Niederreither, Karen; Dollé, Pascal; Moon, Anne; Capecchi, Mario; Zaffran, Stéphane

    2011-01-01

    Much of the heart, including the atria, right ventricle and outflow tract (OFT) is derived from a progenitor cell population termed the second heart field (SHF) that contributes progressively to the embryonic heart during cardiac looping. Several studies have revealed anterior-posterior patterning of the SHF, since the anterior region (anterior heart field) contributes to right ventricular and OFT myocardium whereas the posterior region gives rise to the atria. We have previously shown that Retinoic Acid (RA) signal participates to this patterning. We now show that Hoxb1, Hoxa1, and Hoxa3, as downstream RA targets, are expressed in distinct sub-domains within the SHF. Our genetic lineage tracing analysis revealed that Hoxb1, Hoxa1 and Hoxa3-expressing cardiac progenitor cells contribute to both atria and the inferior wall of the OFT, which subsequently gives rise to myocardium at the base of pulmonary trunk. By contrast to Hoxb1Cre, the contribution of Hoxa1-enhIII-Cre and Hoxa3Cre-labeled cells is restricted to the distal regions of the OFT suggesting that proximo-distal patterning of the OFT is related to SHF sub-domains characterized by combinatorial Hox genes expression. Manipulation of RA signaling pathways showed that RA is required for the correct deployment of Hox-expressing SHF cells. This report provides new insights into the regulatory gene network in SHF cells contributing to the atria and sub-pulmonary myocardium. PMID:21385575

  18. A crack problem with four distinct harmonic functions

    NASA Technical Reports Server (NTRS)

    Sih, G. C.; Kassir, M. K.

    1972-01-01

    The problem of an elastic solid containing a semi-infinite plane crack subjected to concentrated shears parallel to the edge of the crack is considered. A closed form solution using four distinct harmonic functions (none of which can be taken arbitrarily) is found to satisfy the finite displacement and inverse square root stress singularity at the edge of the crack. Explicit expressions in terms of elementary functions are given for the distribution of stress and displacement in the solid. These are obtained by employing Fourier and Kontorovich-Lebedev integral transforms and certain singular solutions of Laplace equations in three dimensions. The variations of the intensity of the local stress field along the crack border are shown graphically. An example is presented, which is in contrast with the conclusion established in the literature that one of the four Papkovich-Neuber functions in three-dimensional elasticity may be arbitrarily set to zero.

  19. Auditory abnormalities in autism: toward functional distinctions among findings.

    PubMed

    Kellerman, Gabriella R; Fan, Jin; Gorman, Jack M

    2005-09-01

    Recently, findings on a wide range of auditory abnormalities among individuals with autism have been reported. To date, functional distinctions among these varied findings are poorly established. Such distinctions should be of interest to clinicians and researchers alike given their potential therapeutic and experimental applications. This review suggests three general trends among these findings as a starting point for future analyses. First, studies of auditory perception of linguistic and social auditory stimuli among individuals with autism generally have found impaired perception versus normal controls. Such findings may correlate with impaired language and communication skills and social isolation observed among individuals with autism. Second, studies of auditory perception of pitch and music among individuals with autism generally have found enhanced perception versus normal controls. These findings may correlate with the restrictive and highly focused behaviors observed among individuals with autism. Third, findings on the auditory perception of non-linguistic, non-musical stimuli among autism patients resist any generalized conclusions. Ultimately, as some researchers have already suggested, the distinction between impaired global processing and enhanced local processing may prove useful in making sense of apparently discordant findings on auditory abnormalities among individuals with autism.

  20. Design of protein function leaps by directed domain interface evolution

    PubMed Central

    Huang, Jin; Koide, Akiko; Makabe, Koki; Koide, Shohei

    2008-01-01

    Most natural proteins performing sophisticated tasks contain multiple domains where an active site is located at the domain interface. Comparative structural analyses suggest that major leaps in protein function occur through gene recombination events that connect two or more protein domains to generate a new active site, frequently occurring at the newly created domain interface. However, such functional leaps by combination of unrelated domains have not been directly demonstrated. Here we show that highly specific and complex protein functions can be generated by joining a low-affinity peptide-binding domain with a functionally inert second domain and subsequently optimizing the domain interface. These directed evolution processes dramatically enhanced both affinity and specificity to a level unattainable with a single domain, corresponding to >500-fold and >2,000-fold increases of affinity and specificity, respectively. An x-ray crystal structure revealed that the resulting “affinity clamp” had clamshell architecture as designed, with large additional binding surface contributed by the second domain. The affinity clamps having a single-nanomolar dissociation constant outperformed a monoclonal antibody in immunochemical applications. This work establishes evolutionary paths from isolated domains with primitive function to multidomain proteins with sophisticated function and introduces a new protein-engineering concept that allows for the generation of highly functional affinity reagents to a predefined target. The prevalence and variety of natural interaction domains suggest that numerous new functions can be designed by using directed domain interface evolution. PMID:18445649

  1. The functional requirement of two structural domains within telomerase RNA emerged early in eukaryotes.

    PubMed

    Podlevsky, Joshua D; Li, Yang; Chen, Julian J-L

    2016-11-16

    Telomerase emerged during evolution as a prominent solution to the eukaryotic linear chromosome end-replication problem. Telomerase minimally comprises the catalytic telomerase reverse transcriptase (TERT) and telomerase RNA (TR) that provides the template for telomeric DNA synthesis. While the TERT protein is well-conserved across taxa, TR is highly divergent amongst distinct groups of species. Herein, we have identified the essential functional domains of TR from the basal eukaryotic species Trypanosoma brucei, revealing the ancestry of TR comprising two distinct structural core domains that can assemble in trans with TERT and reconstitute active telomerase enzyme in vitro The upstream essential domain of T. brucei TR, termed the template core, constitutes three short helices in addition to the 11-nt template. Interestingly, the trypanosome template core domain lacks the ubiquitous pseudoknot found in all known TRs, suggesting later evolution of this critical structural element. The template-distal domain is a short stem-loop, termed equivalent CR4/5 (eCR4/5). While functionally similar to vertebrate and fungal CR4/5, trypanosome eCR4/5 is structurally distinctive, lacking the essential P6.1 stem-loop. Our functional study of trypanosome TR core domains suggests that the functional requirement of two discrete structural domains is a common feature of TRs and emerged early in telomerase evolution.

  2. Functional domains of yeast hexokinase 2

    PubMed Central

    Peláez, Rafael; Herrero, Pilar; Moreno, Fernando

    2010-01-01

    Hkx2 (hexokinase 2) from Saccharomyces cerevisiae was one of the first metabolic enzymes described as a multifunctional protein. Hxk2 has a double subcellular localization: it functions as a glycolytic enzyme in the cytoplasm and as a regulator of gene transcription of several Mig1-regulated genes in the nucleus. To get more insights into the structure–function relationships of the Hxk2 protein, we followed two different approaches. In the first, we deleted the last eight amino acids of Hxk2 and replaced Ser304 with phenylalanine to generate Hxk2wca. Analysis of this mutant demonstrated that these domains play an essential role in the catalytic activity of yeast Hxk2, but has no effect on the regulatory function of this protein. In the second, we analysed whether amino acids from Lys6 to Met15 of Hxk2 (Hxk2wrf) are essential for the regulatory role of Hxk2 and whether there is an effect on the hexose kinase activity of this protein. In the present paper, we report that the Hxk2wca mutant protein interacts with the Mig1 transcriptional repressor and the Snf1 protein kinase in the nucleus at the level of the SUC2–Mig1 repressor complex. We have demonstrated that Hxk2wca maintained full regulatory function because the glucose-repression signalling of the wild-type machinery is maintained. We also report that the Hxk2wrf mutant allele is incapable of glucose repression signalling because it does not interact with Mig1 at the level of the SUC2–Mig1 repressor complex. The two mutants, Hxk2wca and Hxk2wrf retain single functions, as a transcriptional factor or as an enzyme with hexose-phosphorylating activity, but have lost the original bifunctionality of Hxk2. PMID:20815814

  3. Functional domains of yeast hexokinase 2.

    PubMed

    Peláez, Rafael; Herrero, Pilar; Moreno, Fernando

    2010-11-15

    Hkx2 (hexokinase 2) from Saccharomyces cerevisiae was one of the first metabolic enzymes described as a multifunctional protein. Hxk2 has a double subcellular localization: it functions as a glycolytic enzyme in the cytoplasm and as a regulator of gene transcription of several Mig1-regulated genes in the nucleus. To get more insights into the structure-function relationships of the Hxk2 protein, we followed two different approaches. In the first, we deleted the last eight amino acids of Hxk2 and replaced Ser³⁰⁴ with phenylalanine to generate Hxk2(wca). Analysis of this mutant demonstrated that these domains play an essential role in the catalytic activity of yeast Hxk2, but has no effect on the regulatory function of this protein. In the second, we analysed whether amino acids from Lys⁶ to Met¹⁵ of Hxk2 (Hxk2(wrf)) are essential for the regulatory role of Hxk2 and whether there is an effect on the hexose kinase activity of this protein. In the present paper, we report that the Hxk2(wca) mutant protein interacts with the Mig1 transcriptional repressor and the Snf1 protein kinase in the nucleus at the level of the SUC2-Mig1 repressor complex. We have demonstrated that Hxk2(wca) maintained full regulatory function because the glucose-repression signalling of the wild-type machinery is maintained. We also report that the Hxk2(wrf) mutant allele is incapable of glucose repression signalling because it does not interact with Mig1 at the level of the SUC2-Mig1 repressor complex. The two mutants, Hxk2(wca) and Hxk2(wrf) retain single functions, as a transcriptional factor or as an enzyme with hexose-phosphorylating activity, but have lost the original bifunctionality of Hxk2.

  4. Structure and function of WD40 domain proteins.

    PubMed

    Xu, Chao; Min, Jinrong

    2011-03-01

    The WD40 domain exhibits a β-propeller architecture, often comprising seven blades. The WD40 domain is one of the most abundant domains and also among the top interacting domains in eukaryotic genomes. In this review, we will discuss the identification, definition and architecture of the WD40 domains. WD40 domain proteins are involved in a large variety of cellular processes, in which WD40 domains function as a protein-protein or protein-DNA interaction platform. WD40 domain mediates molecular recognition events mainly through the smaller top surface, but also through the bottom surface and sides. So far, no WD40 domain has been found to display enzymatic activity. We will also discuss the different binding modes exhibited by the large versatile family of WD40 domain proteins. In the last part of this review, we will discuss how post-translational modifications are recognized by WD40 domain proteins.

  5. Analysis of Exocyst Subunit EXO70 Family Reveals Distinct Membrane Polar Domains in Tobacco Pollen Tubes1[OPEN

    PubMed Central

    Šantrůček, Jiří; Vukašinović, Nemanja

    2017-01-01

    The vesicle-tethering complex exocyst is one of the crucial cell polarity regulators. The EXO70 subunit is required for the targeting of the complex and is represented by many isoforms in angiosperm plant cells. This diversity could be partly responsible for the establishment and maintenance of membrane domains with different composition. To address this hypothesis, we employed the growing pollen tube, a well-established cell polarity model system, and performed large-scale expression, localization, and functional analysis of tobacco (Nicotiana tabacum) EXO70 isoforms. Various isoforms localized to different regions of the pollen tube plasma membrane, apical vesicle-rich inverted cone region, nucleus, and cytoplasm. The overexpression of major pollen-expressed EXO70 isoforms resulted in growth arrest and characteristic phenotypic deviations of tip swelling and apical invaginations. NtEXO70A1a and NtEXO70B1 occupied two distinct and mutually exclusive plasma membrane domains. Both isoforms partly colocalized with the exocyst subunit NtSEC3a at the plasma membrane, possibly forming different exocyst complex subpopulations. NtEXO70A1a localized to the small area previously characterized as the site of exocytosis in the tobacco pollen tube, while NtEXO70B1 surprisingly colocalized with the zone of clathrin-mediated endocytosis. Both NtEXO70A1a and NtEXO70B1 colocalized to different degrees with markers for the anionic signaling phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. In contrast, members of the EXO70 C class, which are specifically expressed in tip-growing cells, exhibited exocytosis-related functional effects in pollen tubes despite the absence of apparent plasma membrane localization. Taken together, our data support the existence of multiple membrane-trafficking domains regulated by different EXO70-containing exocyst complexes within a single cell. PMID:28082718

  6. Compositionally distinct nuclear pore complexes of functionally distinct dimorphic nuclei in ciliate Tetrahymena.

    PubMed

    Iwamoto, Masaaki; Osakada, Hiroko; Mori, Chie; Fukuda, Yasuhiro; Nagao, Koji; Obuse, Chikashi; Hiraoka, Yasushi; Haraguchi, Tokuko

    2017-04-06

    The nuclear pore complex (NPC), a gateway for nucleocytoplasmic trafficking, is composed of about 30 different proteins called nucleoporins. It remains unknown whether the NPCs within a species are homogeneous or vary depending on the cell type, or physiological condition. Here, we present evidence for compositionally distinct NPCs that form within a single cell in a binucleated ciliate. In Tetrahymena thermophila, each cell contains both a transcriptionally-active macronucleus (MAC) and a germline micronucleus (MIC). By combining in silico analysis, mass spectrometry analysis for immuno-isolated proteins, and subcellular localization analysis of GFP fused proteins, we identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107-160 scaffold complex were enriched in MIC NPCs. Strikingly, two paralogs of Nup214 and of Nup153 localized exclusively to either MAC or MIC NPCs. Furthermore, the transmembrane components Pom121 and Pom82 localize exclusively to MAC and MIC NPCs, respectively. Our results argue that functional nuclear dimorphism in ciliates is likely to depend on compositional and structural specificity of NPCs.

  7. Comparative Analysis of RNA Families Reveals Distinct Repertoires for Each Domain of Life

    PubMed Central

    Hoeppner, Marc P.; Gardner, Paul P.; Poole, Anthony M.

    2012-01-01

    The RNA world hypothesis, that RNA genomes and catalysts preceded DNA genomes and genetically-encoded protein catalysts, has been central to models for the early evolution of life on Earth. A key part of such models is continuity between the earliest stages in the evolution of life and the RNA repertoires of extant lineages. Some assessments seem consistent with a diverse RNA world, yet direct continuity between modern RNAs and an RNA world has not been demonstrated for the majority of RNA families, and, anecdotally, many RNA functions appear restricted in their distribution. Despite much discussion of the possible antiquity of RNA families, no systematic analyses of RNA family distribution have been performed. To chart the broad evolutionary history of known RNA families, we performed comparative genomic analysis of over 3 million RNA annotations spanning 1446 families from the Rfam 10 database. We report that 99% of known RNA families are restricted to a single domain of life, revealing discrete repertoires for each domain. For the 1% of RNA families/clans present in more than one domain, over half show evidence of horizontal gene transfer (HGT), and the rest show a vertical trace, indicating the presence of a complex protein synthesis machinery in the Last Universal Common Ancestor (LUCA) and consistent with the evolutionary history of the most ancient protein-coding genes. However, with limited interdomain transfer and few RNA families exhibiting demonstrable antiquity as predicted under RNA world continuity, our results indicate that the majority of modern cellular RNA repertoires have primarily evolved in a domain-specific manner. PMID:23133357

  8. Different functional modes of BAR domain proteins in formation and plasticity of mammalian postsynapses.

    PubMed

    Kessels, Michael M; Qualmann, Britta

    2015-09-01

    A plethora of cell biological processes involve modulations of cellular membranes. By using extended lipid-binding interfaces, some proteins have the power to shape membranes by attaching to them. Among such membrane shapers, the superfamily of Bin-Amphiphysin-Rvs (BAR) domain proteins has recently taken center stage. Extensive structural work on BAR domains has revealed a common curved fold that can serve as an extended membrane-binding interface to modulate membrane topologies and has allowed the grouping of the BAR domain superfamily into subfamilies with structurally slightly distinct BAR domain subtypes (N-BAR, BAR, F-BAR and I-BAR). Most BAR superfamily members are expressed in the mammalian nervous system. Neurons are elaborately shaped and highly compartmentalized cells. Therefore, analyses of synapse formation and of postsynaptic reorganization processes (synaptic plasticity) - a basis for learning and memory formation - has unveiled important physiological functions of BAR domain superfamily members. These recent advances, furthermore, have revealed that the functions of BAR domain proteins include different aspects. These functions are influenced by the often complex domain organization of BAR domain proteins. In this Commentary, we review these recent insights and propose to classify BAR domain protein functions into (1) membrane shaping, (2) physical integration, (3) action through signaling components, and (4) suppression of other BAR domain functions.

  9. Expression and evolution of functionally distinct haemoglobin genes in plants.

    PubMed

    Hunt, P W; Watts, R A; Trevaskis, B; Llewelyn, D J; Burnell, J; Dennis, E S; Peacock, W J

    2001-11-01

    Haemoglobin genes have been found in a number of plant species, but the number of genes known has been too small to allow effective evolutionary inferences. We present nine new non-symbiotic haemoglobin sequences from a range of plants, including class 1 haemoglobins from cotton, Citrus and tomato, class 2 haemoglobins from cotton, tomato, sugar beet and canola and two haemoglobins from the non-vascular plants, Marchantia polymorpha (a liverwort) and Physcomitrella patens (a moss). Our molecular phylogenetic analysis of all currently known non-symbiotic haemoglobin genes and a selection of symbiotic haemoglobins have confirmed the existence of two distinct classes of haemoglobin genes in the dicots. It is likely that all dicots have both class 1 and class 2 non-symbiotic haemoglobin genes whereas in monocots we have detected only class 1 genes. The symbiotic haemoglobins from legumes and Casuarina are related to the class 2 non-symbiotic haemoglobins, whilst the symbiotic haemoglobin from Parasponia groups with the class 1 non-symbiotic genes. Probably, there have been two independent recruitments of symbiotic haemoglobins. Although the functions of the two non-symbiotic haemoglobins remain unknown, their patterns of expression within plants suggest different functions. We examined the expression in transgenic plants of the two non-symbiotic haemoglobins from Arabidopsis using promoter fusions to a GUS reporter gene. The Arabidopsis GLB1 and GLB2 genes are likely to be functionally distinct. The class 2 haemoglobin gene (GLB2) is expressed in the roots, leaves and inflorescence and can be induced in young plants by cytokinin treatment in contrast to the class 1 gene (GLB1) which is active in germinating seedlings and can be induced by hypoxia and increased sucrose supply, but not by cytokinin treatment.

  10. Automated retinal fovea type distinction in spectral-domain optical coherence tomography of retinal vein occlusion

    NASA Astrophysics Data System (ADS)

    Wu, Jing; Waldstein, Sebastian M.; Gerendas, Bianca S.; Langs, Georg; Simader, Christian; Schmidt-Erfurth, Ursula

    2015-03-01

    Spectral-domain Optical Coherence Tomography (SD-OCT) is a non-invasive modality for acquiring high- resolution, three-dimensional (3D) cross-sectional volumetric images of the retina and the subretinal layers. SD-OCT also allows the detailed imaging of retinal pathology, aiding clinicians in the diagnosis of sight degrading diseases such as age-related macular degeneration (AMD), glaucoma and retinal vein occlusion (RVO). Disease diagnosis, assessment, and treatment will require a patient to undergo multiple OCT scans, possibly using multiple scanners, to accurately and precisely gauge disease activity, progression and treatment success. However, cross-vendor imaging and patient movement may result in poor scan spatial correlation potentially leading to incorrect diagnosis or treatment analysis. The retinal fovea is the location of the highest visual acuity and is present in all patients, thus it is critical to vision and highly suitable for use as a primary landmark for cross-vendor/cross-patient registration for precise comparison of disease states. However, the location of the fovea in diseased eyes is extremely challenging to locate due to varying appearance and the presence of retinal layer destroying pathology. Thus categorising and detecting the fovea type is an important prior stage to automatically computing the fovea position. Presented here is an automated cross-vendor method for fovea distinction in 3D SD-OCT scans of patients suffering from RVO, categorising scans into three distinct types. OCT scans are preprocessed by motion correction and noise filing followed by segmentation using a kernel graph-cut approach. A statistically derived mask is applied to the resulting scan creating an ROI around the probable fovea location from which the uppermost retinal surface is delineated. For a normal appearance retina, minimisation to zero thickness is computed using the top two retinal surfaces. 3D local minima detection and layer thickness analysis are used

  11. Functional regions of the mouse interleukin-10 receptor cytoplasmic domain.

    PubMed Central

    Ho, A S; Wei, S H; Mui, A L; Miyajima, A; Moore, K W

    1995-01-01

    The functions of wild-type and mutant mouse interleukin-10 receptors (mIL-10R) expressed in murine Ba/F3 cells were studied. As observed previously, IL-10 stimulates proliferation of IL-10R-expressing Ba/F3 cells. Accumulation of viable cells in the proliferation assay is to a significant extent balanced by concomitant cell death. Moreover, growth in IL-10 also induces a previously unrecognized response, differentiation of the cells, as evidenced both by formation of large clusters of cells in cultures with IL-10 and by induction or enhancement of expression of several cell surface antigens, including CD32/16, CD2, LECAM-1 (v-selectin), and heat-stable antigen. Two distinct functional regions near the C terminus of the mIL-10R cytoplasmic domain which mediate proliferation were identified; one of these regions also mediates the differentiation response. A third region proximal to the transmembrane domain was identified; removal of this region renders the cell 10- to 100-fold more sensitive to IL-10 in the proliferation assay. In cells expressing both wild-type and mutant IL-10R, stimulation with IL-10 leads to tyrosine phosphorylation of the kinases JAK1 and TYK2 but not JAK2 or JAK3 under the conditions tested. PMID:7544437

  12. The Anthropocene is functionally and stratigraphically distinct from the Holocene.

    PubMed

    Waters, Colin N; Zalasiewicz, Jan; Summerhayes, Colin; Barnosky, Anthony D; Poirier, Clément; Gałuszka, Agnieszka; Cearreta, Alejandro; Edgeworth, Matt; Ellis, Erle C; Ellis, Michael; Jeandel, Catherine; Leinfelder, Reinhold; McNeill, J R; Richter, Daniel deB; Steffen, Will; Syvitski, James; Vidas, Davor; Wagreich, Michael; Williams, Mark; Zhisheng, An; Grinevald, Jacques; Odada, Eric; Oreskes, Naomi; Wolfe, Alexander P

    2016-01-08

    Human activity is leaving a pervasive and persistent signature on Earth. Vigorous debate continues about whether this warrants recognition as a new geologic time unit known as the Anthropocene. We review anthropogenic markers of functional changes in the Earth system through the stratigraphic record. The appearance of manufactured materials in sediments, including aluminum, plastics, and concrete, coincides with global spikes in fallout radionuclides and particulates from fossil fuel combustion. Carbon, nitrogen, and phosphorus cycles have been substantially modified over the past century. Rates of sea-level rise and the extent of human perturbation of the climate system exceed Late Holocene changes. Biotic changes include species invasions worldwide and accelerating rates of extinction. These combined signals render the Anthropocene stratigraphically distinct from the Holocene and earlier epochs.

  13. Functional domains of the floral regulator AGAMOUS: characterization of the DNA binding domain and analysis of dominant negative mutations.

    PubMed Central

    Mizukami, Y; Huang, H; Tudor, M; Hu, Y; Ma, H

    1996-01-01

    The Arabidopsis MADS box gene AGAMOUS (AG) controls reproductive organ identity and floral meristem determinacy. The AG protein binds in vitro to DNA sequences similar to the targets of known MADS domain transcription factors. Whereas most plant MADS domain proteins begin with the MADS domain, AG and its orthologs contain a region N-terminal to the MADS domain. All plant MADS domain proteins share another region with moderate sequence similarity called the K domain. Neither the region (I region) that lies between the MADS and K domains nor the C-terminal region is conserved. We show here that the AG MADS domain and the I region are necessary and sufficient for DNA binding in vitro and that AG binds to DNA as a dimer. To investigate the in vivo function of the regions of AG not required for in vitro DNA binding, we introduced several AG constructs into wild-type plants and characterized their floral phenotypes. We show that transgenic Arabidopsis plants with a 35S-AG construct encoding an AG protein lacking the N-terminal region produced apetala 2 (ap2)-like flowers similar to those ectopically expressing AG proteins retaining the N-terminal region. This result suggests that the N-terminal region is not required to produce the ap2-like phenotype. In addition, transformants with a 35S-AG construct encoding an AG protein lacking the C-terminal region produced ag-like flowers, indicating that this truncated AG protein inhibits normal AG function. Finally, transformants with a 35S-AG construct encoding an AG protein lacking both K and C regions produced flowers with more stamens and carpels. The phenotypes of the AG transformants demonstrate that both the K domain and the C-terminal region have important and distinct in vivo functions. We discuss possible mechanisms through which AG may regulate downstream genes. PMID:8672883

  14. Distinct domains of the limbic system-associated membrane protein (LAMP) mediate discrete effects on neurite outgrowth.

    PubMed

    Eagleson, Kathie L; Pimenta, Aurea F; Burns, Mary M; Fairfull, Liane D; Cornuet, Pamela K; Zhang, Li; Levitt, Pat

    2003-11-01

    The limbic system-associated membrane protein (LAMP) is a glycosylphosphatidylinositol-anchored glycoprotein with three immunoglobulin (Ig) domains that can either enhance or inhibit neurite outgrowth depending upon the neuronal population examined. In the present study, we investigate the domains responsible for these activities. Domain deletion revealed that the N-terminal IgI domain is necessary and sufficient for the neurite-promoting activity observed in hippocampal neurons. In contrast, inhibition of neurite outgrowth in SCG neurons, which is mediated by heterophilic interactions, requires full-length LAMP, although selective inhibition of the second Ig domain, but not the first or third domains, prevented the inhibitory effect. This indicates that the IgII domain of LAMP harbors the neurite-inhibiting activity, but only in the context of the full-length configuration. Covasphere-binding analyses demonstrate IgI/IgI interactions, but no interaction between IgII and any other domain, consistent with the biological activities that each domain mediates. The data suggest that LAMP may serve as a bifunctional guidance molecule, with distinct structural domains contributing to the promotion and inhibition of neurite outgrowth.

  15. Interaction between Functional Domains of Bacillus thuringiensis Insecticidal Crystal Proteins

    PubMed Central

    Rang, Cécile; Vachon, Vincent; de Maagd, Ruud A.; Villalon, Mario; Schwartz, Jean-Louis; Bosch, Dirk; Frutos, Roger; Laprade, Raynald

    1999-01-01

    Interactions among the three structural domains of Bacillus thuringiensis Cry1 toxins were investigated by functional analysis of chimeric proteins. Hybrid genes were prepared by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1C, and Cry1E. The activity of the purified trypsin-activated chimeric toxins was evaluated by testing their effects on the viability and plasma membrane permeability of Sf9 cells. Among the parental toxins, only Cry1C was active against these cells and only chimeras possessing domain II from Cry1C were functional. Combination of domain I from Cry1E with domains II and III from Cry1C, however, resulted in an inactive toxin, indicating that domain II from an active toxin is necessary, but not sufficient, for activity. Pores formed by chimeric toxins in which domain I was from Cry1Ab or Cry1Ac were slightly smaller than those formed by toxins in which domain I was from Cry1C. The properties of the pores formed by the chimeras are therefore likely to result from an interaction between domain I and domain II or III. Domain III appears to modulate the activity of the chimeric toxins: combination of domain III from Cry1Ab with domains I and II of Cry1C gave a protein which was more strongly active than Cry1C. PMID:10388684

  16. Structure and Function of the TIR Domain from the Grape NLR Protein RPV1

    PubMed Central

    Williams, Simon J.; Yin, Ling; Foley, Gabriel; Casey, Lachlan W.; Outram, Megan A.; Ericsson, Daniel J.; Lu, Jiang; Boden, Mikael; Dry, Ian B.; Kobe, Bostjan

    2016-01-01

    The N-terminal Toll/interleukin-1 receptor/resistance protein (TIR) domain has been shown to be both necessary and sufficient for defense signaling in the model plants flax and Arabidopsis. In examples from these organisms, TIR domain self-association is required for signaling function, albeit through distinct interfaces. Here, we investigate these properties in the TIR domain containing resistance protein RPV1 from the wild grapevine Muscadinia rotundifolia. The RPV1 TIR domain, without additional flanking sequence present, is autoactive when transiently expressed in tobacco, demonstrating that the TIR domain alone is capable of cell-death signaling. We determined the crystal structure of the RPV1 TIR domain at 2.3 Å resolution. In the crystals, the RPV1 TIR domain forms a dimer, mediated predominantly through residues in the αA and αE helices (“AE” interface). This interface is shared with the interface discovered in the dimeric complex of the TIR domains from the Arabidopsis RPS4/RRS1 resistance protein pair. We show that surface-exposed residues in the AE interface that mediate the dimer interaction in the crystals are highly conserved among plant TIR domain-containing proteins. While we were unable to demonstrate self-association of the RPV1 TIR domain in solution or using yeast 2-hybrid, mutations of surface-exposed residues in the AE interface prevent the cell-death autoactive phenotype. In addition, mutation of residues known to be important in the cell-death signaling function of the flax L6 TIR domain were also shown to be required for RPV1 TIR domain mediated cell-death. Our data demonstrate that multiple TIR domain surfaces control the cell-death function of the RPV1 TIR domain and we suggest that the conserved AE interface may have a general function in TIR-NLR signaling. PMID:28008335

  17. Functional domains of the poliovirus receptor

    SciTech Connect

    Koike, Satoshi; Ise, Iku; Nomoto, Akio )

    1991-05-15

    A number of mutant cDNAs of the human poliovirus receptor were constructed to identify essential regions of the molecule as the receptor. All mutant cDNAs carrying the sequence coding for the entire N-terminal immunoglobulin-like domain (domain I) confer permissiveness for poliovirus to mouse L cells, but a mutant cDNA lacking the sequence for domain I does not. The transformants permissive for poliovirus were able to bind the virus and were also recognized by monoclonal antibody D171, which competes with poliovirus for the cellular receptor. These results strongly suggest that the poliovirus binding site resides in domain I of the receptor. Mutant cDNAs for the sequence encoding the intracellular peptide were also constructed and expressed in mouse L cells. Susceptibility of these cells to poliovirus revealed that the entire putative cytoplasmic domain is not essential for virus infection. Thus, the cytoplasmic domain of the molecule appears not to play a role in the penetration of poliovirus.

  18. Two distinct domains of Bicoid mediate its transcriptional downregulation by the Torso pathway.

    PubMed

    Janody, F; Sturny, R; Schaeffer, V; Azou, Y; Dostatni, N

    2001-06-01

    The transcriptional activity of the Bicoid morphogen is directly downregulated by the Torso signal transduction cascade at the anterior pole of the Drosophila embryo. This regulation does not involve the homeodomain or direct phosphorylation of Bicoid. We analyse the transcriptional regulation of Bicoid in response to the Torso pathway, using Bicoid variants and fusion proteins between the Bicoid domains and the Gal4 DNA-binding domain. We show that Bicoid possesses three autonomous activation domains. Two of these domains, the serine/threonine-rich and the acidic domains, are downregulated by Torso, whereas the third activation domain, which is rich in glutamine, is not. The alanine-rich domain, previously described as an activation domain in vitro, has a repressive activity that is independent of Torso. Thus, Bicoid downregulation by Torso results from a competition between the glutamine-rich domain that is insensitive to Torso and the serine/threonine-rich and acidic activation domains downregulated by Torso. The alanine-rich domain contributes to this process indirectly by reducing the global activity of the protein and in particular the activity of the glutamine-rich domain that might otherwise prevent downregulation by Torso.

  19. Acetylation mimics within individual core histone tail domains indicate distinct roles in regulating the stability of higher-order chromatin structure.

    PubMed

    Wang, Xiaodong; Hayes, Jeffrey J

    2008-01-01

    Nucleosome arrays undergo salt-dependent self-association into large oligomers in a process thought to recapitulate essential aspects of higher-order tertiary chromatin structure formation. Lysine acetylation within the core histone tail domains inhibits self-association, an effect likely related to its role in facilitating transcription. As acetylation of specific tail domains may encode distinct functions, we investigated biochemical and self-association properties of model nucleosome arrays containing combinations of native and mutant core histones with lysine-to-glutamine substitutions to mimic acetylation. Acetylation mimics within the tail domains of H2B and H4 caused the largest inhibition of array self-association, while modification of the H3 tail uniquely affected the stability of DNA wrapping within individual nucleosomes. In addition, the effect of acetylation mimics on array self-association is inconsistent with a simple charge neutralization mechanism. For example, acetylation mimics within the H2A tail can have either a positive or negative effect on self-association, dependent upon the acetylation state of the other tails and nucleosomal repeat length. Finally, we demonstrate that glutamine substitutions and lysine acetylation within the H4 tail domain have identical effects on nucleosome array self-association. Our results indicate that acetylation of specific tail domains plays distinct roles in the regulation of chromatin structure.

  20. Minicollagen cysteine-rich domains encode distinct modes of polymerization to form stable nematocyst capsules

    PubMed Central

    Tursch, Anja; Mercadante, Davide; Tennigkeit, Jutta; Gräter, Frauke; Özbek, Suat

    2016-01-01

    The stinging capsules of cnidarians, nematocysts, function as harpoon-like organelles with unusual biomechanical properties. The nanosecond discharge of the nematocyst requires a dense protein network of the capsule structure withstanding an internal pressure of up to 150 bar. Main components of the capsule are short collagens, so-called minicollagens, that form extended polymers by disulfide reshuffling of their cysteine-rich domains (CRDs). Although CRDs have identical cysteine patterns, they exhibit different structures and disulfide connectivity at minicollagen N and C-termini. We show that the structurally divergent CRDs have different cross-linking potentials in vitro and in vivo. While the C-CRD can participate in several simultaneous intermolecular disulfides and functions as a cystine knot after minicollagen synthesis, the N-CRD is monovalent. Our combined experimental and computational analyses reveal the cysteines in the C-CRD fold to exhibit a higher structural propensity for disulfide bonding and a faster kinetics of polymerization. During nematocyst maturation, the highly reactive C-CRD is instrumental in efficient cross-linking of minicollagens to form pressure resistant capsules. The higher ratio of C-CRD folding types evidenced in the medusozoan lineage might have fostered the evolution of novel, predatory nematocyst types in cnidarians with a free-swimming medusa stage. PMID:27166560

  1. Specialized Information Processing Deficits and Distinct Metabolomic Profiles Following TM-Domain Disruption of Nrg1.

    PubMed

    O'Tuathaigh, Colm M P; Mathur, Naina; O'Callaghan, Matthew J; MacIntyre, Lynsey; Harvey, Richard; Lai, Donna; Waddington, John L; Pickard, Benjamin S; Watson, David G; Moran, Paula M

    2017-03-11

    Although there is considerable genetic and pathologic evidence for an association between neuregulin 1 (NRG1) dysregulation and schizophrenia, the underlying molecular and cellular mechanisms remain unclear. Mutant mice containing disruption of the transmembrane (TM) domain of the NRG1 gene constitute a heuristic model for dysregulation of NRG1-ErbB4 signaling in schizophrenia. The present study focused on hitherto uncharacterized information processing phenotypes in this mutant line. Using a mass spectrometry-based metabolomics approach, we also quantified levels of unique metabolites in brain. Across 2 different sites and protocols, Nrg1 mutants demonstrated deficits in prepulse inhibition, a measure of sensorimotor gating, that is, disrupted in schizophrenia; these deficits were partially reversed by acute treatment with second, but not first-, generation antipsychotic drugs. However, Nrg1 mutants did not show a specific deficit in latent inhibition, a measure of selective attention that is also disrupted in schizophrenia. In contrast, in a "what-where-when" object recognition memory task, Nrg1 mutants displayed sex-specific (males only) disruption of "what-when" performance, indicative of impaired temporal aspects of episodic memory. Differential metabolomic profiling revealed that these behavioral phenotypes were accompanied, most prominently, by alterations in lipid metabolism pathways. This study is the first to associate these novel physiological mechanisms, previously independently identified as being abnormal in schizophrenia, with disruption of NRG1 function. These data suggest novel mechanisms by which compromised neuregulin function from birth might lead to schizophrenia-relevant behavioral changes in adulthood.

  2. Sequence analysis of the non-recurring C-terminal domains shows that insect lipoprotein receptors constitute a distinct group of LDL receptor family members.

    PubMed

    Rodenburg, Kees W; Smolenaars, Marcel M W; Van Hoof, Dennis; Van der Horst, Dick J

    2006-04-01

    Lipoprotein-mediated delivery of lipids in mammals involves endocytic receptors of the low density lipoprotein (LDL) receptor (LDLR) family. In contrast, in insects, the lipoprotein, lipophorin (Lp), functions as a reusable lipid shuttle in lipid delivery, and these animals, therefore, were not supposed to use endocytic receptors. However, recent data indicate additional endocytic uptake of Lp, mediated by a Lp receptor (LpR) of the LDLR family. The two N-terminal domains of LDLR family members are involved in ligand binding and dissociation, respectively, and are composed of a mosaic of multiple repeats. The three C-terminal domains, viz., the optional O-linked glycosylation domain, the transmembrane domain, and the intracellular domain, are of a non-repetitive sequence. The present classification of newly discovered LDLR family members, including the LpRs, bears no relevance to physiological function. Therefore, as a novel approach, the C-terminal domains of LDLR family members across the entire animal kingdom were used to perform a sequence comparison analysis in combination with a phylogenetic tree analysis. The LpRs appeared to segregate into a specific group distinct from the groups encompassing the other family members, and each of the three C-terminal domains of the insect receptors is composed of unique set of sequence motifs. Based on conservation of sequence motifs and organization of these motifs in the domains, LpR resembles most the groups of the LDLRs, very low density lipoprotein (VLDL) receptors, and vitellogenin receptors. However, in sequence aspects in which LpR deviates from these three receptor groups, it most notably resembles LDLR-related protein-2, or megalin. These features might explain the functional differences disclosed between insect and mammalian lipoprotein receptors.

  3. 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...

  4. Synthetic actin-binding domains reveal compositional constraints for function.

    PubMed

    Lorenzi, Maria; Gimona, Mario

    2008-01-01

    The actin-binding domains of many proteins consist of a canonical type 1/type 2 arrangement of the structurally conserved calponin homology domain. Using the actin-binding domain of alpha-actinin-1 as a scaffold we have generated synthetic actin-binding domains by altering position and composition of the calponin homology domains. We show that the presence of two calponin homology domains alone and in the context of an actin-binding domain is not sufficient for actin-binding, and that both single and homotypic type 2 calponin homology domain tandems fail to bind to actin in vitro and in transfected cells. In contrast, single and tandem type 1 calponin homology domain arrays bind actin directly but result in defective turnover rates on actin filaments, and in aberrant actin bundling when introduced into the full-length alpha-actinin molecule. An actin-binding domain harboring the calponin homology domains in an inverted position, however, functions both in isolation and in the context of the dimeric alpha-actinin molecule. Our data demonstrate that the dynamics and specificity of actin-binding via actin-binding domains requires both the filament binding properties of the type 1, and regulation by type 2 calponin homology domains, and appear independent of their position.

  5. CBS domains: structure, function, and pathology in human proteins.

    PubMed

    Ignoul, Sofie; Eggermont, Jan

    2005-12-01

    The cystathionine-beta-synthase (CBS) domain is an evolutionarily conserved protein domain that is present in the proteome of archaebacteria, prokaryotes, and eukaryotes. CBS domains usually come in tandem repeats and are found in cytosolic and membrane proteins performing different functions (metabolic enzymes, kinases, and channels). Crystallographic studies of bacterial CBS domains have shown that two CBS domains form an intramolecular dimeric structure (CBS pair). Several human hereditary diseases (homocystinuria, retinitis pigmentosa, hypertrophic cardiomyopathy, myotonia congenital, etc.) can be caused by mutations in CBS domains of, respectively, cystathionine-beta-synthase, inosine 5'-monophosphate dehydrogenase, AMP kinase, and chloride channels. Despite their clinical relevance, it remains to be established what the precise function of CBS domains is and how they affect the structural and/or functional properties of an enzyme, kinase, or channel. Depending on the protein in which they occur, CBS domains have been proposed to affect multimerization and sorting of proteins, channel gating, and ligand binding. However, recent experiments revealing that CBS domains can bind adenosine-containing ligands such ATP, AMP, or S-adenosylmethionine have led to the hypothesis that CBS domains function as sensors of intracellular metabolites.

  6. 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

  7. 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

  8. 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.

  9. Distinct self-interaction domains promote Multi Sex Combs accumulation in and formation of the Drosophila histone locus body.

    PubMed

    Terzo, Esteban A; Lyons, Shawn M; Poulton, John S; Temple, Brenda R S; Marzluff, William F; Duronio, Robert J

    2015-04-15

    Nuclear bodies (NBs) are structures that concentrate proteins, RNAs, and ribonucleoproteins that perform functions essential to gene expression. How NBs assemble is not well understood. We studied the Drosophila histone locus body (HLB), a NB that concentrates factors required for histone mRNA biosynthesis at the replication-dependent histone gene locus. We coupled biochemical analysis with confocal imaging of both fixed and live tissues to demonstrate that the Drosophila Multi Sex Combs (Mxc) protein contains multiple domains necessary for HLB assembly. An important feature of this assembly process is the self-interaction of Mxc via two conserved N-terminal domains: a LisH domain and a novel self-interaction facilitator (SIF) domain immediately downstream of the LisH domain. Molecular modeling suggests that the LisH and SIF domains directly interact, and mutation of either the LisH or the SIF domain severely impairs Mxc function in vivo, resulting in reduced histone mRNA accumulation. A region of Mxc between amino acids 721 and 1481 is also necessary for HLB assembly independent of the LisH and SIF domains. Finally, the C-terminal 195 amino acids of Mxc are required for recruiting FLASH, an essential histone mRNA-processing factor, to the HLB. We conclude that multiple domains of the Mxc protein promote HLB assembly in order to concentrate factors required for histone mRNA biosynthesis.

  10. Recombinant spider silk genetically functionalized with affinity domains.

    PubMed

    Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-Åke; Hedhammar, My

    2014-05-12

    Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

  11. Modular protein domains: an engineering approach toward functional biomaterials.

    PubMed

    Lin, Charng-Yu; Liu, Julie C

    2016-08-01

    Protein domains and peptide sequences are a powerful tool for conferring specific functions to engineered biomaterials. Protein sequences with a wide variety of functionalities, including structure, bioactivity, protein-protein interactions, and stimuli responsiveness, have been identified, and advances in molecular biology continue to pinpoint new sequences. Protein domains can be combined to make recombinant proteins with multiple functionalities. The high fidelity of the protein translation machinery results in exquisite control over the sequence of recombinant proteins and the resulting properties of protein-based materials. In this review, we discuss protein domains and peptide sequences in the context of functional protein-based materials, composite materials, and their biological applications.

  12. 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.

  13. Dynamic remodeling of microbial biofilms by functionally distinct exopolysaccharides.

    PubMed

    Chew, Su Chuen; Kundukad, Binu; Seviour, Thomas; van der Maarel, Johan R C; Yang, Liang; Rice, Scott A; Doyle, Patrick; Kjelleberg, Staffan

    2014-08-05

    Biofilms are densely populated communities of microbial cells protected and held together by a matrix of extracellular polymeric substances. The structure and rheological properties of the matrix at the microscale influence the retention and transport of molecules and cells in the biofilm, thereby dictating population and community behavior. Despite its importance, quantitative descriptions of the matrix microstructure and microrheology are limited. Here, particle-tracking microrheology in combination with genetic approaches was used to spatially and temporally study the rheological contributions of the major exopolysaccharides Pel and Psl in Pseudomonas aeruginosa biofilms. Psl increased the elasticity and effective cross-linking within the matrix, which strengthened its scaffold and appeared to facilitate the formation of microcolonies. Conversely, Pel reduced effective cross-linking within the matrix. Without Psl, the matrix becomes more viscous, which facilitates biofilm spreading. The wild-type biofilm decreased in effective cross-linking over time, which would be advantageous for the spreading and colonization of new surfaces. This suggests that there are regulatory mechanisms to control production of the exopolysaccharides that serve to remodel the matrix of developing biofilms. The exopolysaccharides were also found to have profound effects on the spatial organization and integration of P. aeruginosa in a mixed-species biofilm model of P. aeruginosa-Staphylococcus aureus. Pel was required for close association of the two species in mixed-species microcolonies. In contrast, Psl was important for P. aeruginosa to form single-species biofilms on top of S. aureus biofilms. Our results demonstrate that Pel and Psl have distinct physical properties and functional roles during biofilm formation. Importance: Most bacteria grow as biofilms in the environment or in association with eukaryotic hosts. Removal of biofilms that form on surfaces is a challenge in clinical

  14. Extensions of PDZ domains as important structural and functional elements.

    PubMed

    Wang, Conan K; Pan, Lifeng; Chen, Jia; Zhang, Mingjie

    2010-08-01

    'Divide and conquer' has been the guiding strategy for the study of protein structure and function. Proteins are divided into domains with each domain having a canonical structural definition depending on its type. In this review, we push forward with the interesting observation that many domains have regions outside of their canonical definition that affect their structure and function; we call these regions 'extensions'. We focus on the highly abundant PDZ (PSD-95, DLG1 and ZO-1) domain. Using bioinformatics, we find that many PDZ domains have potential extensions and we developed an openly-accessible website to display our results ( http://bcz102.ust.hk/pdzex/ ). We propose, using well-studied PDZ domains as illustrative examples, that the roles of PDZ extensions can be classified into at least four categories: 1) protein dynamics-based modulation of target binding affinity, 2) provision of binding sites for macro-molecular assembly, 3) structural integration of multi-domain modules, and 4) expansion of the target ligand-binding pocket. Our review highlights the potential structural and functional importance of domain extensions, highlighting the significance of looking beyond the canonical boundaries of protein domains in general.

  15. ABCA1, ABCG1, and ABCG4 are distributed to distinct membrane meso-domains and disturb detergent-resistant domains on the plasma membrane.

    PubMed

    Sano, Osamu; Ito, Shiho; Kato, Reiko; Shimizu, Yuji; Kobayashi, Aya; Kimura, Yasuhisa; Kioka, Noriyuki; Hanada, Kentaro; Ueda, Kazumitsu; Matsuo, Michinori

    2014-01-01

    ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters.

  16. Direct activation and anti-repression functions of GAL4-VP16 use distinct molecular mechanisms.

    PubMed Central

    Lyons, J G; Chambon, P

    1995-01-01

    In order to determine whether the molecular mechanisms used for direct activation by GAL4-VP16 are the same as those used for anti-repression, we have employed monoclonal antibodies specific for the VP16 activation domain. In the absence of added repressors, GAL4-VP16 was able to stimulate transcription from a template containing GAL4-binding sites, and the antibodies raised against the VP16 activation domain failed to inhibit this direct activation. GAL4-VP16 also was able to prevent histone H1-mediated repression by a mechanism that was strongly dependent on the presence of specific GAL4-binding elements in the promoter. However, in contrast to the assays conducted in the absence of repressors, the antibodies were strong inhibitors of GAL4-VP16-activated transcription in the presence of histone H1. Thus the binding of the antibodies distinguished between the direct activation and anti-repression functions of GAL4-VP16, indicating that these functions operate through distinct molecular mechanisms. The anti-repression-specific mechanism that is inhibitable by the antibodies acted at an early stage of preinitiation complex formation. Deletions of individual subdomains of the VP16 activation domain demonstrated that there was not a discrete subdomain responsible for the anti-repression function of GAL4-VP16. Thus, the inhibitory effect of the antibodies appeared to be due to the location of the epitope within the activator protein rather than to some inherent biochemical property of that region of the protein that is required specifically for anti-repression. The inhibitory effect of the antibodies also ruled out the possibility that steric exclusion of repressor proteins from the promoter was the sole means of anti-repression by the transcriptional activator. Images Figure 1 Figure 2 PMID:8554536

  17. Variable-Domain Functional Regression for Modeling ICU Data.

    PubMed

    Gellar, Jonathan E; Colantuoni, Elizabeth; Needham, Dale M; Crainiceanu, Ciprian M

    2014-12-01

    We introduce a class of scalar-on-function regression models with subject-specific functional predictor domains. The fundamental idea is to consider a bivariate functional parameter that depends both on the functional argument and on the width of the functional predictor domain. Both parametric and nonparametric models are introduced to fit the functional coefficient. The nonparametric model is theoretically and practically invariant to functional support transformation, or support registration. Methods were motivated by and applied to a study of association between daily measures of the Intensive Care Unit (ICU) Sequential Organ Failure Assessment (SOFA) score and two outcomes: in-hospital mortality, and physical impairment at hospital discharge among survivors. Methods are generally applicable to a large number of new studies that record a continuous variables over unequal domains.

  18. Membrane-bound mucin modular domains: from structure to function.

    PubMed

    Jonckheere, Nicolas; Skrypek, Nicolas; Frénois, Frédéric; Van Seuningen, Isabelle

    2013-06-01

    Mucins belong to a heterogeneous family of large O-glycoproteins composed of a long peptidic chain called apomucin on which are linked hundreds of oligosaccharidic chains. Among mucins, membrane-bound mucins are modular proteins and have a structural organization usually containing Pro/Thr/Ser-rich O-glycosylated domains (PTS), EGF-like and SEA domains. Via these modular domains, the membrane-bound mucins participate in cell signalling and cell interaction with their environment in normal and pathological conditions. Moreover, the recent knowledge of these domains and their biological activities led to the development of new therapeutic approaches involving mucins. In this review, we show 3D structures of EGF and SEA domains. We also describe the functional features of the evolutionary conserved domains of membrane-bound mucins and discuss consequences of splice events.

  19. Domain mobility in proteins: functional and evolutionary implications.

    PubMed

    Basu, Malay Kumar; Poliakov, Eugenia; Rogozin, Igor B

    2009-05-01

    A substantial fraction of eukaryotic proteins contains multiple domains, some of which show a tendency to occur in diverse domain architectures and can be considered mobile (or 'promiscuous'). These promiscuous domains are typically involved in protein-protein interactions and play crucial roles in interaction networks, particularly those contributing to signal transduction. They also play a major role in creating diversity of protein domain architecture in the proteome. It is now apparent that promiscuity is a volatile and relatively fast-changing feature in evolution, and that only a few domains retain their promiscuity status throughout evolution. Many such domains attained their promiscuity status independently in different lineages. Only recently, we have begun to understand the diversity of protein domain architectures and the role the promiscuous domains play in evolution of this diversity. However, many of the biological mechanisms of protein domain mobility remain shrouded in mystery. In this review, we discuss our present understanding of protein domain promiscuity, its evolution and its role in cellular function.

  20. Aperiodic topological order in the domain configurations of functional materials

    NASA Astrophysics Data System (ADS)

    Huang, Fei-Ting; Cheong, Sang-Wook

    2017-03-01

    In numerous functional materials, such as steels, ferroelectrics and magnets, new functionalities can be achieved through the engineering of the domain structures, which are associated with the ordering of certain parameters within the material. The recent progress in technologies that enable imaging at atomic-scale spatial resolution has transformed our understanding of domain topology, revealing that, along with simple stripe-like or irregularly shaped domains, intriguing vortex-type topological domain configurations also exist. In this Review, we present a new classification scheme of 'Zm Zn domains with Zl vortices' for 2D macroscopic domain structures with m directional variants and n translational antiphases. This classification, together with the concepts of topological protection and topological charge conservation, can be applied to a wide range of materials, such as multiferroics, improper ferroelectrics, layered transition metal dichalcogenides and magnetic superconductors, as we discuss using selected examples. The resulting topological considerations provide a new basis for the understanding of the formation, kinetics, manipulation and property optimization of domains and domain boundaries in functional materials.

  1. Distinct enzyme combinations in AKAP signalling complexes permit functional diversity.

    PubMed

    Hoshi, Naoto; Langeberg, Lorene K; Scott, John D

    2005-11-01

    Specificity in cell signalling can be influenced by the targeting of different enzyme combinations to substrates. The A-kinase anchoring protein AKAP79/150 is a multivalent scaffolding protein that coordinates the subcellular localization of second-messenger-regulated enzymes, such as protein kinase A, protein kinase C and protein phosphatase 2B. We developed a new strategy that combines RNA interference of the endogenous protein with a protocol that selects cells that have been rescued with AKAP79/150 forms that are unable to anchor selected enzymes. Using this approach, we show that AKAP79/150 coordinates different enzyme combinations to modulate the activity of two distinct neuronal ion channels: AMPA-type glutamate receptors and M-type potassium channels. Utilization of distinct enzyme combinations in this manner provides a means to expand the repertoire of cellular events that the same AKAP modulates.

  2. Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases.

    PubMed

    Panigrahi, Rashmi; Lemieux, M Joanne

    2015-01-01

    Intramembrane proteases are membrane embedded enzymes that cleave transmembrane substrates. This interesting class of enzyme and its water mediated substrate cleavage mechanism occurring within the hydrophobic lipid bilayer has drawn the attention of researchers. Rhomboids are a family of ubiquitous serine intramembrane proteases. Bacterial forms of rhomboid proteases are mainly composed of six transmembrane helices that are preceded by a soluble N-terminal domain. Several crystal structures of the membrane domain of the E. coli rhomboid protease ecGlpG have been solved. Independently, the ecGlpG N-terminal cytoplasmic domain structure was solved using both NMR and protein crystallography. Despite these structures, we still do not know the structure of the full-length protein, nor do we know the functional role of these domains in the cell. This chapter will review the structural and functional roles of the different domains associated with prokaryotic rhomboid proteases. Lastly, we will address questions remaining in the field.

  3. Disgust trait modulates frontal-posterior coupling as a function of disgust domain.

    PubMed

    Borg, Charmaine; de Jong, Peter J; Renken, Remco J; Georgiadis, Janniko R

    2013-03-01

    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.

  4. Chlamydia trachomatis Tarp harbors distinct G and F actin binding domains that bundle actin filaments.

    PubMed

    Jiwani, Shahanawaz; Alvarado, Stephenie; Ohr, Ryan J; Romero, Adriana; Nguyen, Brenda; Jewett, Travis J

    2013-02-01

    All species of Chlamydia undergo a unique developmental cycle that transitions between extracellular and intracellular environments and requires the capacity to invade new cells for dissemination. A chlamydial protein called Tarp has been shown to nucleate actin in vitro and is implicated in bacterial entry into human cells. Colocalization studies of ectopically expressed enhanced green fluorescent protein (EGFP)-Tarp indicate that actin filament recruitment is restricted to the C-terminal half of the effector protein. Actin filaments are presumably associated with Tarp via an actin binding alpha helix that is also required for actin nucleation in vitro, but this has not been investigated. Tarp orthologs from C. pneumoniae, C. muridarum, and C. caviae harbor between 1 and 4 actin binding domains located in the C-terminal half of the protein, but C. trachomatis serovar L2 has only one characterized domain. In this work, we examined the effects of domain-specific mutations on actin filament colocalization with EGFP-Tarp. We now demonstrate that actin filament colocalization with Tarp is dependent on two novel F-actin binding domains that endow the Tarp effector with actin-bundling activity. Furthermore, Tarp-mediated actin bundling did not require actin nucleation, as the ability to bundle actin filaments was observed in mutant Tarp proteins deficient in actin nucleation. These data shed molecular insight on the complex cytoskeletal rearrangements required for C. trachomatis entry into host cells.

  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.

  6. Two Distinct Central Serotonin Receptors with Different Physiological Functions

    NASA Astrophysics Data System (ADS)

    Peroutka, Stephen J.; Lebovitz, Richard M.; Snyder, Solomon H.

    1981-05-01

    Two distinct serotonin (5-hydroxytryptamine) receptors designated serotonin 1 and serotonin 2 bind tritium-labeled serotonin and tritium-labeled spiroperidol, respectively. Drug potencies at serotonin 2 sites, but not at serotonin 1 sites, predict their effects on the ``serotonin behavioral syndrome,'' indicating that serotonin 2 sites mediate these behaviors. The limited correlation of drug effects with regulation by guanine nucleotides suggests that serotonin 1 sites might be linked to adenylate cyclase. Drug specificities of serotonin-elicited synaptic inhibition and excitation may reflect serotonin 1 and serotonin 2 receptor interactions, respectively.

  7. MLLT1 YEATS domain mutations in clinically distinctive Favourable Histology Wilms tumours | Office of Cancer Genomics

    Cancer.gov

    Wilms tumour is an embryonal tumour of childhood that closely resembles the developing kidney. Genomic changes responsible for the development of the majority of Wilms tumours remain largely unknown. Here we identify recurrent mutations within Wilms tumours that involve the highly conserved YEATS domain of MLLT1 (ENL), a gene known to be involved in transcriptional elongation during early development. The mutant MLLT1 protein shows altered binding to acetylated histone tails.

  8. Identical folds used for distinct mechanical functions of the bacterial flagellar rod and hook

    PubMed Central

    Fujii, Takashi; Kato, Takayuki; Hiraoka, Koichi D.; Miyata, Tomoko; Minamino, Tohru; Chevance, Fabienne F. V.; Hughes, Kelly T.; Namba, Keiichi

    2017-01-01

    The bacterial flagellum is a motile organelle driven by a rotary motor, and its axial portions function as a drive shaft (rod), a universal joint (hook) and a helical propeller (filament). The rod and hook are directly connected to each other, with their subunit proteins FlgG and FlgE having 39% sequence identity, but show distinct mechanical properties; the rod is straight and rigid as a drive shaft whereas the hook is flexible in bending as a universal joint. Here we report the structure of the rod and comparison with that of the hook. While these two structures have the same helical symmetry and repeat distance and nearly identical folds of corresponding domains, the domain orientations differ by ∼7°, resulting in tight and loose axial subunit packing in the rod and hook, respectively, conferring the rigidity on the rod and flexibility on the hook. This provides a good example of versatile use of a protein structure in biological organisms. PMID:28120828

  9. Structural similarities and functional diversity of eukaryotic discoidin-like domains.

    PubMed

    Kiedzierska, A; Smietana, K; Czepczynska, H; Otlewski, J

    2007-09-01

    The discoidin domain is a approximately 150 amino acid motif common in both eukaryotic and prokaryotic proteins. It is found in a variety of extracellular, intracellular and transmembrane multidomain proteins characterized by a considerable functional diversity, mostly involved in developmental processes. The biological role of the domain depends on its interactions with different molecules, including growth factors, phospholipids and lipids, galactose or its derivatives, and collagen. The conservation of the motif, as well as the serious physiological consequences of discoidin domain disorders underscore the importance of the fold, while the ability to accommodate such an extraordinarily broad range of ligand molecules makes it a fascinating research target. In present review we characterize the distinctive features of discoidin domains and briefly outline the biological role of this module in various eukaryotic proteins.

  10. Limited proteolysis differentially modulates the stability and subcellular localization of domains of RPGRIP1 that are distinctly affected by mutations in Leber's congenital amaurosis.

    PubMed

    Lu, Xinrong; Guruju, Mallikarjuna; Oswald, John; Ferreira, Paulo A

    2005-05-15

    The retinitis pigmentosa GTPase regulator (RPGR) protein interacts with the retinitis pigmentosa GTPase regulator interacting protein-1 (RPGRIP1). Genetic lesions in the cognate genes lead to distinct and severe human retinal dystrophies. The biological role of these proteins in retinal function and pathogenesis of retinal diseases is elusive. Here, we present the first physiological assay of the role of RPGRIP1 and mutations therein. We found that the monoallelic and homozygous mutations, DeltaE1279 and D1114G, in the RPGR-interacting domain (RID) of RPGRIP1, enhance and abolish, respectively, its interaction in vivo with RPGR without affecting the stability of RID. In contrast to RID(WT) and RID(D1114G), chemical genetics shows that the interaction of RID(DeltaE1279) with RPGR is resistant to various stress treatments such as osmotic, pH and heat-shock stimuli. Hence, RID(D1114G) and RID(DeltaE1279) constitute loss- and gain-of-function mutations. Moreover, we find that the isoforms, bRPGRIP1 and bRPGRIP1b, undergo limited proteolysis constitutively in vivo in the cytoplasm compartment. This leads to the relocation and accumulation of a small and stable N-terminal domain of approximately 7 kDa to the nucleus, whereas the cytosolic C-terminal domain of RPGRIP1 is degraded and short-lived. The RID(D1114G) and RID(DeltaE1279) mutations exhibit strong cis-acting and antagonistic biological effects on the nuclear relocation, subcellular distribution and proteolytic cleavage of RPGRIP1 and/or domains thereof. These data support distinct and spatiotemporal subcellular-specific roles to RPGRIP1. A novel RPGRIP1-mediated nucleocytoplasmic crosstalk and transport pathway regulated by RID, and hence by RPGR, emerges with implications in the molecular pathogenesis of retinopathies, and a model to other diseases.

  11. The putative LEF-1 proteins from two distinct Choristoneura fumiferana multiple nucleopolyhedroviruses share domain homology to eukaryotic primases.

    PubMed

    Barrett, J W; Lauzon, H A; Mercuri, P S; Krell, P J; Sohi, S S; Arif, B M

    1996-01-01

    We have identified the lef-1 genes from two multiple nucleopolyhedroviruses that infect natural populations of Choristoneura fumiferana. The lef-1 genes in both viruses are directly upstream and in the opposite orientation of their respective ecdysteroid UDP-glucosyltransferase (egt) genes. This gene organization pattern is similar to that found in the genomes of AcMNPV and of OpMNPV. As well, the coding regions and putative protein sequences share a high degree of similarity. Alignment of the predicted amino acid sequences of all known baculovirus lef-1 genes suggests that the LEF-1 proteins have a relatively high degree of conservation, particularly at four identified and distinct domains. Moreover, LEF-1 proteins bear clear similarity to some eukaryotic primases, predominately at three of the four domains where certain amino acids are absolutely conserved.

  12. Differential antibiosis against Helicoverpa armigera exerted by distinct inhibitory repeat domains of Capsicum annuum proteinase inhibitors.

    PubMed

    Joshi, Rakesh S; Gupta, Vidya S; Giri, Ashok P

    2014-05-01

    Plant defensive serine proteinase inhibitors (PIs) are known to have negative impact on digestive physiology of herbivore insects and thus have a crucial role in plant protection. Here, we have assessed the efficacy and specificity of three previously characterized inhibitory repeat domain (IRD) variants from Capsicum annuum PIs viz., IRD-7, -9 and -12 against gut proteinases from Helicoverpa armigera. Comparative study of in silico binding energy revealed that IRD-9 possesses higher affinity towards H. armigera serine proteinases as compared to IRD-7 and -12. H. armigera fed on artificial diet containing 5 TIU/g of recombinant IRD proteins exhibited differential effects on larval growth, survival rate and other nutritional parameters. Major digestive gut trypsin and chymotrypsin genes were down regulated in the IRD fed larvae, while few of them were up-regulated, this indicate alterations in insect digestive physiology. The results corroborated with proteinase activity assays and zymography. These findings suggest that the sequence variations among PIs reflect in their efficacy against proteinases in vitro and in vivo, which also could be used for developing tailor-made multi-domain inhibitor gene(s).

  13. Coordinated and Distinct Functions of Velvet Proteins in Fusarium verticillioides

    PubMed Central

    Lan, Nan; Zhang, Hanxing; Hu, Chengcheng; Wang, Wenzhao; Calvo, Ana M.; Harris, Steven D.; Chen, She

    2014-01-01

    Velvet-domain-containing proteins are broadly distributed within the fungal kingdom. In the corn pathogen Fusarium verticillioides, previous studies showed that the velvet protein F. verticillioides VE1 (FvVE1) is critical for morphological development, colony hydrophobicity, toxin production, and pathogenicity. In this study, tandem affinity purification of FvVE1 revealed that FvVE1 can form a complex with the velvet proteins F. verticillioides VelB (FvVelB) and FvVelC. Phenotypic characterization of gene knockout mutants showed that, as in the case of FvVE1, FvVelB regulated conidial size, hyphal hydrophobicity, fumonisin production, and oxidant resistance, while FvVelC was dispensable for these biological processes. Comparative transcriptional analysis of eight genes involved in the ROS (reactive oxygen species) removal system revealed that both FvVE1 and FvVelB positively regulated the transcription of a catalase-encoding gene, F. verticillioides CAT2 (FvCAT2). Deletion of FvCAT2 resulted in reduced oxidant resistance, providing further explanation of the regulation of oxidant resistance by velvet proteins in the fungal kingdom. PMID:24792348

  14. KRAS insertion mutations are oncogenic and exhibit distinct functional properties

    PubMed Central

    White, Yasmine; Bagchi, Aditi; Van Ziffle, Jessica; Inguva, Anagha; Bollag, Gideon; Zhang, Chao; Carias, Heidi; Dickens, David; Loh, Mignon; Shannon, Kevin; Firestone, Ari J.

    2016-01-01

    Oncogenic KRAS mutations introduce discrete amino acid substitutions that reduce intrinsic Ras GTPase activity and confer resistance to GTPase-activating proteins (GAPs). Here we discover a partial duplication of the switch 2 domain of K-Ras encoding a tandem repeat of amino acids G60_A66dup in a child with an atypical myeloproliferative neoplasm. K-Ras proteins containing this tandem duplication or a similar five amino acid E62_A66dup mutation identified in lung and colon cancers transform the growth of primary myeloid progenitors and of Ba/F3 cells. Recombinant K-RasG60_A66dup and K-RasE62_A66dup proteins display reduced intrinsic GTP hydrolysis rates, accumulate in the GTP-bound conformation and are resistant to GAP-mediated GTP hydrolysis. Remarkably, K-Ras proteins with switch 2 insertions are impaired for PI3 kinase binding and Akt activation, and are hypersensitive to MEK inhibition. These studies illuminate a new class of oncogenic KRAS mutations and reveal unexpected plasticity in oncogenic Ras proteins that has diagnostic and therapeutic implications. PMID:26854029

  15. Distinct Brca1 Mutations Differentially Reduce Hematopoietic Stem Cell Function.

    PubMed

    Mgbemena, Victoria E; Signer, Robert A J; Wijayatunge, Ranjula; Laxson, Travis; Morrison, Sean J; Ross, Theodora S

    2017-01-24

    BRCA1 is a well-known DNA repair pathway component and a tissue-specific tumor suppressor. However, its role in hematopoiesis is uncertain. Here, we report that a cohort of patients heterozygous for BRCA1 mutations experienced more hematopoietic toxicity from chemotherapy than those with BRCA2 mutations. To test whether this reflects a requirement for BRCA1 in hematopoiesis, we generated mice with Brca1 mutations in hematopoietic cells. Mice homozygous for a null Brca1 mutation in the embryonic hematopoietic system (Vav1-iCre;Brca1(F22-24/F22-24)) developed hematopoietic defects in early adulthood that included reduced hematopoietic stem cells (HSCs). Although mice homozygous for a huBRCA1 knockin allele (Brca1(BRCA1/BRCA1)) were normal, mice with a mutant huBRCA1/5382insC allele and a null allele (Mx1-Cre;Brca1(F22-24/5382insC)) had severe hematopoietic defects marked by a complete loss of hematopoietic stem and progenitor cells. Our data show that Brca1 is necessary for HSC maintenance and normal hematopoiesis and that distinct mutations lead to different degrees of hematopoietic dysfunction.

  16. The distinctive role of executive functions in implicit emotion regulation.

    PubMed

    Sperduti, Marco; Makowski, Dominique; Arcangeli, Margherita; Wantzen, Prany; Zalla, Tiziana; Lemaire, Stéphane; Dokic, Jérôme; Pelletier, Jérôme; Piolino, Pascale

    2017-02-01

    Several theoretical models stress the role of executive functions in emotion regulation (ER). However, most of the previous studies on ER employed explicit regulatory strategies that could have engaged executive functions, beyond regulatory processes per se. Recently, there has been renewed interest in implicit forms of ER, believed to be closer to daily-life requirements. While various studies have shown that implicit and explicit ER engage partially overlapping neurocognitive processes, the contribution of different executive functions in implicit ER has not been investigated. In the present study, we presented participants with negatively valenced pictures of varying emotional intensity preceded by short texts describing them as either fictional or real. This manipulation was meant to induce a spontaneous emotional down-regulation. We recorded electrodermal activity (EDA) and subjective reports of emotion arousal. Executive functions (updating, switching, and inhibition) were also assessed. No difference was found between the fictional and real condition on EDA. A diminished self-reported arousal was observed, however, when pictures were described as fictional for high- and mild-intensity material, but not for neutral material. The amount of down-regulation in the fictional condition was found to be predicted by interindividual variability in updating performances, but not by the other measures of executive functions, suggesting its implication even in implicit forms of ER. The relationship between down-regulation and updating was significant only for high-intensity material. We discuss the role of updating in relation to the consciousness of one's emotional state.

  17. 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

  18. The distinction in radiobiology between medical and public health functions

    SciTech Connect

    Bond, V.P.; Wielopolski, L.

    1995-12-31

    Starting with the classical threshold-sigmoid Medical-Toxicological plot, reasons are advanced for why the coordinates of this function are not appropriate for the analysis of Public Health-Epidemiological data. Misunderstanding with respect to both the level of biological organization and the word ``dose`` are pointed out, which explain why Public Health-Epidemiological data, anomalously, yield linear functions on medical-toxicological coordinates. It is then shown why substantially different coordinates must be used to obtain a function that describes properly and completely the cancer data obtained from epidemiological studies on the atomic bomb survivors. Arguments are put forth that seriously weaken the current interpretation of the ``linear, no-threshold hypothesis``. Reasons are advanced for why, if the amount of radiation energy is expressed in the proper terms, the numerical value for the cancer ``risk coefficient`` becomes substantially smaller than it now is.

  19. Nuclear stability and transcriptional directionality separate functionally distinct RNA species.

    PubMed

    Andersson, Robin; Refsing Andersen, Peter; Valen, Eivind; Core, Leighton J; Bornholdt, Jette; Boyd, Mette; Heick Jensen, Torben; Sandelin, Albin

    2014-11-12

    Mammalian genomes are pervasively transcribed, yielding a complex transcriptome with high variability in composition and cellular abundance. Although recent efforts have identified thousands of new long non-coding (lnc) RNAs and demonstrated a complex transcriptional repertoire produced by protein-coding (pc) genes, limited progress has been made in distinguishing functional RNA from spurious transcription events. This is partly due to present RNA classification, which is typically based on technical rather than biochemical criteria. Here we devise a strategy to systematically categorize human RNAs by their sensitivity to the ribonucleolytic RNA exosome complex and by the nature of their transcription initiation. These measures are surprisingly effective at correctly classifying annotated transcripts, including lncRNAs of known function. The approach also identifies uncharacterized stable lncRNAs, hidden among a vast majority of unstable transcripts. The predictive power of the approach promises to streamline the functional analysis of known and novel RNAs.

  20. The mitochondrial elongation factors MIEF1 and MIEF2 exert partially distinct functions in mitochondrial dynamics

    SciTech Connect

    Liu, Tong; Yu, Rong; Jin, Shao-Bo; Han, Liwei; Lendahl, Urban; Zhao, Jian; Nistér, Monica

    2013-11-01

    Mitochondria are dynamic organelles whose morphology is regulated by a complex balance of fission and fusion processes, and we still know relatively little about how mitochondrial dynamics is regulated. MIEF1 (also called MiD51) has recently been characterized as a key regulator of mitochondrial dynamics and in this report we explore the functions of its paralog MIEF2 (also called MiD49), to learn to what extent MIEF2 is functionally distinct from MIEF1. We show that MIEF1 and MIEF2 have many functions in common. Both are anchored in the mitochondrial outer membrane, recruit Drp1 from the cytoplasm to the mitochondrial surface and cause mitochondrial fusion, and MIEF2, like MIEF1, can interact with Drp1 and hFis1. MIEF1 and MIEF2, however, also differ in certain aspects. MIEF1 and MIEF2 are differentially expressed in human tissues during development. When overexpressed, MIEF2 exerts a stronger fusion-promoting effect than MIEF1, and in line with this, hFis1 and Mff can only partially revert the MIEF2-induced fusion phenotype, whereas MIEF1-induced fusion is reverted to a larger extent by hFis1 and Mff. MIEF2 forms high molecular weight oligomers, while MIEF1 is largely present as a dimer. Furthermore, MIEF1 and MIEF2 use distinct domains for oligomerization: in MIEF1, the region from amino acid residues 109–154 is required, whereas oligomerization of MIEF2 depends on amino acid residues 1 to 49, i.e. the N-terminal end. We also show that oligomerization of MIEF1 is not required for its mitochondrial localization and interaction with Drp1. In conclusion, our data suggest that the mitochondrial regulators MIEF1 and MIEF2 exert partially distinct functions in mitochondrial dynamics. - Highlights: • MIEF1 and MIEF2 recruit Drp1 to mitochondria and cause mitochondrial fusion. • MIEF2, like MIEF1, can interact with Drp1 and hFis1. • MIEF1 and MIEF2 are differentially expressed in human tissues during development. • MIEF2 exerts a stronger fusion

  1. VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System

    PubMed Central

    Cianfanelli, Francesca R.; Alcoforado Diniz, Juliana; Guo, Manman; De Cesare, Virginia; Trost, Matthias; Coulthurst, Sarah J.

    2016-01-01

    The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently (‘specialised’) or non-covalently (‘cargo’ effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a ‘core’ T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the

  2. MLLT1 YEATS domain mutations in clinically distinctive Favourable Histology Wilms tumours

    PubMed Central

    Perlman, Elizabeth J.; Gadd, Samantha; Arold, Stefan T.; Radhakrishnan, Anand; Gerhard, Daniela S.; Jennings, Lawrence; Huff, Vicki; Guidry Auvil, Jaime M.; Davidsen, Tanja M.; Dome, Jeffrey S.; Meerzaman, Daoud; Hsu, Chih Hao; Nguyen, Cu; Anderson, James; Ma, Yussanne; Mungall, Andrew J.; Moore, Richard A.; Marra, Marco A.; Mullighan, Charles G.; Ma, Jing; Wheeler, David A.; Hampton, Oliver A.; Gastier-Foster, Julie M.; Ross, Nicole; Smith, Malcolm A.

    2015-01-01

    Wilms tumour is an embryonal tumour of childhood that closely resembles the developing kidney. Genomic changes responsible for the development of the majority of Wilms tumours remain largely unknown. Here we identify recurrent mutations within Wilms tumours that involve the highly conserved YEATS domain of MLLT1 (ENL), a gene known to be involved in transcriptional elongation during early development. The mutant MLLT1 protein shows altered binding to acetylated histone tails. Moreover, MLLT1-mutant tumours show an increase in MYC gene expression and HOX dysregulation. Patients with MLLT1-mutant tumours present at a younger age and have a high prevalence of precursor intralobar nephrogenic rests. These data support a model whereby activating MLLT1 mutations early in renal development result in the development of Wilms tumour. PMID:26635203

  3. A novel functionally distinct subtype of striatal neuropeptide Y interneuron.

    PubMed

    Ibáñez-Sandoval, Osvaldo; Tecuapetla, Fatuel; Unal, Bengi; Shah, Fulva; Koós, Tibor; Tepper, James M

    2011-11-16

    We investigated the properties of neostriatal neuropeptide Y (NPY)-expressing interneurons in transgenic GFP (green fluorescent protein)-NPY reporter mice. In vitro whole-cell recordings and biocytin staining demonstrated the existence of a novel class of neostriatal NPY-expressing GABAergic interneurons that exhibit electrophysiological, neurochemical, and morphological properties strikingly different from those of previously described NPY-containing, plateau-depolarization low-threshold spike (NPY-PLTS) interneurons. The novel NPY interneuron type (NPY-neurogliaform) differed from previously described NPY-PLTS interneurons by exhibiting a significantly lower input resistance and hyperpolarized membrane potential, regular, nonaccommodating spiking in response to depolarizing current injections, and an absence of plateau depolarizations or low-threshold spikes. NPY-neurogliaform interneurons were also easily distinguished morphologically by their dense, compact, and highly branched dendritic and local axonal arborizations that contrasted sharply with the sparse and extended axonal and dendritic arborizations of NPY-PLTS interneurons. Furthermore, NPY-neurogliaform interneurons did not express immunofluorescence for somatostatin or nitric oxide synthase that was ubiquitous in NPY-PLTS interneurons. IPSP/Cs could only rarely be elicited in spiny projection neurons (SPNs) in paired recordings with NPY-PLTS interneurons. In contrast, the probability of SPN innervation by NPY-neurogliaform interneurons was extremely high, the synapse very reliable (no failures were observed), and the resulting postsynaptic response was a slow, GABA(A) receptor-mediated IPSC that has not been previously described in striatum but that has been elicited from NPY-GABAergic neurogliaform interneurons in cortex and hippocampus. These properties suggest unique and distinctive roles for NPY-PLTS and NPY-neurogliaform interneurons in the integrative properties of the neostriatum.

  4. Tryptophan Scanning Mutagenesis Identifies the Molecular Determinants of Distinct Barttin Functions*

    PubMed Central

    Wojciechowski, Daniel; Fischer, Martin; Fahlke, Christoph

    2015-01-01

    CLC-K chloride channels are expressed in the kidney and in the inner ear and require the accessory subunit barttin for proper function and membrane insertion. Barttin exerts multiple functions on CLC-proteins: it modifies protein stability and intracellular trafficking as well as channel activity, ion conduction, and gating. So far, the molecular determinants of these distinct barttin functions have remained elusive. Here we performed serial perturbation mutagenesis to identify the sequence determinants of barttin function. Barttin consists of two transmembrane helices followed by a long intracellular carboxyl terminus, and earlier work demonstrated that the transmembrane core of barttin suffices for most effects on the α-subunit. We individually substituted every amino acid of the predicted transmembrane core (amino acids 9–26 and 35–55) with tryptophan, co-expressed mutant barttin with hClC-Ka or V166E rClC-K1, and characterized CLC-K/barttin channels by patch clamp techniques, biochemistry, and confocal microscopy. The majority of mutations left the chaperone function of barttin, i.e. the effects on endoplasmic reticulum exit and surface membrane insertion, unaffected. In contrast, tryptophan insertion at multiple positions resulted in impaired activity of hClC-Ka/barttin and changes in gating of V166E rClC-K1/barttin. These results demonstrate that mutations in a cluster of hydrophobic residues within transmembrane domain 1 affect barttin-CLC-K interaction and impair gating modification by the accessory subunit. Whereas tight interaction is necessary for functional modification, even impaired association of barttin and CLC-K suffices for normal intracellular trafficking. Our findings allow definition of a likely interaction surface and clarify the mechanisms underlying CLC-K channel modification by barttin. PMID:26063802

  5. TLR-exosomes exhibit distinct kinetics and effector function

    PubMed Central

    Srinivasan, Swetha; Su, Michelle; Ravishankar, Shashidhar; Moore, James; Head, PamelaSara; Dixon, J. Brandon; Vannberg, Fredrik

    2017-01-01

    The innate immune system is vital to rapidly responding to pathogens and Toll-like receptors (TLRs) are a critical component of this response. Nanovesicular exosomes play a role in immunity, but to date their exact contribution to the dissemination of the TLR response is unknown. Here we show that exosomes from TLR stimulated cells can largely recapitulate TLR activation in distal cells in vitro. We can abrogate the action-at-a-distance signaling of exosomes by UV irradiation, demonstrating that RNA is crucial for their effector function. We are the first to show that exosomes derived from poly(I:C) stimulated cells induce in vivo macrophage M1-like polarization within murine lymph nodes. These poly(I:C) exosomes demonstrate enhanced trafficking to the node and preferentially recruit neutrophils as compared to control exosomes. This work definitively establishes the differential effector function for exosomes in communicating the TLR activation state of the cell of origin. PMID:28290538

  6. 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

  7. 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.

  8. Diversity of Structure and Function of Response Regulator Output Domains

    PubMed Central

    Galperin, Michael Y.

    2011-01-01

    Summary Response regulators (RRs) within two-component signal transduction systems control a variety of cellular processes. Most RRs contain DNA-binding output domains and serve as transcriptional regulators. Other RR types contain RNA-binding, ligand-binding, protein-binding or transporter output domains and exert regulation at the transcriptional, post-transcriptional or post-translational levels. In a significant fraction of RRs, output domains are enzymes that themselves participate in signal transduction: methylesterases, adenylate or diguanylate cyclases, c-di-GMP-specific phosphodiesterases, histidine kinases, serine/threonine protein kinases and protein phosphatases. In addition, there remain output domains whose functions are still unknown. Patterns of the distribution of various RR families are generally conserved within key microbial lineages and can be used to trace adaptations of various species to their unique ecological niches. PMID:20226724

  9. Diversity of structure and function of response regulator output domains.

    PubMed

    Galperin, Michael Y

    2010-04-01

    Response regulators (RRs) within two-component signal transduction systems control a variety of cellular processes. Most RRs contain DNA-binding output domains and serve as transcriptional regulators. Other RR types contain RNA-binding, ligand-binding, protein-binding or transporter output domains and exert regulation at the transcriptional, post-transcriptional or post-translational levels. In a significant fraction of RRs, output domains are enzymes that themselves participate in signal transduction: methylesterases, adenylate or diguanylate cyclases, c-di-GMP-specific phosphodiesterases, histidine kinases, serine/threonine protein kinases and protein phosphatases. In addition, there remain output domains whose functions are still unknown. Patterns of the distribution of various RR families are generally conserved within key microbial lineages and can be used to trace adaptations of various species to their unique ecological niches.

  10. 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

  11. Functional diversification of hsp40: distinct j-protein functional requirements for two prions allow for chaperone-dependent prion selection.

    PubMed

    Harris, Julia M; Nguyen, Phil P; Patel, Milan J; Sporn, Zachary A; Hines, Justin K

    2014-07-01

    Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or 'strains'. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have

  12. 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

  13. Phylogeography of Y-Chromosome Haplogroup I Reveals Distinct Domains of Prehistoric Gene Flow in Europe

    PubMed Central

    Rootsi, Siiri; Magri, Chiara; Kivisild, Toomas; Benuzzi, Giorgia; Help, Hela; Bermisheva, Marina; Kutuev, Ildus; Barać, Lovorka; Peričić, Marijana; Balanovsky, Oleg; Pshenichnov, Andrey; Dion, Daniel; Grobei, Monica; Zhivotovsky, Lev A.; Battaglia, Vincenza; Achilli, Alessandro; Al-Zahery, Nadia; Parik, Jüri; King, Roy; Cinnioğlu, Cengiz; Khusnutdinova, Elsa; Rudan, Pavao; Balanovska, Elena; Scheffrahn, Wolfgang; Simonescu, Maya; Brehm, Antonio; Goncalves, Rita; Rosa, Alexandra; Moisan, Jean-Paul; Chaventre, Andre; Ferak, Vladimir; Füredi, Sandor; Oefner, Peter J.; Shen, Peidong; Beckman, Lars; Mikerezi, Ilia; Terzić, Rifet; Primorac, Dragan; Cambon-Thomsen, Anne; Krumina, Astrida; Torroni, Antonio; Underhill, Peter A.; Santachiara-Benerecetti, A. Silvana; Villems, Richard; Semino, Ornella

    2004-01-01

    To investigate which aspects of contemporary human Y-chromosome variation in Europe are characteristic of primary colonization, late-glacial expansions from refuge areas, Neolithic dispersals, or more recent events of gene flow, we have analyzed, in detail, haplogroup I (Hg I), the only major clade of the Y phylogeny that is widespread over Europe but virtually absent elsewhere. The analysis of 1,104 Hg I Y chromosomes, which were identified in the survey of 7,574 males from 60 population samples, revealed several subclades with distinct geographic distributions. Subclade I1a accounts for most of Hg I in Scandinavia, with a rapidly decreasing frequency toward both the East European Plain and the Atlantic fringe, but microsatellite diversity reveals that France could be the source region of the early spread of both I1a and the less common I1c. Also, I1b*, which extends from the eastern Adriatic to eastern Europe and declines noticeably toward the southern Balkans and abruptly toward the periphery of northern Italy, probably diffused after the Last Glacial Maximum from a homeland in eastern Europe or the Balkans. In contrast, I1b2 most likely arose in southern France/Iberia. Similarly to the other subclades, it underwent a postglacial expansion and marked the human colonization of Sardinia ∼9,000 years ago. PMID:15162323

  14. Phospholipid binding to the FAK catalytic domain impacts function

    PubMed Central

    Schaller, Michael D.

    2017-01-01

    Focal adhesion kinase is an essential nonreceptor tyrosine kinase that plays an important role in development, in homeostasis and in the progression of human disease. Multiple stimuli activate FAK, which requires a change in structure from an autoinhibited to activated conformation. In the autoinhibited conformation the FERM domain associates with the catalytic domain of FAK and PI(4,5)P2 binding to the FERM domain plays a role in the release of autoinhibition, activating the enzyme. An in silico model of FAK/PI(4,5)P2 interaction suggests that residues on the catalytic domain interact with PI(4,5)P2, in addition to the known FERM domain PI(4,5)P2 binding site. This study was undertaken to test the significance of this in silico observation. Mutations designed to disrupt the putative PI(4,5)P2 binding site were engineered into FAK. These mutants exhibited defects in phosphorylation and failed to completely rescue the phenotype associated with fak -/- phenotype fibroblasts demonstrating the importance of these residues in FAK function. The catalytic domain of FAK exhibited PI(4,5)P2 binding in vitro and binding activity was lost upon mutation of putative PI(4,5)P2 binding site basic residues. However, binding was not selective for PI(4,5)P2, and the catalytic domain bound to several phosphatidylinositol phosphorylation variants. The mutant exhibiting the most severe biological defect was defective for phosphatidylinositol phosphate binding, supporting the model that catalytic domain phospholipid binding is important for biochemical and biological function. PMID:28222177

  15. Distinct Functional Connectivities Predict Clinical Response with Emotion Regulation Therapy

    PubMed Central

    Fresco, David M.; Roy, Amy K.; Adelsberg, Samantha; Seeley, Saren; García-Lesy, Emmanuel; Liston, Conor; Mennin, Douglas S.

    2017-01-01

    Despite the success of available medical and psychosocial treatments, a sizable subgroup of individuals with commonly co-occurring disorders, generalized anxiety disorder (GAD) and major depressive disorder (MDD), fail to make sufficient treatment gains thereby prolonging their deficits in life functioning and satisfaction. Clinically, these patients often display temperamental features reflecting heightened sensitivity to underlying motivational systems related to threat/safety and reward/loss (e.g., somatic anxiety) as well as inordinate negative self-referential processing (e.g., worry, rumination). This profile may reflect disruption in two important neural networks associated with emotional/motivational salience (e.g., salience network) and self-referentiality (e.g., default network, DN). Emotion Regulation Therapy (ERT) was developed to target this hypothesized profile and its neurobehavioral markers. In the present study, 22 GAD patients (with and without MDD) completed resting state MRI scans before receiving 16 sessions of ERT. To test study these hypotheses, we examined the associations between baseline patterns of intrinsic functional connectivity (iFC) of the insula and of hubs within the DN (anterior and dorsal medial prefrontal cortex [MPFC] and posterior cingulate cortex [PCC]) and treatment-related changes in worry, somatic anxiety symptoms and decentering. Results suggest that greater treatment linked reductions in worry were associated with iFC clusters in both the insular and parietal cortices. Greater treatment linked gains in decentering, a metacognitive process that involves the capacity to observe items that arise in the mind with healthy psychological distance that is targeted by ERT, was associated with iFC clusters in the anterior and posterior DN. The current study adds to the growing body of research implicating disruptions in the default and salience networks as promising targets of treatment for GAD with and without co-occurring MDD

  16. Distinct Functional Connectivities Predict Clinical Response with Emotion Regulation Therapy.

    PubMed

    Fresco, David M; Roy, Amy K; Adelsberg, Samantha; Seeley, Saren; García-Lesy, Emmanuel; Liston, Conor; Mennin, Douglas S

    2017-01-01

    Despite the success of available medical and psychosocial treatments, a sizable subgroup of individuals with commonly co-occurring disorders, generalized anxiety disorder (GAD) and major depressive disorder (MDD), fail to make sufficient treatment gains thereby prolonging their deficits in life functioning and satisfaction. Clinically, these patients often display temperamental features reflecting heightened sensitivity to underlying motivational systems related to threat/safety and reward/loss (e.g., somatic anxiety) as well as inordinate negative self-referential processing (e.g., worry, rumination). This profile may reflect disruption in two important neural networks associated with emotional/motivational salience (e.g., salience network) and self-referentiality (e.g., default network, DN). Emotion Regulation Therapy (ERT) was developed to target this hypothesized profile and its neurobehavioral markers. In the present study, 22 GAD patients (with and without MDD) completed resting state MRI scans before receiving 16 sessions of ERT. To test study these hypotheses, we examined the associations between baseline patterns of intrinsic functional connectivity (iFC) of the insula and of hubs within the DN (anterior and dorsal medial prefrontal cortex [MPFC] and posterior cingulate cortex [PCC]) and treatment-related changes in worry, somatic anxiety symptoms and decentering. Results suggest that greater treatment linked reductions in worry were associated with iFC clusters in both the insular and parietal cortices. Greater treatment linked gains in decentering, a metacognitive process that involves the capacity to observe items that arise in the mind with healthy psychological distance that is targeted by ERT, was associated with iFC clusters in the anterior and posterior DN. The current study adds to the growing body of research implicating disruptions in the default and salience networks as promising targets of treatment for GAD with and without co-occurring MDD.

  17. Spatial Segregation of γ-Secretase and Substrates in Distinct Membrane Domains*

    PubMed Central

    Vetrivel, Kulandaivelu S.; Cheng, Haipeng; Kim, Seong-Hun; Chen, Ying; Barnes, Natalie Y.; Parent, Ange‘le T.; Sisodia, Sangram S.; Thinakaran, Gopal

    2005-01-01

    γ-Secretase facilitates the regulated intramembrane proteolysis of select type I membrane proteins that play diverse physiological roles in multiple cell types and tissue. In this study, we used biochemical approaches to examine the distribution of amyloid precursor protein (APP) and several additional γ-secretase substrates in membrane microdomains. We report that APP C-terminal fragments (CTFs) and γ-secretase reside in Lubrol WX detergent-insoluble membranes (DIM) of cultured cells and adult mouse brain. APP CTFs that accumulate in cells lacking γ-secretase activity preferentially associate with DIM. Cholesterol depletion and magnetic im-munoisolation studies indicate recruitment of APP CTFs into cholesterol- and sphingolipid-rich lipid rafts, and co-residence of APP CTFs, PS1, and syntaxin 6 in DIM patches derived from the trans-Golgi network. Photoaffinity cross-linking studies provided evidence for the preponderance of active γ-secretase in lipid rafts of cultured cells and adult brain. Remarkably, unlike the case of APP, CTFs derived from Notch1, Jagged2, deleted in colorectal cancer (DCC), and N-cadherin remain largely detergent-soluble, indicative of their spatial segregation in non-raft domains. In embryonic brain, the majority of PS1 and nicastrin is present in Lubrol WX-soluble membranes, wherein the CTFs derived from APP, Notch1, DCC, and N-cadherin also reside. We suggest that γ-secretase residence in non-raft membranes facilitates proteolysis of diverse substrates during embryonic development but that the translocation of γ-secretase to lipid rafts in adults ensures processing of certain substrates, including APP CTFs, while limiting processing of other potential substrates. PMID:15886206

  18. Distinct Neurobehavioural Effects of Cannabidiol in Transmembrane Domain Neuregulin 1 Mutant Mice

    PubMed Central

    Long, Leonora E.; Chesworth, Rose; Huang, Xu-Feng; Wong, Alexander; Spiro, Adena; McGregor, Iain S.; Arnold, Jonathon C.; Karl, Tim

    2012-01-01

    The cannabis constituent cannabidiol (CBD) possesses anxiolytic and antipsychotic properties. We have previously shown that transmembrane domain neuregulin 1 mutant (Nrg1 TM HET) mice display altered neurobehavioural responses to the main psychoactive constituent of cannabis, Δ9-tetrahydrocannabinol. Here we investigated whether Nrg1 TM HET mice respond differently to CBD and whether CBD reverses schizophrenia-related phenotypes expressed by these mice. Adult male Nrg1 TM HET and wild type-like littermates (WT) received vehicle or CBD (1, 50 or 100 mg/kg i.p.) for 21 days. During treatment and 48 h after withdrawal we measured behaviour, whole blood CBD concentrations and autoradiographic receptor binding. Nrg1 HET mice displayed locomotor hyperactivity, PPI deficits and reduced 5-HT2A receptor binding density in the substantia nigra, but these phenotypes were not reversed by CBD. However, long-term CBD (50 and 100 mg/kg) selectively enhanced social interaction in Nrg1 TM HET mice. Furthermore, acute CBD (100 mg/kg) selectively increased PPI in Nrg1 TM HET mice, although tolerance to this effect was manifest upon repeated CBD administration. Long-term CBD (50 mg/kg) also selectively increased GABAA receptor binding in the granular retrosplenial cortex in Nrg1 TM HET mice and reduced 5-HT2A binding in the substantia nigra in WT mice. Nrg1 appears necessary for CBD-induced anxiolysis since only WT mice developed decreased anxiety-related behaviour with repeated CBD treatment. Altered pharmacokinetics in mutant mice could not explain our findings since no genotype differences existed in CBD blood concentrations. Here we demonstrate that Nrg1 modulates acute and long-term neurobehavioural effects of CBD, which does not reverse the schizophrenia-relevant phenotypes. PMID:22509273

  19. Structural and Functional Relationships between the Lectin and Arm Domains of Calreticulin*

    PubMed Central

    Pocanschi, Cosmin L.; Kozlov, Guennadi; Brockmeier, Ulf; Brockmeier, Achim; Williams, David B.; Gehring, Kalle

    2011-01-01

    Calreticulin and calnexin are key components in maintaining the quality control of glycoprotein folding within the endoplasmic reticulum. Although their lectin function of binding monoglucosylated sugar moieties of glycoproteins is well documented, their chaperone activity in suppressing protein aggregation is less well understood. Here, we use a series of deletion mutants of calreticulin to demonstrate that its aggregation suppression function resides primarily within its lectin domain. Using hydrophobic peptides as substrate mimetics, we show that aggregation suppression is mediated through a single polypeptide binding site that exhibits a Kd for peptides of 0.5–1 μm. This site is distinct from the oligosaccharide binding site and differs from previously identified sites of binding to thrombospondin and GABARAP (4-aminobutyrate type A receptor-associated protein). Although the arm domain of calreticulin was incapable of suppressing aggregation or binding hydrophobic peptides on its own, it did contribute to aggregation suppression in the context of the whole molecule. The high resolution x-ray crystal structure of calreticulin with a partially truncated arm domain reveals a marked difference in the relative orientations of the arm and lectin domains when compared with calnexin. Furthermore, a hydrophobic patch was detected on the arm domain that mediates crystal packing and may contribute to calreticulin chaperone function. PMID:21652723

  20. Domain-specific functions of Stardust in Drosophila embryonic development

    PubMed Central

    Koch, Leonie; Feicht, Sabine; Sun, Rui; Sen, Arnab

    2016-01-01

    In Drosophila, the adaptor protein Stardust is essential for the stabilization of the polarity determinant Crumbs in various epithelial tissues, including the embryonic epidermis, the follicular epithelium and photoreceptor cells of the compound eye. In turn, Stardust recruits another adaptor protein, PATJ, to the subapical region to support adherens junction formation and morphogenetic events. Moreover, Stardust binds to Lin-7, which is dispensable in epithelial cells but functions in postsynaptic vesicle fusion. Finally, Stardust has been reported to bind directly to PAR-6, thereby linking the Crumbs–Stardust–PATJ complex to the PAR-6/aPKC complex. PAR-6 and aPKC are also capable of directly binding Bazooka (the Drosophila homologue of PAR-3) to form the PAR/aPKC complex, which is essential for apical–basal polarity and cell–cell contact formation in most epithelia. However, little is known about the physiological relevance of these interactions in the embryonic epidermis of Drosophila in vivo. Thus, we performed a structure–function analysis of the annotated domains with GFP-tagged Stardust and evaluated the localization and function of the mutant proteins in epithelial cells of the embryonic epidermis. The data presented here confirm a crucial role of the PDZ domain in binding Crumbs and recruiting the protein to the subapical region. However, the isolated PDZ domain is not capable of being recruited to the cortex, and the SH3 domain is essential to support the binding to Crumbs. Notably, the conserved N-terminal regions (ECR1 and ECR2) are not crucial for epithelial polarity. Finally, the GUK domain plays an important role for the protein's function, which is not directly linked to Crumbs stabilization, and the L27N domain is essential for epithelial polarization independently of recruiting PATJ. PMID:28018665

  1. Clarifying the Nature of the Distinctiveness by Domain Interaction in Conceptual Structure: Comment on Cree, McNorgan, and McRae (2006)

    ERIC Educational Resources Information Center

    Taylor, Kirsten I.; Salamoura, Angeliki; Randall, Billi; Moss, Helen; Tyler, Lorraine K.

    2008-01-01

    The conceptual structure account of semantic memory (CSA; L. K. Tyler & H. E. Moss, 2001) claims that feature correlation (the degree to which features co-occur) and feature distinctiveness (the number of concepts in which a feature occurs) interact with domains of knowledge (e.g., living vs. nonliving) such that the distinctive features of…

  2. Distinct transcriptional regulation and function of the human BACE2 and BACE1 genes.

    PubMed

    Sun, Xiulian; Wang, Yingcheng; Qing, Hong; Christensen, Michelle A; Liu, Yunqiang; Zhou, Weihui; Tong, Yigang; Xiao, Cuiying; Huang, Yi; Zhang, Sizhong; Liu, Xiehe; Song, Weihong

    2005-05-01

    Amyloid beta protein (Abeta) is the principal component of neuritic plaques in Alzheimer's disease (AD). Abeta is derived from beta amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-site APP cleaving enzyme 1 (BACE1) has been identified as the major beta-secretase. BACE2 is the homolog of BACE1. The BACE2 gene is on chromosome 21 and has been implicated in the pathogenesis of AD. However, the function of BACE2 in Abeta generation is controversial. Some studies have shown that BACE2 cleaved APP at the beta-site whereas other studies showed it cleaved around the alpha-secretase site. To elucidate the involvement of BACE2 in AD pathogenesis, we compared BACE2 and BACE1 gene regulation and their functions in Abeta generation. We cloned and functionally characterized the human BACE2 promoter. The BACE2 gene is controlled by a TATA-less promoter. Though Sp1 can regulate both BACE1 and BACE2 genes, comparative sequence analysis and transcription factor prediction showed little similarity between the two promoters. BACE1 increased APP cleavage at the beta-site and Abeta production whereas BACE2 did not. Overexpression of BACE2 significantly increased sAPP levels in conditioned media but markedly reduced Abeta production. Knockdown of BACE2 resulted in increased APP C83. Our data indicate that despite being homologous in amino acid sequence, BACE2 and BACE1 have distinct functions and transcriptional regulation. BACE2 is not a beta-secretase, but processes APP within the Abeta domain at a site downstream of the alpha-secretase cleavage site. Our data argue against BACE2 being involved in the formation of neuritic plaques in AD.

  3. Interdependence of the rad50 hook and globular domain functions.

    PubMed

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

    2015-02-05

    Rad50 contains a conserved Zn(2+) 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 Zn(2+)-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 domains, 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.

  4. 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.

  5. 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

  6. Functional properties of extracellular domains of transducer receptor gp130.

    PubMed

    Kostjukova, M N; Tupitsyn, N N

    2011-04-01

    Cytokine receptor molecules have been shown to have extracellular domains of complex structure and a multi-step activation system. Glycoprotein gp130 is a typical transducer of cytokine signal; it functions by forming multicomponent receptor complexes and transferring signals of tens of cytokines from the IL-6 family. Structural organization and basic functioning principles of gp130 are well known, as well as related signal pathways, which function during normal differentiation and are involved in pathogenesis of many tumors. The role of gp130 in IL-6-dependent tumors is best studied. In this review, based on extensive accumulated data, we examine the functional significance of certain parts of gp130 extracellular domains. Potentials of a recently developed method for estimation of receptor activation at the level of epitope structure are discussed.

  7. Distinct Prion Domain Sequences Ensure Efficient Amyloid Propagation by Promoting Chaperone Binding or Processing In Vivo

    PubMed Central

    Langlois, Christine R.; Serio, Tricia R.

    2016-01-01

    Prions are a group of proteins that can adopt a spectrum of metastable conformations in vivo. These alternative states change protein function and are self-replicating and transmissible, creating protein-based elements of inheritance and infectivity. Prion conformational flexibility is encoded in the amino acid composition and sequence of the protein, which dictate its ability not only to form an ordered aggregate known as amyloid but also to maintain and transmit this structure in vivo. But, while we can effectively predict amyloid propensity in vitro, the mechanism by which sequence elements promote prion propagation in vivo remains unclear. In yeast, propagation of the [PSI+] prion, the amyloid form of the Sup35 protein, has been linked to an oligopeptide repeat region of the protein. Here, we demonstrate that this region is composed of separable functional elements, the repeats themselves and a repeat proximal region, which are both required for efficient prion propagation. Changes in the numbers of these elements do not alter the physical properties of Sup35 amyloid, but their presence promotes amyloid fragmentation, and therefore maintenance, by molecular chaperones. Rather than acting redundantly, our observations suggest that these sequence elements make complementary contributions to prion propagation, with the repeat proximal region promoting chaperone binding to and the repeats promoting chaperone processing of Sup35 amyloid. PMID:27814358

  8. Concurrent Vaccination with two distinct vaccine platforms targeting the same antigen generates phenotypically and functionally distinct T-cell populations

    PubMed Central

    Boehm, Amanda L.; Higgins, Jack; Franzusoff, Alex; Schlom, Jeffrey; Hodge, James W.

    2009-01-01

    Purpose Studies comparing two or more vaccine platforms have historically evaluated each platform based on its ability to induce an immune response and may conclude that one vaccine is more efficacious than the other(s), leading to a recommendation for development of the more effective vaccine for clinical studies. Alternatively, these studies have documented the advantages of a diversified prime and boost regimen due to amplification of the antigen-specific T-cell population. We hypothesize here that two vaccine platforms targeting the same antigen might induce shared and distinct antigen-specific T-cell populations, and examined the possibility that two distinct vaccines could be used concomitantly. Experimental design Using recombinant poxvirus and yeast vaccines, we compared the T-cell populations induced by these two platforms in terms of serum cytokine response, T-cell gene expression, T-cell receptor phenotype, antigen-specific cytokine expression, T-cell avidity, and T-cell antigen-specific tumor cell lysis. Results These studies demonstrate for the first time that vaccination with a recombinant poxvirus platform (rV/F-CEA/TRICOM) or a heat-killed yeast vaccine platform (yeast-CEA) elicits T-cell populations with both shared and unique phenotypic and functional characteristics. Furthermore, both the antigen and the vector play a role in the induction of distinct T-cell populations. Conclusions In this study, we demonstrate that concurrent administration of two vaccines targeting the same antigen induces a more diverse T-cell population that leads to enhanced antitumor efficacy. These studies provide the rationale for future clinical studies investigating concurrent administration of vaccine platforms targeting a single antigen to enhance the antigen-specific immune response. PMID:19756595

  9. 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.

  10. From shared to distinct self-other representations in empathy: evidence from neurotypical function and socio-cognitive disorders.

    PubMed

    Lamm, C; Bukowski, H; Silani, G

    2016-01-19

    Neuroscientific research has identified two fundamental components of empathy: shared emotional representations between self and other, and self-other distinction. The concept of shared representations suggests that during empathy, we co-represent another person's affect by engaging brain and bodily functions underpinning the first-hand experience of the emotion we are empathizing with. This possible grounding of empathy in our own emotional experiences explains the necessity for self-other distinction, which is the capacity to correctly distinguish between our own affective representations and those related to the other. In spite of the importance of these two components in empathy, several aspects still remain controversial. This paper addresses some of them and focuses on (i) the distinction between shared activations versus representations, raising the question what shared representations entail in terms of the underlying neural mechanisms, (ii) the possible mechanisms behind self-other distinction in the cognitive and the affective domains, and whether they have distinct neural underpinnings and (iii) the consequences associated with a selective impairment of one of the two components, thereby addressing their importance in mental disorders such as autism spectrum disorders, psychopathy and alexithymia.

  11. From shared to distinct self–other representations in empathy: evidence from neurotypical function and socio-cognitive disorders

    PubMed Central

    2016-01-01

    Neuroscientific research has identified two fundamental components of empathy: shared emotional representations between self and other, and self–other distinction. The concept of shared representations suggests that during empathy, we co-represent another person's affect by engaging brain and bodily functions underpinning the first-hand experience of the emotion we are empathizing with. This possible grounding of empathy in our own emotional experiences explains the necessity for self–other distinction, which is the capacity to correctly distinguish between our own affective representations and those related to the other. In spite of the importance of these two components in empathy, several aspects still remain controversial. This paper addresses some of them and focuses on (i) the distinction between shared activations versus representations, raising the question what shared representations entail in terms of the underlying neural mechanisms, (ii) the possible mechanisms behind self–other distinction in the cognitive and the affective domains, and whether they have distinct neural underpinnings and (iii) the consequences associated with a selective impairment of one of the two components, thereby addressing their importance in mental disorders such as autism spectrum disorders, psychopathy and alexithymia. PMID:26644601

  12. Structure and function of regulator of G protein signaling homology domains.

    PubMed

    Tesmer, John J G

    2009-01-01

    All regulator of G protein signaling (RGS) proteins contain a conserved domain of approximately 130 amino acids that binds to activated heterotrimeric G protein α subunits (Gα) and accelerates their rate of GTP hydrolysis. Homologous domains are found in at least six other protein families, including a family of Rho guanine nucleotide exchange factors (RhoGEFs) and the G protein-coupled receptor kinases (GRKs). Although some of the RhoGEF and GRK RGS-like domains can also bind to activated Gα subunits, they do so in distinct ways and with much lower levels of GTPase activation. In other protein families, the domains have as of yet no obvious relationship to heterotrimeric G protein signaling. These RGS homology (RH) domains are now recognized as mediators of extraordinarily diverse protein-protein interactions. Through these interactions, they play roles that range from enzyme to molecular scaffold to signal transducing module. In this review, the atomic structures of RH domains from RGS proteins, Axins, RhoGEFs, and GRKs are compared in light of what is currently known about their functional roles.

  13. Identification of distinct biological functions for four 3′-5′ RNA polymerases

    PubMed Central

    Long, Yicheng; Abad, Maria G.; Olson, Erik D.; Carrillo, Elisabeth Y.; Jackman, Jane E.

    2016-01-01

    The superfamily of 3′-5′ polymerases synthesize RNA in the opposite direction to all other DNA/RNA polymerases, and its members include eukaryotic tRNAHis guanylyltransferase (Thg1), as well as Thg1-like proteins (TLPs) of unknown function that are broadly distributed, with family members in all three domains of life. Dictyostelium discoideum encodes one Thg1 and three TLPs (DdiTLP2, DdiTLP3 and DdiTLP4). Here, we demonstrate that depletion of each of the genes results in a significant growth defect, and that each protein catalyzes a unique biological reaction, taking advantage of specialized biochemical properties. DdiTLP2 catalyzes a mitochondria-specific tRNAHis maturation reaction, which is distinct from the tRNAHis maturation reaction typically catalyzed by Thg1 enzymes on cytosolic tRNA. DdiTLP3 catalyzes tRNA repair during mitochondrial tRNA 5′-editing in vivo and in vitro, establishing template-dependent 3′-5′ polymerase activity of TLPs as a bona fide biological activity for the first time since its unexpected discovery more than a decade ago. DdiTLP4 is cytosolic and, surprisingly, catalyzes robust 3′-5′ polymerase activity on non-tRNA substrates, strongly implying further roles for TLP 3′-5′ polymerases in eukaryotes. PMID:27484477

  14. Functional diversity of potassium channel voltage-sensing domains.

    PubMed

    Islas, León D

    2016-01-01

    Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology.

  15. Functional diversity of potassium channel voltage-sensing domains

    PubMed Central

    Islas, León D.

    2016-01-01

    Abstract Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology. PMID:26794852

  16. Diet-induced docosahexaenoic acid non-raft domains and lymphocyte function.

    PubMed

    Raza Shaikh, Saame

    2010-01-01

    Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that generally suppresses the function of T lymphocytes and antigen presenting cells (APCs). An emerging mechanism by which DHA modifies lymphocyte function is through changes in the organization of sphingolipid/cholesterol lipid raft membrane domains. Two contradictory models have been proposed to explain how DHA exerts its effects through changes in raft organization. The biophysical model, developed in model membranes, shows that DHA-containing phospholipids form unique non-raft membrane domains, that are organizationally distinct from lipid rafts, which serve to alter the conformation and/or lateral organization of lymphocyte proteins. In contrast, the cellular model on DHA and rafts shows that DHA suppresses lymphocyte function, in part, by directly incorporating into lipid rafts and altering protein activity. To reconcile opposing biophysical and cellular viewpoints, a major revision to existing models is presented herein. Based largely on quantitative microscopy data, it is proposed that DHA, consumed through the diet, modifies lymphocyte function, in part, through the formation of nanometer scale DHA-rich domains. These nano-scale domains disrupt the optimal raft-dependent clustering of proteins necessary for initial signaling. The data covered in this review highlights the importance of understanding how dietary n-3 PUFAs modify lymphocyte membranes, which is essential toward developing these fatty acids as therapeutic agents for treating inflammatory diseases.

  17. Measles virus recognizes its receptor, CD46, via two distinct binding domains within SCR1-2.

    PubMed

    Manchester, M; Gairin, J E; Patterson, J B; Alvarez, J; Liszewski, M K; Eto, D S; Atkinson, J P; Oldstone, M B

    1997-06-23

    Measles virus (MV) enters cells by attachment of the viral hemagglutinin to the major cell surface receptor CD46 (membrane cofactor protein). CD46 is a transmembrane glycoprotein whose ectodomain is largely composed of four conserved modules called short consensus repeats (SCRs). We have previously shown that MV interacts with SCR1 and SCR2 of CD46. (M. Manchester et al. (1995) Proc. Natl. Acad. Sci. USA 92, 2303-2307) Here we report mapping the MV interaction with SCR1 and SCR2 of CD46 using a combination of peptide inhibition and mutagenesis studies. By testing a series of overlapping peptides corresponding to the 126 amino acid SCR1-2 region for inhibition of MV infection, two domains were identified that interacted with MV. One domain was found within SCR1 (amino acids 37-56) and another within SCR2 (amino acids 85-104). These results were confirmed by constructing chimeras with complementary regions from structurally similar, but non-MV-binding, SCRs of decay accelerating factor (DAF; CD55). These results indicate that MV contacts at least two distinct sites within SCR1-2.

  18. Wind-instrument reflection function measurements in the time domain.

    PubMed

    Keefe, D H

    1996-04-01

    Theoretical and computational analyses of wind-instrument sound production in the time domain have emerged as useful tools for understanding musical instrument acoustics, yet there exist few experimental measurements of the air-column response directly in the time domain. A new experimental, time-domain technique is proposed to measure the reflection function response of woodwind and brass-instrument air columns. This response is defined at the location of sound regeneration in the mouthpiece or double reed. A probe assembly comprised of an acoustic source and microphone is inserted directly into the air column entryway using a foam plug to ensure a leak-free fit. An initial calibration phase involves measurements on a single cylindrical tube of known dimensions. Measurements are presented on an alto saxophone and euphonium. The technique has promise for testing any musical instrument air columns using a single probe assembly and foam plugs over a range of diameters typical of air-column entryways.

  19. 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.

  20. Functional analysis of the nuclear LIM domain interactor NLI.

    PubMed Central

    Jurata, L W; Gill, G N

    1997-01-01

    LIM homeodomain and LIM-only (LMO) transcription factors contain two tandemly arranged Zn2+-binding LIM domains capable of mediating protein-protein interactions. These factors have restricted patterns of expression, are found in invertebrates as well as vertebrates, and are required for cell type specification in a variety of developing tissues. A recently identified, widely expressed protein, NLI, binds with high affinity to the LIM domains of LIM homeodomain and LMO proteins in vitro and in vivo. In this study, a 38-amino-acid fragment of NLI was found to be sufficient for the association of NLI with nuclear LIM domains. In addition, NLI was shown to form high affinity homodimers through the amino-terminal 200 amino acids, but dimerization of NLI was not required for association with the LIM homeodomain protein Lmxl. Chemical cross-linking analysis revealed higher-order complexes containing multiple NLI molecules bound to Lmx1, indicating that dimerization of NLI does not interfere with LIM domain interactions. Additionally, NLI formed complexes with Lmx1 on the rat insulin I promoter and inhibited the LIM domain-dependent synergistic transcriptional activation by Lmx1 and the basic helix-loop-helix protein E47 from the rat insulin I minienhancer. These studies indicate that NLI contains at least two functionally independent domains and may serve as a negative regulator of synergistic transcriptional responses which require direct interaction via LIM domains. Thus, NLI may regulate the transcriptional activity of LIM homeodomain proteins by determining specific partner interactions. PMID:9315627

  1. 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

  2. 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.

  3. Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors.

    PubMed

    Hopf, Thomas A; Morinaga, Satoshi; Ihara, Sayoko; Touhara, Kazushige; Marks, Debora S; Benton, Richard

    2015-01-13

    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.

  4. Fcp1 directly recognizes the C-terminal domain (CTD) and interacts with a site on RNA polymerase II distinct from the CTD

    PubMed Central

    Suh, Man-Hee; Ye, Ping; Zhang, Mincheng; Hausmann, Stéphane; Shuman, Stewart; Gnatt, Averell L.; Fu, Jianhua

    2005-01-01

    Fcp1 is an essential protein phosphatase that hydrolyzes phosphoserines within the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). Fcp1 plays a major role in the regulation of CTD phosphorylation and, hence, critically influences the function of Pol II throughout the transcription cycle. The basic understanding of Fcp1–CTD interaction has remained ambiguous because two different modes have been proposed: the “dockingsite” model versus the “distributive” mechanism. Here we demonstrate biochemically that Fcp1 recognizes and dephosphorylates the CTD directly, independent of the globular non-CTD part of the Pol II structure. We point out that the recognition of CTD by the phosphatase is based on random access and is not driven by Pol II conformation. Results from three different types of experiments reveal that the overall interaction between Fcp1 and Pol II is not stable but dynamic. In addition, we show that Fcp1 also interacts with a region on the polymerase distinct from the CTD. We emphasize that this non-CTD site is functionally distinct from the docking site invoked previously as essential for the CTD phosphatase activity of Fcp1. We speculate that Fcp1 interaction with the non-CTD site may mediate its stimulatory effect on transcription elongation reported previously. PMID:16301539

  5. Independent transport and sorting of functionally distinct protein families in Tetrahymena thermophila dense core secretory granules.

    PubMed

    Rahaman, Abdur; Miao, Wei; Turkewitz, Aaron P

    2009-10-01

    Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal beta/gamma-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, DeltaGRT1 DeltaGRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in DeltaGRT1 DeltaGRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from DeltaGRT1 DeltaGRT2 cells appear less adhesive than those from the wild type.

  6. Engagement of two distinct binding domains on CCL17 is required for signaling through CCR4 and establishment of localized inflammatory conditions in the lung.

    PubMed

    Santulli-Marotto, Sandra; Boakye, Ken; Lacy, Eilyn; Wu, Sheng-Jiun; Luongo, Jennifer; Kavalkovich, Karl; Coelho, Ana; Hogaboam, Cory M; Ryan, Mary

    2013-01-01

    CCL17 (TARC) function can be completely abolished by mAbs that block either one of two distinct sites required for CCR4 signaling. This chemokine is elevated in sera of asthma patients and is responsible for establishing inflammatory sites through CCR4-mediated recruitment of immune cells. CCL17 shares the GPCR CCR4, with CCL22 (MDC) but these two chemokines differentially affect the immune response. To better understand chemokine mediated effects through CCR4, we have generated chimeric anti-mouse CCL17 surrogate antibodies that inhibit function of this ligand in vitro and in vivo. The affinities of the surrogate antibodies for CCL17 range from 685 pM for B225 to 4.9 nM for B202. One antibody, B202, also exhibits weak binding to CCL22 (KD∼2 µM) and no binding to CCL22 is detectable with the second antibody, B225. In vitro, both antibodies inhibit CCL17-mediated calcium mobilization, β-arrestin recruitment and chemotaxis; B202 can also partially inhibit CCL22-mediated β-arrestin recruitment. Both B202 and B225 antibodies neutralize CCL17 in vivo as demonstrated by reduction of methacholine-induced airway hyperreactivity in the A. fumigatus model of asthma. That both antibodies block CCL17 function but only B202 shows any inhibition of CCL22 function suggests that they bind CCL17 at different sites. Competition binding studies confirm that these two antibodies recognize unique epitopes that are non-overlapping despite the small size of CCL17. Taking into consideration the data from both the functional and binding studies, we propose that effective engagement of CCR4 by CCL17 involves two distinct binding domains and interaction with both is required for signaling.

  7. Evolution of function in the "two dinucleotide binding domains" flavoproteins.

    PubMed

    Ojha, Sunil; Meng, Elaine C; Babbitt, Patricia C

    2007-07-01

    Structural and biochemical constraints force some segments of proteins to evolve more slowly than others, often allowing identification of conserved structural or sequence motifs that can be associated with substrate binding properties, chemical mechanisms, and molecular functions. We have assessed the functional and structural constraints imposed by cofactors on the evolution of new functions in a superfamily of flavoproteins characterized by two-dinucleotide binding domains, the "two dinucleotide binding domains" flavoproteins (tDBDF) superfamily. Although these enzymes catalyze many different types of oxidation/reduction reactions, each is initiated by a stereospecific hydride transfer reaction between two cofactors, a pyridine nucleotide and flavin adenine dinucleotide (FAD). Sequence and structural analysis of more than 1,600 members of the superfamily reveals new members and identifies details of the evolutionary connections among them. Our analysis shows that in all of the highly divergent families within the superfamily, these cofactors adopt a conserved configuration optimal for stereospecific hydride transfer that is stabilized by specific interactions with amino acids from several motifs distributed among both dinucleotide binding domains. The conservation of cofactor configuration in the active site restricts the pyridine nucleotide to interact with FAD from the re-side, limiting the flow of electrons from the re-side to the si-side. This directionality of electron flow constrains interactions with the different partner proteins of different families to occur on the same face of the cofactor binding domains. As a result, superimposing the structures of tDBDFs aligns not only these interacting proteins, but also their constituent electron acceptors, including heme and iron-sulfur clusters. Thus, not only are specific aspects of the cofactor-directed chemical mechanism conserved across the superfamily, the constraints they impose are manifested in the

  8. 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

  9. Genomic microarray analysis reveals distinct locations for the CENP-A binding domains in three human chromosome 13q32 neocentromeres.

    PubMed

    Alonso, Alicia; Mahmood, Radma; Li, Shulan; Cheung, Fanny; Yoda, Kinya; Warburton, Peter E

    2003-10-15

    Human neocentromeres are fully functional centromeres that provide mitotic stability to rearranged chromosomes that have separated from endogenous centromeres. A disproportionate number of neocentromeres has been observed in certain regions such as chromosome 3q (n=6), 15q (n=9) and 13q32 (n=7), suggesting that these regions contain DNA sequences with a high propensity for neocentromere formation. Therefore, we have addressed the role of primary DNA sequence in neocentromere formation by asking whether multiple independent neocentromeres that were cytologically localized to chromosome 13q32 are in fact localized to the same underlying genomic DNA. Analysis of four independent 13q32 neocentromeres using simultaneous FISH with ordered YAC probes and immunofluorescence with antibodies to CENP-C have localized three neocentromeres to a distal approximately 7 Mb domain in chromosome 13q32, and one to an overlapping proximal domain of approximately 7 Mb. DNA was obtained from three of these neocentromeres by CENP-A chromatin immunoprecipitation (ChIP) and used to screen ordered BACs using both a slot-blotted BAC pool approach and a genomic microarray that contiguously spans 13q31.3-13q33.1. The CENP-A binding domains from each of these neocentromeres was identified to distinct genomic locations of approximately 130, 215 and 275 kb within an approximately 6.5 Mb region. Thus, the lack of coincidence of these neocentromeres to the same underlying DNA sequence refutes the idea of a DNA sequence based neocentromere 'hotspot' in 13q32 and further supports the sequence-independent epigenetic formation of human neocentromeres. The screening of genomic microarrays with ChIP DNA provides a powerful method to identify mammalian DNA sequences associated with particular functional chromatin states.

  10. Functional Analysis of GLRX5 Mutants Reveals Distinct Functionalities of GLRX5 Protein.

    PubMed

    Liu, Gang; Wang, Yongwei; Anderson, Gregory J; Camaschella, Clara; Chang, Yanzhong; Nie, Guangjun

    2016-01-01

    Glutaredoxin 5 (GLRX5) is a 156 amino acid mitochondrial protein that plays an essential role in mitochondrial iron-sulfur cluster transfer. Mutations in this protein were reported to result in sideroblastic anemia and variant nonketotic hyperglycinemia in human. Recently, we have characterized a Chinese congenital sideroblastic anemia patient who has two compound heterozygous missense mutations (c. 301 A>C and c. 443 T>C) in his GLRX5 gene. Herein, we developed a GLRX5 knockout K562 cell line and studied the biochemical functions of the identified pathogenic mutations and other conserved amino acids with predicted essential functions. We observed that the K101Q mutation (due to c. 301 A>C mutation) may prevent the binding of [Fe-S] to GLRX5 protein, while L148S (due to c. 443 T>C mutation) may interfere with [Fe-S] transfer from GLRX5 to iron regulatory protein 1 (IRP1), mitochondrial aconitase (m-aconitase) and ferrochelatase. We also demonstrated that L148S is functionally complementary to the K51del mutant with respect to Fe/S-ferrochelatase, Fe/S-IRP1, Fe/S-succinate dehydrogenase, and Fe/S-m-aconitase biosynthesis and lipoylation of pyruvate dehydrogenase complex and α-ketoglutarate dehydrogenase complex. Furthermore, we demonstrated that the mutations of highly conserved amino acid residues in GLRX5 protein can have different effects on downstream Fe/S proteins. Collectively, our current work demonstrates that GLRX5 protein is multifunctional in [Fe-S] protein synthesis and maturation and defects of the different amino acids of the protein will lead to distinct effects on downstream Fe/S biosynthesis.

  11. Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase.

    PubMed

    Clark, Daniel N; Flanagan, John M; Hu, Jianming

    2017-02-01

    Hepatitis B virus (HBV) encodes a multifunction reverse transcriptase or polymerase (P), which is composed of several domains. The terminal protein (TP) domain is unique to HBV and related hepadnaviruses and is required for specifically binding to the viral pregenomic RNA (pgRNA). Subsequently, the TP domain is necessary for pgRNA packaging into viral nucleocapsids and the initiation of viral reverse transcription for conversion of the pgRNA to viral DNA. Uniquely, the HBV P protein initiates reverse transcription via a protein priming mechanism using the TP domain as a primer. No structural homologs or high-resolution structure exists for the TP domain. Secondary structure prediction identified three disordered loops in TP with highly conserved sequences. A meta-analysis of mutagenesis studies indicated these predicted loops are almost exclusively where functionally important residues are located. Newly constructed TP mutations revealed a priming loop in TP which plays a specific role in protein-primed DNA synthesis beyond simply harboring the site of priming. Substitutions of potential sites of phosphorylation surrounding the priming site demonstrated that these residues are involved in interactions critical for priming but are unlikely to be phosphorylated during viral replication. Furthermore, the first 13 and 66 TP residues were shown to be dispensable for protein priming and pgRNA binding, respectively. Combining current and previous mutagenesis work with sequence analysis has increased our understanding of TP structure and functions by mapping specific functions to distinct predicted secondary structures and will facilitate antiviral targeting of this unique domain.

  12. Crystal structure of a functional dimer of the PhoQ sensor domain.

    PubMed

    Cheung, Jonah; Bingman, Craig A; Reyngold, Marsha; Hendrickson, Wayne A; Waldburger, Carey D

    2008-05-16

    The PhoP-PhoQ two-component system is a well studied bacterial signaling system that regulates virulence and stress response. Catalytic activity of the histidine kinase sensor protein PhoQ is activated by low extracellular concentrations of divalent cations such as Mg2+, and subsequently the response regulator PhoP is activated in turn through a classic phosphotransfer pathway that is typical in such systems. The PhoQ sensor domains of enteric bacteria contain an acidic cluster of residues (EDDDDAE) that has been implicated in direct binding to divalent cations. We have determined crystal structures of the wild-type Escherichia coli PhoQ periplasmic sensor domain and of a mutant variant in which the acidic cluster was neutralized to conservative uncharged residues (QNNNNAQ). The PhoQ domain structure is similar to that of DcuS and CitA sensor domains, and this PhoQ-DcuS-CitA (PDC) sensor fold is seen to be distinct from the superficially similar PAS domain fold. Analysis of the wild-type structure reveals a dimer that allows for the formation of a salt bridge across the dimer interface between Arg-50' and Asp-179 and with nickel ions bound to aspartate residues in the acidic cluster. The physiological importance of the salt bridge to in vivo PhoQ function has been confirmed by mutagenesis. The mutant structure has an alternative, non-physiological dimeric association.

  13. Structural and functional analysis of domains of the progesterone receptor.

    PubMed

    Hill, Krista K; Roemer, Sarah C; Churchill, Mair E A; Edwards, Dean P

    2012-01-30

    Steroid hormone receptors are multi-domain proteins composed of conserved well-structured regions, such as ligand (LBD) and DNA binding domains (DBD), plus other naturally unstructured regions including the amino-terminal domain (NTD) and the hinge region between the LBD and DBD. The hinge is more than just a flexible region between the DBD and LBD and is capable of binding co-regulatory proteins and the minor groove of DNA flanking hormone response elements. Because the hinge can directly participate in DNA binding it has also been termed the carboxyl terminal extension (CTE) of the DNA binding domain. The CTE and NTD are dynamic regions of the receptor that can adopt multiple conformations depending on the environment of interacting proteins and DNA. Both regions have important regulatory roles for multiple receptor functions that are related to the ability of the CTE and NTD to form multiple active conformations. This review focuses on studies of the CTE and NTD of progesterone receptor (PR), as well as related work with other steroid/nuclear receptors.

  14. [Respiratory domain of revised amyotrophic lateral sclerosis. Functional Rating Scale].

    PubMed

    Lima, Sandra E; Pessolano, Fernando A; Monteiro, Sergio G; De Vito, Eduardo L

    2009-01-01

    Virtually all patients with amyotrophic lateral sclerosis will complain of dyspnea, which is perhaps the most distressing symptom of this devastating disease. The objective was to correlate respiratory domain of ALSFRS-R with forced vital capacity and maximal static pressures in the mouth. We designed a prospective study in 20 consecutive patients without dyspnea during 24 months. The global decline of ALSFRS-R was from 34.3 +/- 10.3 to 22.1 +/- 8.0 (p = 0.0325), the contribution of respiratory domain was irrelevant. Those who referred dyspnea (n: 12), forced vital capacity fell 41 +/- 21% of the initial value but with similar value of fall (46 +/- 23%) 8 patients did not referred dyspnea. Total score of ALSFRS-R correlated with forced vital capacity (litres), r: 0.73, p = 0.0016 and maximal inspiratory pressure (cm H2O), r: 0.84, p = 0.0038, but the fall of the forced vital capacity (%) did not correlate with dyspnea (r(s): 0.23, p = 0.1400). There was a moderate correlation between dyspnea and maximal inspiratory pressure (%), r(s): 0.58, p = 0.0300 and between dyspnea and maximal expiratory pressure (%), r(s): 0.49, p = 0.0400. We concluded that the respiratory functional deterioration could not be predicted using respiratory domain of ALSFRS-R. This suggests that respiratory domain of this scale does not replace to respiratory function testing measurements and, due to the respiratory insufficiency could not be clinically evident; performing pulmonary function tests provides an objective view and permit to make anticipatory actions.

  15. Yeast DNA ligase IV mutations reveal a nonhomologous end joining function of BRCT1 distinct from XRCC4/Lif1 binding.

    PubMed

    Chiruvella, Kishore K; Renard, Brian M; Birkeland, Shanda R; Sunder, Sham; Liang, Zhuobin; Wilson, Thomas E

    2014-12-01

    LIG4/Dnl4 is the DNA ligase that (re)joins DNA double-strand breaks (DSBs) via nonhomologous end joining (NHEJ), an activity supported by binding of its tandem BRCT domains to the ligase accessory protein XRCC4/Lif1. We screened a panel of 88 distinct ligase mutants to explore the structure–function relationships of the yeast Dnl4 BRCT domains and inter-BRCT linker in NHEJ. Screen results suggested two distinct classes of BRCT mutations with differential effects on Lif1 interaction as compared to NHEJ completion. Validated constructs confirmed that D800K and GG(868:869)AA mutations, which target the Lif1 binding interface, showed a severely defective Dnl4–Lif1 interaction but a less consistent and often small decrease in NHEJ activity in some assays, as well as nearly normal levels of Dnl4 accumulation at DSBs. In contrast, mutants K742A and KTT(742:744)ATA, which target the β3-α2 region of the first BRCT domain, substantially decreased NHEJ function commensurate with a large defect in Dnl4 recruitment to DSBs, despite a comparatively greater preservation of the Lif1 interaction. Together, these separation-of-function mutants indicate that Dnl4 BRCT1 supports DSB recruitment and NHEJ in a manner distinct from Lif1 binding and reveal a complexity of Dnl4 BRCT domain functions in support of stable DSB association.

  16. Human DNA-Damage-Inducible 2 Protein Is Structurally and Functionally Distinct from Its Yeast Ortholog

    PubMed Central

    Sivá, Monika; Svoboda, Michal; Veverka, Václav; Trempe, Jean-François; Hofmann, Kay; Kožíšek, Milan; Hexnerová, Rozálie; Sedlák, František; Belza, Jan; Brynda, Jiří; Šácha, Pavel; Hubálek, Martin; Starková, Jana; Flaisigová, Iva; Konvalinka, Jan; Šašková, Klára Grantz

    2016-01-01

    Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor. All Ddi1-like family members also contain a highly conserved retroviral protease-like (RVP) domain with unknown substrate specificity. While the structure and biological function of yeast Ddi1 have been investigated, no such analysis is available for the human homologs. To address this, we solved the 3D structures of the human Ddi2 UBL and RVP domains and identified a new helical domain that extends on either side of the RVP dimer. While Ddi1-like proteins from all vertebrates lack a UBA domain, we identify a novel ubiquitin-interacting motif (UIM) located at the C-terminus of the protein. The UIM showed a weak yet specific affinity towards ubiquitin, as did the Ddi2 UBL domain. However, the full-length Ddi2 protein is unable to bind to di-ubiquitin chains. While proteomic analysis revealed no activity, implying that the protease requires other factors for activation, our structural characterization of all domains of human Ddi2 sets the stage for further characterization. PMID:27461074

  17. Crystal structure of the human, FIC-domain containing protein HYPE and implications for its functions.

    PubMed

    Bunney, Tom D; Cole, Ambrose R; Broncel, Malgorzata; Esposito, Diego; Tate, Edward W; Katan, Matilda

    2014-12-02

    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 auto AMPylation 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.

  18. Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

    SciTech Connect

    Dahms, Sven O.; Mayer, Magnus C.; Roeser, Dirk; Multhaup, Gerd; Than, Manuel E.

    2015-03-01

    Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects various (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.

  19. Phenotypic lentivirus screens to identify functional single domain antibodies

    PubMed Central

    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. While 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 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

  20. Human GATA-3 trans-activation, DNA-binding, and nuclear localization activities are organized into distinct structural domains.

    PubMed Central

    Yang, Z; Gu, L; Romeo, P H; Bories, D; Motohashi, H; Yamamoto, M; Engel, J D

    1994-01-01

    GATA-3 is a zinc finger transcription factor which is expressed in a highly restricted and strongly conserved tissue distribution pattern in vertebrate organisms, specifically, in a subset of hematopoietic cells, in cells within the central and peripheral nervous systems, in the kidney, and in placental trophoblasts. Tissue-specific cellular genes regulated by GATA-3 have been identified in T lymphocytes and the placenta, while GATA-3-regulated genes in the nervous system and kidney have not yet been defined. We prepared monoclonal antibodies with which we could dissect the biochemical and functional properties of human GATA-3. The results of these experiments show some anticipated phenotypes, for example, the definition of discrete domains required for specific DNA-binding site recognition (amino acids 303 to 348) and trans activation (amino acids 30 to 74). The signaling sequence for nuclear localization of human GATA-3 is a property conferred by sequences within and surrounding the amino finger (amino acids 249 to 311) of the protein, thereby assigning a function to this domain and thus explaining the curious observation that this zinc finger is dispensable for DNA binding by the GATA family of transcription factors. Images PMID:8114750

  1. Distinct Quantitative Computed Tomography Emphysema Patterns Are Associated with Physiology and Function in Smokers

    PubMed Central

    San José Estépar, Raúl; Mendoza, Carlos S.; Hersh, Craig P.; Laird, Nan; Crapo, James D.; Lynch, David A.; Silverman, Edwin K.; Washko, George R.

    2013-01-01

    Rationale: Emphysema occurs in distinct pathologic patterns, but little is known about the epidemiologic associations of these patterns. Standard quantitative measures of emphysema from computed tomography (CT) do not distinguish between distinct patterns of parenchymal destruction. Objectives: To study the epidemiologic associations of distinct emphysema patterns with measures of lung-related physiology, function, and health care use in smokers. Methods: Using a local histogram-based assessment of lung density, we quantified distinct patterns of low attenuation in 9,313 smokers in the COPDGene Study. To determine if such patterns provide novel insights into chronic obstructive pulmonary disease epidemiology, we tested for their association with measures of physiology, function, and health care use. Measurements and Main Results: Compared with percentage of low-attenuation area less than −950 Hounsfield units (%LAA-950), local histogram-based measures of distinct CT low-attenuation patterns are more predictive of measures of lung function, dyspnea, quality of life, and health care use. These patterns are strongly associated with a wide array of measures of respiratory physiology and function, and most of these associations remain highly significant (P < 0.005) after adjusting for %LAA-950. In smokers without evidence of chronic obstructive pulmonary disease, the mild centrilobular disease pattern is associated with lower FEV1 and worse functional status (P < 0.005). Conclusions: Measures of distinct CT emphysema patterns provide novel information about the relationship between emphysema and key measures of physiology, physical function, and health care use. Measures of mild emphysema in smokers with preserved lung function can be extracted from CT scans and are significantly associated with functional measures. PMID:23980521

  2. Defining the boundaries: structure and function of LOB domain proteins.

    PubMed

    Majer, Christine; Hochholdinger, Frank

    2011-01-01

    The plant-specific LBD (Lateral Organ Boundaries Domain) gene family is essential in the regulation of plant lateral organ development and is involved in the regulation of anthocyanin and nitrogen metabolism. LBD proteins contain a characteristic LOB domain composed of a C-motif required for DNA-binding, a conserved glycine residue, and a leucine-zipper-like sequence required for protein-protein interactions. Recently, several LBD genes associated with mutant phenotypes related to almost all aspects of plant development, including embryo, root, leaf, and inflorescence development have been functionally characterized. These novel insights contribute to a better understanding of the molecular definition of boundaries between organs or boundaries between organs and meristems and the regulation of these processes by environmental cues and phytohormones.

  3. 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

  4. Structure of N-Terminal Domain of NPC1 Reveals Distinct Subdomains for Binding and Transfer of Cholesterol

    SciTech Connect

    Kwon, Hyock Joo; Abi-Mosleh, Lina; Wang, Michael L.; Deisenhofer, Johann; Goldstein, Joseph L.; Brown, Michael S.; Infante, Rodney E.

    2010-09-21

    LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3{beta}-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3{beta}-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from the binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model wherein after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.

  5. Natural-like function in artificial WW domains.

    PubMed

    Russ, William P; Lowery, Drew M; Mishra, Prashant; Yaffe, Michael B; Ranganathan, Rama

    2005-09-22

    Protein sequences evolve through random mutagenesis with selection for optimal fitness. Cooperative folding into a stable tertiary structure is one aspect of fitness, but evolutionary selection ultimately operates on function, not on structure. In the accompanying paper, we proposed a model for the evolutionary constraint on a small protein interaction module (the WW domain) through application of the SCA, a statistical analysis of multiple sequence alignments. Construction of artificial protein sequences directed only by the SCA showed that the information extracted by this analysis is sufficient to engineer the WW fold at atomic resolution. Here, we demonstrate that these artificial WW sequences function like their natural counterparts, showing class-specific recognition of proline-containing target peptides. Consistent with SCA predictions, a distributed network of residues mediates functional specificity in WW domains. The ability to recapitulate natural-like function in designed sequences shows that a relatively small quantity of sequence information is sufficient to specify the global energetics of amino acid interactions.

  6. 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.

  7. The Dof domain, a zinc finger DNA-binding domain conserved only in higher plants, truly functions as a Cys2/Cys2 Zn finger domain.

    PubMed

    Umemura, Yoshimi; Ishiduka, Tomoko; Yamamoto, Rie; Esaka, Muneharu

    2004-03-01

    The Dof (DNA-binding with one finger) proteins are plant transcription factors that have a highly conserved DNA-binding domain, called the Dof domain. The Dof domain, which is composed of 52 amino acid residues, is similar to the Cys2/Cys2 zinc finger DNA-binding domain of GATA1 and steroid hormone receptors, but has a longer putative loop than that in the case of these zinc finger domains. The DNA-binding function of ascorbate oxidase gene binding protein (AOBP), a Dof protein, was investigated by gel retardation analysis. When Cys was replaced by His, the Dof domain could not function as a Cys3/His- or a Cys2/His2-type zinc finger. The characteristic longer loop was essential for DNA-binding activity. Furthermore, heavy metals such as Co(II), Ni(II), Cd(II), Cu(II), Hg(II), Fe(II), and Fe(III) inhibited the DNA-binding activity of the Dof domain. Manganese ion as well as zinc ion was coordinated by the Dof domain in vitro. On the other hand, the analysis using inductively coupled argon plasma mass spectrometry (ICP-MS) showed that the Dof domain contained zinc ion but not manganese ion. Thus, the Dof domain was proved to function as a Cys2/Cys2 zinc finger domain.

  8. What is your patient’s cognitive profile? Three distinct subgroups of cognitive function in persons with heart failure

    PubMed Central

    Hawkins, Misty A.W.; Schaefer, Julie T.; Gunstad, John; Dolansky, Mary A.; Redle, Joseph D.; Josephson, Richard; Moore, Shirley M.; Hughes, Joel W.

    2014-01-01

    Purpose To determine whether patients with heart failure (HF) have distinct profiles of cognitive impairment. Background Cognitive impairment is common in HF. Recent work found three cognitive profiles in HF patients— (1) intact, (2) impaired, and (3) memory-impaired. We examined the reproducibility of these profiles and clarified mechanisms. Methods HF patients (68.6±9.7years; N=329) completed neuropsychological testing. Composite scores were created for cognitive domains and used to identify clusters via agglomerative-hierarchical cluster analysis. Results A 3-cluster solution emerged. Cluster 1 (n=109) had intact cognition. Cluster 2 (n=123) was impaired across all domains. Cluster 3 (n=97) had impaired memory only. Clusters differed in age, race, education, SES, IQ, BMI, and diabetes (ps ≤.026) but not in mood, anxiety, cardiovascular, or pulmonary disease (ps≥.118). Conclusions We replicated three distinct patterns of cognitive function in persons with HF. These profiles may help providers offer tailored care to patients with different cognitive and clinical needs. PMID:25510559

  9. Structure-function analysis of the NB-ARC domain of plant disease resistance proteins.

    PubMed

    van Ooijen, Gerben; Mayr, Gabriele; Kasiem, Mobien M A; Albrecht, Mario; Cornelissen, Ben J C; Takken, Frank L W

    2008-01-01

    Resistance (R) proteins in plants are involved in pathogen recognition and subsequent activation of innate immune responses. Most resistance proteins contain a central nucleotide-binding domain. This so-called NB-ARC domain consists of three subdomains: NB, ARC1, and ARC2. The NB-ARC domain is a functional ATPase domain, and its nucleotide-binding state is proposed to regulate activity of the R protein. A highly conserved methionine-histidine-aspartate (MHD) motif is present at the carboxy-terminus of ARC2. An extensive mutational analysis of the MHD motif in the R proteins I-2 and Mi-1 is reported. Several novel autoactivating mutations of the MHD invariant histidine and conserved aspartate were identified. The combination of MHD mutants with autoactivating hydrolysis mutants in the NB subdomain showed that the autoactivation phenotypes are not additive. This finding indicates an important regulatory role for the MHD motif in the control of R protein activity. To explain these observations, a three-dimensional model of the NB-ARC domain of I-2 was built, based on the APAF-1 template structure. The model was used to identify residues important for I-2 function. Substitution of the selected residues resulted in the expected distinct phenotypes. Based on the model, it is proposed that the MHD motif fulfils the same function as the sensor II motif found in AAA+ proteins (ATPases associated with diverse cellular activities)-co-ordination of the nucleotide and control of subdomain interactions. The presented 3D model provides a framework for the formulation of hypotheses on how mutations in the NB-ARC exert their effects.

  10. Functional domains within the human immunodeficiency virus type 2 envelope protein required to enhance virus production.

    PubMed

    Abada, Paolo; Noble, Beth; Cannon, Paula M

    2005-03-01

    Primate lentiviruses code for a protein that stimulates virus production. In human immunodeficiency virus type 1 (HIV-1), the activity is provided by the accessory protein, Vpu, while in HIV-2 and simian immunodeficiency virus it is a property of the envelope (Env) glycoprotein. Using a group of diverse retroviruses and cell types, we have confirmed the functional equivalence of the two proteins. However, despite these similarities, the two proteins have markedly different functional domains. While the Vpu activity is associated primarily with its membrane-spanning region, we have determined that the HIV-2 Env activity requires both the cytoplasmic tail and ectodomain of the protein, with the membrane-spanning domain being less important. Within the Env cytoplasmic tail, we further defined the necessary sequence as a membrane-proximal tyrosine-based motif. Providing the two Env regions separately as distinct CD8 chimeric proteins did not increase virus release. This suggests that the two domains must be either contained within a single protein or closely associated within a multiprotein oligomer, such as the Env trimer, in order to function. Finally, we observed that wild-type levels of incorporation of the HIV-2 Env into budding viruses were not required for this activity.

  11. Functional Domains within the Human Immunodeficiency Virus Type 2 Envelope Protein Required To Enhance Virus Production

    PubMed Central

    Abada, Paolo; Noble, Beth; Cannon, Paula M.

    2005-01-01

    Primate lentiviruses code for a protein that stimulates virus production. In human immunodeficiency virus type 1 (HIV-1), the activity is provided by the accessory protein, Vpu, while in HIV-2 and simian immunodeficiency virus it is a property of the envelope (Env) glycoprotein. Using a group of diverse retroviruses and cell types, we have confirmed the functional equivalence of the two proteins. However, despite these similarities, the two proteins have markedly different functional domains. While the Vpu activity is associated primarily with its membrane-spanning region, we have determined that the HIV-2 Env activity requires both the cytoplasmic tail and ectodomain of the protein, with the membrane-spanning domain being less important. Within the Env cytoplasmic tail, we further defined the necessary sequence as a membrane-proximal tyrosine-based motif. Providing the two Env regions separately as distinct CD8 chimeric proteins did not increase virus release. This suggests that the two domains must be either contained within a single protein or closely associated within a multiprotein oligomer, such as the Env trimer, in order to function. Finally, we observed that wild-type levels of incorporation of the HIV-2 Env into budding viruses were not required for this activity. PMID:15731257

  12. Evolutionary Algorithms for Boolean Functions in Diverse Domains of Cryptography.

    PubMed

    Picek, Stjepan; Carlet, Claude; Guilley, Sylvain; Miller, Julian F; Jakobovic, Domagoj

    2016-01-01

    The role of Boolean functions is prominent in several areas including cryptography, sequences, and coding theory. Therefore, various methods for the construction of Boolean functions with desired properties are of direct interest. New motivations on the role of Boolean functions in cryptography with attendant new properties have emerged over the years. There are still many combinations of design criteria left unexplored and in this matter evolutionary computation can play a distinct role. This article concentrates on two scenarios for the use of Boolean functions in cryptography. The first uses Boolean functions as the source of the nonlinearity in filter and combiner generators. Although relatively well explored using evolutionary algorithms, it still presents an interesting goal in terms of the practical sizes of Boolean functions. The second scenario appeared rather recently where the objective is to find Boolean functions that have various orders of the correlation immunity and minimal Hamming weight. In both these scenarios we see that evolutionary algorithms are able to find high-quality solutions where genetic programming performs the best.

  13. Distinct Growth Factor Families Are Recruited in Unique Spatiotemporal Domains during Long-Term Memory Formation in Aplysia californica.

    PubMed

    Kopec, Ashley M; Philips, Gary T; Carew, Thomas J

    2015-06-03

    Several growth factors (GFs) have been implicated in long-term memory (LTM), but no single GF can support all of the plastic changes that occur during memory formation. Because GFs engage highly convergent signaling cascades that often mediate similar functional outcomes, the relative contribution of any particular GF to LTM is difficult to ascertain. To explore this question, we determined the unique contribution of distinct GF families (signaling via TrkB and TGF-βr-II) to LTM formation in Aplysia. We demonstrate that TrkB and TGF-βr-II signaling are differentially recruited during two-trial training in both time (by trial 1 or 2, respectively) and space (in distinct subcellular compartments). These GFs independently regulate MAPK activation and synergistically regulate gene expression. We also show that trial 1 TrkB and trial 2 TGF-βr-II signaling are required for LTM formation. These data support the view that GFs engaged in LTM formation are interactive components of a complex molecular network.

  14. Identification of distinct SET/TAF-Iβ domains required for core histone binding and quantitative characterisation of the interaction

    PubMed Central

    Karetsou, Zoe; Emmanouilidou, Anastasia; Sanidas, Ioannis; Liokatis, Stamatis; Nikolakaki, Eleni; Politou, Anastasia S; Papamarcaki, Thomais

    2009-01-01

    Background The assembly of nucleosomes to higher-order chromatin structures is finely tuned by the relative affinities of histones for chaperones and nucleosomal binding sites. The myeloid leukaemia protein SET/TAF-Iβ belongs to the NAP1 family of histone chaperones and participates in several chromatin-based mechanisms, such as chromatin assembly, nucleosome reorganisation and transcriptional activation. To better understand the histone chaperone function of SET/TAF-Iβ, we designed several SET/TAF-Iβ truncations, examined their structural integrity by circular Dichroism and assessed qualitatively and quantitatively the histone binding properties of wild-type protein and mutant forms using GST-pull down experiments and fluorescence spectroscopy-based binding assays. Results Wild type SET/TAF-Iβ binds to histones H2B and H3 with Kd values of 2.87 and 0.15 μM, respectively. The preferential binding of SET/TAF-Iβ to histone H3 is mediated by its central region and the globular part of H3. On the contrary, the acidic C-terminal tail and the amino-terminal dimerisation domain of SET/TAF-Iβ, as well as the H3 amino-terminal tail, are dispensable for this interaction. Conclusion This type of analysis allowed us to assess the relative affinities of SET/TAF-Iβ for different histones and identify the domains of the protein required for effective histone recognition. Our findings are consistent with recent structural studies of SET/TAF-Iβ and can be valuable to understand the role of SET/TAF-Iβ in chromatin function. PMID:19358706

  15. Strong functional patterns in the evolution of eukaryotic genomes revealed by the reconstruction of ancestral protein domain repertoires

    PubMed Central

    2011-01-01

    Background Genome size and complexity, as measured by the number of genes or protein domains, is remarkably similar in most extant eukaryotes and generally exhibits no correlation with their morphological complexity. Underlying trends in the evolution of the functional content and capabilities of different eukaryotic genomes might be hidden by simultaneous gains and losses of genes. Results We reconstructed the domain repertoires of putative ancestral species at major divergence points, including the last eukaryotic common ancestor (LECA). We show that, surprisingly, during eukaryotic evolution domain losses in general outnumber domain gains. Only at the base of the animal and the vertebrate sub-trees do domain gains outnumber domain losses. The observed gain/loss balance has a distinct functional bias, most strikingly seen during animal evolution, where most of the gains represent domains involved in regulation and most of the losses represent domains with metabolic functions. This trend is so consistent that clustering of genomes according to their functional profiles results in an organization similar to the tree of life. Furthermore, our results indicate that metabolic functions lost during animal evolution are likely being replaced by the metabolic capabilities of symbiotic organisms such as gut microbes. Conclusions While protein domain gains and losses are common throughout eukaryote evolution, losses oftentimes outweigh gains and lead to significant differences in functional profiles. Results presented here provide additional arguments for a complex last eukaryotic common ancestor, but also show a general trend of losses in metabolic capabilities and gain in regulatory complexity during the rise of animals. PMID:21241503

  16. Capturing distinct KCNQ2 channel resting states by metal ion bridges in the voltage-sensor domain.

    PubMed

    Gourgy-Hacohen, Orit; Kornilov, Polina; Pittel, Ilya; Peretz, Asher; Attali, Bernard; Paas, Yoav

    2014-12-01

    Although crystal structures of various voltage-gated K(+) (Kv) and Na(+) channels have provided substantial information on the activated conformation of the voltage-sensing domain (VSD), the topology of the VSD in its resting conformation remains highly debated. Numerous studies have investigated the VSD resting state in the Kv Shaker channel; however, few studies have explored this issue in other Kv channels. Here, we investigated the VSD resting state of KCNQ2, a K(+) channel subunit belonging to the KCNQ (Kv7) subfamily of Kv channels. KCNQ2 can coassemble with the KCNQ3 subunit to mediate the IM current that regulates neuronal excitability. In humans, mutations in KCNQ2 are associated with benign neonatal forms of epilepsy or with severe epileptic encephalopathy. We introduced cysteine mutations into the S4 transmembrane segment of the KCNQ2 VSD and determined that external application of Cd(2+) profoundly reduced the current amplitude of S4 cysteine mutants S195C, R198C, and R201C. Based on reactivity with the externally accessible endogenous cysteine C106 in S1, we infer that each of the above S4 cysteine mutants forms Cd(2+) bridges to stabilize a channel closed state. Disulfide bonds and metal bridges constrain the S4 residues S195, R198, and R201 near C106 in S1 in the resting state, and experiments using concatenated tetrameric constructs indicate that this occurs within the same VSD. KCNQ2 structural models suggest that three distinct resting channel states have been captured by the formation of different S4-S1 Cd(2+) bridges. Collectively, this work reveals that residue C106 in S1 can be very close to several N-terminal S4 residues for stabilizing different KCNQ2 resting conformations.

  17. Capturing distinct KCNQ2 channel resting states by metal ion bridges in the voltage-sensor domain

    PubMed Central

    Gourgy-Hacohen, Orit; Kornilov, Polina; Pittel, Ilya; Peretz, Asher

    2014-01-01

    Although crystal structures of various voltage-gated K+ (Kv) and Na+ channels have provided substantial information on the activated conformation of the voltage-sensing domain (VSD), the topology of the VSD in its resting conformation remains highly debated. Numerous studies have investigated the VSD resting state in the Kv Shaker channel; however, few studies have explored this issue in other Kv channels. Here, we investigated the VSD resting state of KCNQ2, a K+ channel subunit belonging to the KCNQ (Kv7) subfamily of Kv channels. KCNQ2 can coassemble with the KCNQ3 subunit to mediate the IM current that regulates neuronal excitability. In humans, mutations in KCNQ2 are associated with benign neonatal forms of epilepsy or with severe epileptic encephalopathy. We introduced cysteine mutations into the S4 transmembrane segment of the KCNQ2 VSD and determined that external application of Cd2+ profoundly reduced the current amplitude of S4 cysteine mutants S195C, R198C, and R201C. Based on reactivity with the externally accessible endogenous cysteine C106 in S1, we infer that each of the above S4 cysteine mutants forms Cd2+ bridges to stabilize a channel closed state. Disulfide bonds and metal bridges constrain the S4 residues S195, R198, and R201 near C106 in S1 in the resting state, and experiments using concatenated tetrameric constructs indicate that this occurs within the same VSD. KCNQ2 structural models suggest that three distinct resting channel states have been captured by the formation of different S4–S1 Cd2+ bridges. Collectively, this work reveals that residue C106 in S1 can be very close to several N-terminal S4 residues for stabilizing different KCNQ2 resting conformations. PMID:25385787

  18. Purification, Cellular Levels, and Functional Domains of LMF1

    PubMed Central

    Babilonia-Rosa, Melissa; Neher, Saskia B.

    2014-01-01

    Over a third of the US adult population has hypertriglyceridemia, resulting in an increased risk of atherosclerosis, pancreatitis, and metabolic syndrome. Lipoprotein lipase (LPL)1, a dimeric enzyme, is the main lipase responsible for TG clearance from the blood after food intake. LPL requires an endoplasmic reticulum (ER)-resident, transmembrane protein known as lipase maturation factor 1 (LMF1) for secretion and enzymatic activity. LMF1 is believed to act as a client specific chaperone for dimeric lipases, but the precise mechanism by which LMF1 functions is not understood. Here, we examine which domains of LMF1 contribute to dimeric lipase maturation by assessing the function of truncation variants. N-terminal truncations of LMF1 show that all the domains are necessary for LPL maturation. Fluorescence microscopy and protease protection assays confirmed that these variants were properly oriented in the ER. We measured cellular levels of LMF1 and found that it is expressed at low levels and each molecule of LMF1 promotes the maturation of 50 or more molecules of LPL. Thus we provide evidence for the critical role of the N-terminus of LMF1 for the maturation of LPL and relevant ratio of chaperone to substrate. PMID:24909692

  19. 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.

  20. 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.

  1. Guiding Cell Attachment in 3D Microscaffolds Selectively Functionalized with Two Distinct Adhesion Proteins.

    PubMed

    Richter, Benjamin; Hahn, Vincent; Bertels, Sarah; Claus, Tanja K; Wegener, Martin; Delaittre, Guillaume; Barner-Kowollik, Christopher; Bastmeyer, Martin

    2017-02-01

    The combination of three different photoresists into a single direct laser written 3D microscaffold permits functionalization with two bioactive full-length proteins. The cell-instructive microscaffolds consist of a passivating framework equipped with light activatable constituents featuring distinct protein-binding properties. This allows directed cell attachment of epithelial or fibroblast cells in 3D.

  2. On Determinatives and the Category-Function Distinction: A Reply to Brett Reynolds

    ERIC Educational Resources Information Center

    Lenchuk, Iryna; Ahmed, Amer

    2014-01-01

    This article examines the arguments made in the article "Determiners, Feline Marsupials, and the Category-Function Distinction: A Critique of ELT Grammars" by Brett Reynolds recently published in the "TESL Canada Journal" (2013). In our response, we demonstrate that the author's arguments are problematic on both…

  3. Distinct Patterns of Grey Matter Abnormality in High-Functioning Autism and Asperger's Syndrome

    ERIC Educational Resources Information Center

    McAlonan, Grainne M.; Suckling, John; Wong, Naikei; Cheung, Vinci; Lienenkaemper, Nina; Cheung, Charlton; Chua, Siew E.

    2008-01-01

    Background: Autism exists across a wide spectrum and there is considerable debate as to whether children with Asperger's syndrome, who have normal language milestones, should be considered to comprise a subgroup distinct other from high-functioning children with autism (HFA), who have a history of delayed language development. Magnetic resonance…

  4. The HIV-1 Envelope Transmembrane Domain Binds TLR2 through a Distinct Dimerization Motif and Inhibits TLR2-Mediated Responses

    PubMed Central

    Rotem, Etai; Schwarzter, Roland; Gramatica, Andrea; Futerman, Anthony H.; Shai, Yechiel

    2014-01-01

    HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation. PMID:25121610

  5. The HIV-1 envelope transmembrane domain binds TLR2 through a distinct dimerization motif and inhibits TLR2-mediated responses.

    PubMed

    Reuven, Eliran Moshe; Ali, Mohammad; Rotem, Etai; Schwarzer, Roland; Schwarzter, Roland; Gramatica, Andrea; Futerman, Anthony H; Shai, Yechiel

    2014-08-01

    HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.

  6. Modular structure of chromosomal proteins HMG-14 and HMG-17: Definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain

    SciTech Connect

    Trieschmann, L.; Postnikov, Y.V.; Rickers, A.; Bustin, M.

    1995-12-01

    This report describes how deletion mutants and peptides were used to identify the transcriptional enhancement domain and the nucleosome binding domain of two chromosomal proteins, HMG-14 and HMG-17. The research indicates that mutations involving C-terminal amino acids significantly reduces the ability of the nucleoproteins to enhance transcription from chromatin templates. 42 refs., 6 figs., 1 tab.

  7. A selective screen reveals discrete functional domains in Drosophila Nanos.

    PubMed Central

    Arrizabalaga, G; Lehmann, R

    1999-01-01

    The Drosophila protein Nanos encodes an evolutionarily conserved protein with two zinc finger motifs. In the embryo, Nanos protein function is required for establishment of the anterior-posterior body pattern and for the migration of primordial germ cells. During oogenesis, Nanos protein is involved in the establishment and maintenance of germ-line stem cells and the differentiation of oocyte precursor cells. To establish proper embryonic patterning, Nanos acts as a translational regulator of hunchback RNA. Nanos' targets for germ cell migration and development are not known. Here, we describe a selective genetic screen aimed at isolating new nanos alleles. The molecular and genetic analysis of 68 new alleles has allowed us to identify amino acids critical for nanos function. This analysis shows that the CCHC motifs, which coordinate two metal ions, are essential for all known functions of Nanos protein. Furthermore, a region C-terminal to the zinc fingers seems to constitute a novel functional domain within the Nanos protein. This "tail region" of Nanos is required for abdomen formation and germ cell migration, but not for oogenesis. PMID:10581288

  8. Differential Loss of Prolyl Isomerase or Chaperone Activity of Ran-binding Protein 2 (Ranbp2) Unveils Distinct Physiological Roles of Its Cyclophilin Domain in Proteostasis*

    PubMed Central

    Cho, Kyoung-in; Patil, Hemangi; Senda, Eugene; Wang, Jessica; Yi, Haiqing; Qiu, Sunny; Yoon, Dosuk; Yu, Minzhong; Orry, Andrew; Peachey, Neal S.; Ferreira, Paulo A.

    2014-01-01

    The immunophilins, cyclophilins, catalyze peptidyl cis-trans prolyl-isomerization (PPIase), a rate-limiting step in protein folding and a conformational switch in protein function. Cyclophilins are also chaperones. Noncatalytic mutations affecting the only cyclophilins with known but distinct physiological substrates, the Drosophila NinaA and its mammalian homolog, cyclophilin-B, impair opsin biogenesis and cause osteogenesis imperfecta, respectively. However, the physiological roles and substrates of most cyclophilins remain unknown. It is also unclear if PPIase and chaperone activities reflect distinct cyclophilin properties. To elucidate the physiological idiosyncrasy stemming from potential cyclophilin functions, we generated mice lacking endogenous Ran-binding protein-2 (Ranbp2) and expressing bacterial artificial chromosomes of Ranbp2 with impaired C-terminal chaperone and with (Tg-Ranbp2WT-HA) or without PPIase activities (Tg-Ranbp2R2944A-HA). The transgenic lines exhibit unique effects in proteostasis. Either line presents selective deficits in M-opsin biogenesis with its accumulation and aggregation in cone photoreceptors but without proteostatic impairment of two novel Ranbp2 cyclophilin partners, the cytokine-responsive effectors, STAT3/STAT5. Stress-induced STAT3 activation is also unaffected in Tg-Ranbp2R2944A-HA::Ranbp2−/−. Conversely, proteomic analyses found that the multisystem proteinopathy/amyotrophic lateral sclerosis proteins, heterogeneous nuclear ribonucleoproteins A2/B1, are down-regulated post-transcriptionally only in Tg-Ranbp2R2944A-HA::Ranbp2−/−. This is accompanied by the age- and tissue-dependent reductions of diubiquitin and ubiquitylated proteins, increased deubiquitylation activity, and accumulation of the 26 S proteasome subunits S1 and S5b. These manifestations are absent in another line, Tg-Ranbp2CLDm-HA::Ranbp2−/−, harboring SUMO-1 and S1-binding mutations in the Ranbp2 cyclophilin-like domain. These results unveil

  9. Distinct functional properties of isoamylase-type starch debranching enzymes in monocot and dicot leaves.

    PubMed

    Facon, Maud; Lin, Qiaohui; Azzaz, Abdelhamid M; Hennen-Bierwagen, Tracie A; Myers, Alan M; Putaux, Jean-Luc; Roussel, Xavier; D'Hulst, Christophe; Wattebled, Fabrice

    2013-11-01

    Isoamylase-type starch debranching enzymes (ISA) play important roles in starch biosynthesis in chloroplast-containing organisms, as shown by the strict conservation of both catalytically active ISA1 and the noncatalytic homolog ISA2. Functional distinctions exist between species, although they are not understood yet. Numerous plant tissues require both ISA1 and ISA2 for normal starch biosynthesis, whereas monocot endosperm and leaf exhibit nearly normal starch metabolism without ISA2. This study took in vivo and in vitro approaches to determine whether organism-specific physiology or evolutionary divergence between monocots and dicots is responsible for distinctions in ISA function. Maize (Zea mays) ISA1 was expressed in Arabidopsis (Arabidopsis thaliana) lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functioned in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2. Analysis of recombinant enzymes showed that Arabidopsis ISA1 requires ISA2 as a partner for enzymatic function, whereas maize ISA1 was active by itself. The electrophoretic mobility of recombinant and native maize ISA differed, suggestive of posttranslational modifications in vivo. Sedimentation equilibrium measurements showed recombinant maize ISA1 to be a dimer, in contrast to previous gel permeation data that estimated the molecular mass as a tetramer. These data demonstrate that evolutionary divergence between monocots and dicots is responsible for the distinctions in ISA1 function.

  10. Pathway logic modeling of protein functional domains in signal transduction.

    PubMed

    Talcott, C; Eker, S; Knapp, M; Lincoln, P; Laderoute, K

    2004-01-01

    Protein functional domains (PFDs) are consensus sequences within signaling molecules that recognize and assemble other signaling components into complexes. Here we describe the application of an approach called Pathway Logic to the symbolic modeling signal transduction networks at the level of PFDs. These models are developed using Maude, a symbolic language founded on rewriting logic. Models can be queried (analyzed) using the execution, search and model-checking tools of Maude. We show how signal transduction processes can be modeled using Maude at very different levels of abstraction involving either an overall state of a protein or its PFDs and their interactions. The key insight for the latter is our algebraic representation of binding interactions as a graph.

  11. Diagnosis of Autism Spectrum Disorders Using Temporally-Distinct Resting-State Functional Connectivity Networks

    PubMed Central

    Wee, Chong-Yaw; Yap, Pew-Thian; Shen, Dinggang

    2016-01-01

    Introduction Resting-state functional magnetic resonance imaging (R-fMRI) is dynamic in nature since neural activities constantly change over the time and are dominated by repeating brief activations and deactivations involving many brain regions. Each region participates in multiple brain functions and is part of various functionally distinct but spatially overlapping networks. Functional connectivity computed as correlations over the entire time series always overlooks inter-region interactions that often occur repeatedly and dynamically in time, limiting its application to disease diagnosis. Aims We develop a novel framework that uses short-time activation patterns of brain connectivity to better detect subtle disease-induced disruptions of brain connectivity. A clustering algorithm is first used to temporally decompose R-fMRI time series into distinct clusters with similar spatial distribution of neural activity based on the assumption that functionally distinct networks should be largely temporally distinct since brain states do not simultaneously coexist in general. A Pearson correlation-based functional connectivity network is then constructed for each cluster to allow for better exploration of spatiotemporal dynamics of individual neural activity. To reduce significant inter-subject variability and to remove possible spurious connections, we use a group-constrained sparse regression model to construct a backbone sparse network for each cluster and use it to weight the corresponding Pearson correlation network. Results The proposed method outperforms the conventional static, temporally-dependent fully-connected correlation-based networks by at least 7% on a publicly available autism dataset. We were able to reproduce similar results using data from other centers. Conclusions By combining the advantages of temporal independence and group-constrained sparse regression, our method improves autism diagnosis. PMID:26821773

  12. 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

  13. AUX/LAX Genes Encode a Family of Auxin Influx Transporters That Perform Distinct Functions during Arabidopsis Development[C][W

    PubMed Central

    Péret, Benjamin; Swarup, Kamal; Ferguson, Alison; Seth, Malvika; Yang, Yaodong; Dhondt, Stijn; James, Nicholas; Casimiro, Ilda; Perry, Paula; Syed, Adnan; Yang, Haibing; Reemmer, Jesica; Venison, Edward; Howells, Caroline; Perez-Amador, Miguel A.; Yun, Jeonga; Alonso, Jose; Beemster, Gerrit T.S.; Laplaze, Laurent; Murphy, Angus; Bennett, Malcolm J.; Nielsen, Erik; Swarup, Ranjan

    2012-01-01

    Auxin transport, which is mediated by specialized influx and efflux carriers, plays a major role in many aspects of plant growth and development. AUXIN1 (AUX1) has been demonstrated to encode a high-affinity auxin influx carrier. In Arabidopsis thaliana, AUX1 belongs to a small multigene family comprising four highly conserved genes (i.e., AUX1 and LIKE AUX1 [LAX] genes LAX1, LAX2, and LAX3). We report that all four members of this AUX/LAX family display auxin uptake functions. Despite the conservation of their biochemical function, AUX1, LAX1, and LAX3 have been described to regulate distinct auxin-dependent developmental processes. Here, we report that LAX2 regulates vascular patterning in cotyledons. We also describe how regulatory and coding sequences of AUX/LAX genes have undergone subfunctionalization based on their distinct patterns of spatial expression and the inability of LAX sequences to rescue aux1 mutant phenotypes, respectively. Despite their high sequence similarity at the protein level, transgenic studies reveal that LAX proteins are not correctly targeted in the AUX1 expression domain. Domain swapping studies suggest that the N-terminal half of AUX1 is essential for correct LAX localization. We conclude that Arabidopsis AUX/LAX genes encode a family of auxin influx transporters that perform distinct developmental functions and have evolved distinct regulatory mechanisms. PMID:22773749

  14. Functional expression of Phanerochaete chrysosporium cellobiose dehydrogenase flavin domain in Escherichia coli.

    PubMed

    Desriani; Ferri, Stefano; Sode, Koji

    2010-06-01

    Cellobiose dehydrogenase (CDH; EC 1.1.99.18) is an extracellular glycosylated protein composed of two distinct domains, a C-terminal catalytic flavin domain and an N-terminal cytochrome-b-type heme domain, which transfers electrons from the flavin domain to external electron acceptors. The soluble flavin domain of the Phanerochaete chrysosporium CDH was successfully expressed in Escherichia coli. The enzyme showed dye-mediated CDH activity higher than that of the complete CDH, composed of flavin domain and heme domain, prepared using Pichia pastoris as the host microorganism. The ability to conveniently express the recombinant CDH flavin domain in E. coli provides great opportunities for the molecular engineering of the catalytic properties of CDH.

  15. 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.

  16. PAR-3 oligomerization may provide an actin-independent mechanism to maintain distinct par protein domains in the early Caenorhabditis elegans embryo.

    PubMed

    Dawes, Adriana T; Munro, Edwin M

    2011-09-21

    Par proteins establish discrete intracellular spatial domains to polarize many different cell types. In the single-cell embryo of the nematode worm Caenorhabditis elegans, the segregation of Par proteins is crucial for proper division and cell fate specification. Actomyosin-based cortical flows drive the initial formation of anterior and posterior Par domains, but cortical actin is not required for the maintenance of these domains. Here we develop a model of interactions between the Par proteins that includes both mutual inhibition and PAR-3 oligomerization. We show that this model gives rise to a bistable switch mechanism, allowing the Par proteins to occupy distinct anterior and posterior domains seen in the early C. elegans embryo, independent of dynamics or asymmetries in the actin cortex. The model predicts a sharp loss of cortical Par protein asymmetries during gradual depletion of the Par protein PAR-6, and we confirm this prediction experimentally. Together, these results suggest both mutual inhibition and PAR-3 oligomerization are sufficient to maintain distinct Par protein domains in the early C. elegans embryo.

  17. Profound regulation of neonatal CA1 rat hippocampal GABAergic transmission by functionally distinct kainate receptor populations

    PubMed Central

    Maingret, François; Lauri, Sari E; Taira, Tomi; Isaac, John TR

    2005-01-01

    Neonatal hippocampus exhibits distinct patterns of network activity that are dependent on the interaction between inhibitory and excitatory transmission. Kainate receptors are ideally positioned to regulate this activity by virtue of their ability to regulate presynaptic function in GABAergic interneurones. Indeed, kainate receptors are highly expressed in neonatal hippocampal interneurones, yet the role and mechanisms by which they might regulate neonatal circuitry are unexplored. To address this we investigated the kainate receptor-dependent regulation of GABAergic transmission onto neonatal CA1 pyramidal neurones. Kainate receptor activation produced two distinct opposing effects, a very large increase in the frequency of spontaneous IPSCs, and a robust depression of evoked GABAergic transmission. The up-regulation of spontaneous transmission was due to activation of somatodendritic and axonal receptors while the depression of evoked transmission could be fully accounted for by a direct regulation of GABA release by kainate receptors located at the terminals. None of the effects of kainate receptor agonists were sensitive to GABAB receptor antagonists, nor was there any postsynaptic kainate receptor-dependent effects observed in CA1 pyramidal cells that could account for our findings. Our data demonstrate that kainate receptors profoundly regulate neonatal CA1 GABAergic circuitry by two distinct opposing mechanisms, and indicate that these two effects are mediated by functionally distinct populations of receptors. Thus kainate receptors are strategically located to play a critical role in shaping early hippocampal network activity and by virtue of this have a key role in hippocampal development. PMID:15946969

  18. Human germline and pan-cancer variomes and their distinct functional profiles.

    PubMed

    Pan, Yang; Karagiannis, Konstantinos; Zhang, Haichen; Dingerdissen, Hayley; Shamsaddini, Amirhossein; Wan, Quan; Simonyan, Vahan; Mazumder, Raja

    2014-10-01

    Identification of non-synonymous single nucleotide variations (nsSNVs) has exponentially increased due to advances in Next-Generation Sequencing technologies. The functional impacts of these variations have been difficult to ascertain because the corresponding knowledge about sequence functional sites is quite fragmented. It is clear that mapping of variations to sequence functional features can help us better understand the pathophysiological role of variations. In this study, we investigated the effect of nsSNVs on more than 17 common types of post-translational modification (PTM) sites, active sites and binding sites. Out of 1 705 285 distinct nsSNVs on 259 216 functional sites we identified 38 549 variations that significantly affect 10 major functional sites. Furthermore, we found distinct patterns of site disruptions due to germline and somatic nsSNVs. Pan-cancer analysis across 12 different cancer types led to the identification of 51 genes with 106 nsSNV affected functional sites found in 3 or more cancer types. 13 of the 51 genes overlap with previously identified Significantly Mutated Genes (Nature. 2013 Oct 17;502(7471)). 62 mutations in these 13 genes affecting functional sites such as DNA, ATP binding and various PTM sites occur across several cancers and can be prioritized for additional validation and investigations.

  19. Single-cell analysis reveals functionally distinct classes within the planarian stem cell compartment

    PubMed Central

    van Wolfswinkel, Josien C.; Wagner, Daniel E.; Reddien, Peter W.

    2014-01-01

    Planarians are flatworms capable of regenerating any missing body region. This capacity is mediated by neoblasts, a proliferative cell population that contains pluripotent stem cells. Although population-based studies have revealed many neoblast characteristics, whether functionally distinct classes exist within this population is unclear. Here, we used high-dimensional single-cell transcriptional profiling from over a thousand individual neoblasts to directly compare gene expression fingerprints during homeostasis and regeneration. We identified two prominent neoblast classes that we named ζ (zeta) and σ (sigma). Zeta-neoblasts encompass specified cells that give rise to an abundant postmitotic lineage including epidermal cells, and are not required for regeneration. By contrast, sigma-neoblasts proliferate in response to injury, possess broad lineage capacity, and can give rise to zeta-neoblasts. These findings present a new view of planarian neoblasts, in which the population is comprised of two major and functionally distinct cellular compartments. PMID:25017721

  20. Distinct functions and regulation of epithelial progesterone receptor in the mouse cervix, vagina, and uterus.

    PubMed

    Mehta, Fabiola F; Son, Jieun; Hewitt, Sylvia C; Jang, Eunjung; Lydon, John P; Korach, Kenneth S; Chung, Sang-Hyuk

    2016-04-05

    While the function of progesterone receptor (PR) has been studied in the mouse vagina and uterus, its regulation and function in the cervix has not been described. We selectively deleted epithelial PR in the female reproductive tracts using the Cre/LoxP recombination system. We found that epithelial PR was required for induction of apoptosis and suppression of cell proliferation by progesterone (P4) in the cervical and vaginal epithelium. We also found that epithelial PR was dispensable for P4 to suppress apoptosis and proliferation in the uterine epithelium. PR is encoded by the Pgr gene, which is regulated by estrogen receptor α (ERα) in the female reproductive tracts. Using knock-in mouse models expressing ERα mutants, we determined that the DNA-binding domain (DBD) and AF2 domain of ERα were required for upregulation of Pgr in the cervix and vagina as well as the uterine stroma. The ERα AF1 domain was required for upregulation of Pgr in the vaginal stroma and epithelium and cervical epithelium, but not in the uterine and cervical stroma. ERα DBD, AF1, and AF2 were required for suppression of Pgr in the uterine epithelium, which was mediated by stromal ERα. Epithelial ERα was responsible for upregulation of epithelial Pgr in the cervix and vagina. Our results indicate that regulation and functions of epithelial PR are different in the cervix, vagina, and uterus.

  1. Distinct circuit-dependent functions of presynaptic neurexin-3 at GABAergic and glutamatergic synapses

    PubMed Central

    Aoto, Jason; Földy, Csaba; Ilcus, Silviana Maria Ciurea; Tabuchi, Katsuhiko; Südhof, Thomas C.

    2015-01-01

    α- and β-neurexins are presynaptic cell-adhesion molecules whose general importance for synaptic transmission is well documented. The specific functions of neurexins, however, remain largely unknown because no conditional neurexin knockouts are available, and because targeting all α- and β-neurexins produced by a particular gene is challenging. Using newly-generated constitutive and conditional knockout mice that target all neurexin-3α and -3β isoforms, we here show that neurexin-3 is differentially required for distinct synaptic functions in different brain regions. Specifically, we find that in cultured neurons and acute slices of the hippocampus, presynaptic neurexin-3 mediates trans-synaptic regulation of postsynaptic AMPA-receptors via its extracellular sequences. In cultured neurons and acute slices of the olfactory bulb, however, presynaptic neurexin-3 is selectively required for GABA release by a mechanism involving its intracellular sequences. Thus, our data demonstrate that neurexin-3 performs distinct essential pre- or postsynaptic functions in different brain regions by distinct mechanisms. PMID:26030848

  2. 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.

  3. Organized living: formation mechanisms and functions of plasma membrane domains in yeast.

    PubMed

    Ziółkowska, Natasza E; Christiano, Romain; Walther, Tobias C

    2012-03-01

    Plasma membrane proteins and lipids organize into lateral domains of specific composition. Domain formation is achieved by a combination of lipid-lipid and lipid-protein interactions, membrane-binding protein scaffolds and protein fences. The resulting domains function in membrane protein turnover and homeostasis, as well as in cell signaling. We review the mechanisms generating plasma membrane domains and the functional consequences of this organization, focusing on recent findings from research on the yeast model system.

  4. Binding of the cSH3 domain of Grb2 adaptor to two distinct RXXK motifs within Gab1 docker employs differential mechanisms.

    PubMed

    McDonald, Caleb B; Seldeen, Kenneth L; Deegan, Brian J; Bhat, Vikas; Farooq, Amjad

    2011-01-01

    A ubiquitous component of cellular signaling machinery, Gab1 docker plays a pivotal role in routing extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. Here, using isothermal titration calorimetry (ITC) in combination with macromolecular modeling (MM), we show that although Gab1 contains four distinct RXXK motifs, designated G1, G2, G3, and G4, only G1 and G2 motifs bind to the cSH3 domain of Grb2 adaptor and do so with distinct mechanisms. Thus, while the G1 motif strictly requires the PPRPPKP consensus sequence for high-affinity binding to the cSH3 domain, the G2 motif displays preference for the PXVXRXLKPXR consensus. Such sequential differences in the binding of G1 and G2 motifs arise from their ability to adopt distinct polyproline type II (PPII)- and 3(10) -helical conformations upon binding to the cSH3 domain, respectively. Collectively, our study provides detailed biophysical insights into a key protein-protein interaction involved in a diverse array of signaling cascades central to health and disease.

  5. Binding of the cSH3 Domain of Grb2 Adaptor to Two Distinct RXXK Motifs within Gab1 Docker Employs Differential Mechanisms

    PubMed Central

    McDonald, Caleb B.; Seldeen, Kenneth L.; Deegan, Brian J.; Bhat, Vikas; Farooq, Amjad

    2010-01-01

    A ubiquitous component of cellular signaling machinery, Gab1 docker plays a pivotal role in routing extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. Here, using isothermal titration calorimetry (ITC) in combination with macromolecular modeling (MM), we show that although Gab1 contains four distinct RXXK motifs, designated G1, G2, G3 and G4, only G1 and G2 motifs bind to the cSH3 domain of Grb2 adaptor and do so with distinct mechanisms. Thus, while the G1 motif strictly requires the PPRPPKP consensus sequence for high-affinity binding to the cSH3 domain, the G2 motif displays preference for the PXVXRXLKPXR consensus. Such sequential differences in the binding of G1 and G2 motifs arise from their ability to adopt distinct polyproline type II (PPII)- and 310-helical conformations upon binding to the cSH3 domain, respectively. Collectively, our study provides detailed biophysical insights into a key protein-protein interaction involved in a diverse array of signaling cascades central to health and disease. PMID:21472810

  6. 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.

  7. Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain

    PubMed Central

    Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J.; Polo, Simona

    2016-01-01

    Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VIshort and myosin VIlong, which differ in the C-terminal region. Their physiological and pathological role remains unknown. Here we identified an isoform-specific regulatory helix, named α2-linker that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a novel clathrin-binding domain that is unique to myosin VIlong and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, where alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VIshort for tumor cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VIshort. Thus the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VIlong) or migratory (myosin VIshort) functional roles. PMID:26950368

  8. Systematic Mapping of WNT-FZD Protein Interactions Reveals Functional Selectivity by Distinct WNT-FZD Pairs*

    PubMed Central

    Dijksterhuis, Jacomijn P.; Baljinnyam, Bolormaa; Stanger, Karen; Sercan, Hakki O.; Ji, Yun; Andres, Osler; Rubin, Jeffrey S.; Hannoush, Rami N.; Schulte, Gunnar

    2015-01-01

    The seven-transmembrane-spanning receptors of the FZD1–10 class are bound and activated by the WNT family of lipoglycoproteins, thereby inducing a complex network of signaling pathways. However, the specificity of the interaction between mammalian WNT and FZD proteins and the subsequent signaling cascade downstream of the different WNT-FZD pairs have not been systematically addressed to date. In this study, we determined the binding affinities of various WNTs for different members of the FZD family by using bio-layer interferometry and characterized their functional selectivity in a cell system. Using purified WNTs, we show that different FZD cysteine-rich domains prefer to bind to distinct WNTs with fast on-rates and slow off-rates. In a 32D cell-based system engineered to overexpress FZD2, FZD4, or FZD5, we found that WNT-3A (but not WNT-4, -5A, or -9B) activated the WNT-β-catenin pathway through FZD2/4/5 as measured by phosphorylation of LRP6 and β-catenin stabilization. Surprisingly, different WNT-FZD pairs showed differential effects on phosphorylation of DVL2 and DVL3, revealing a previously unappreciated DVL isoform selectivity by different WNT-FZD pairs in 32D cells. In summary, we present extensive mapping of WNT-FZD cysteine-rich domain interactions complemented by analysis of WNT-FZD pair functionality in a unique cell system expressing individual FZD isoforms. Differential WNT-FZD binding and selective functional readouts suggest that endogenous WNT ligands evolved with an intrinsic natural bias toward different downstream signaling pathways, a phenomenon that could be of great importance in the design of FZD-targeting drugs. PMID:25605717

  9. Transiently populated intermediate functions as a branching point of the FF domain folding pathway.

    PubMed

    Korzhnev, Dmitry M; Religa, Tomasz L; Kay, Lewis E

    2012-10-30

    Studies of protein folding and the intermediates that are formed along the folding pathway provide valuable insights into the process by which an unfolded ensemble forms a functional native conformation. However, because intermediates on folding pathways can serve as initiation points of aggregation (implicated in a number of diseases), their characterization assumes an even greater importance. Establishing the role of such intermediates in folding, misfolding, and aggregation remains a major challenge due to their often low populations and short lifetimes. We recently used NMR relaxation dispersion methods and computational techniques to determine an atomic resolution structure of the folding intermediate of a small protein module--the FF domain--with an equilibrium population of 2-3% and a millisecond lifetime, 25 °C. Based on this structure a variant FF domain has been designed in which the native state is selectively destabilized by removing the carboxyl-terminal helix in the native structure to produce a highly populated structural mimic of the intermediate state. Here, we show via solution NMR studies of the designed mimic that the mimic forms distinct conformers corresponding to monomeric and dimeric (K(d) = 0.2 mM) forms of the protein. The conformers exchange on the seconds timescale with a monomer association rate of 1.1 · 10(4) M(-1) s(-1) and with a region responsible for dimerization localized to the amino-terminal residues of the FF domain. This study establishes the FF domain intermediate as a central player in both folding and misfolding pathways and illustrates how incomplete folding can lead to the formation of higher-order structures.

  10. FUNCTIONALLY AND SPATIALLY DISTINCT MODES OF MUNC18-SYNTAXIN 1 INTERACTION*

    PubMed Central

    Rickman, Colin; Medine, Claire N.; Bergmann, Axel; Duncan, Rory R.

    2007-01-01

    Eukaryotic membrane trafficking is a conserved process under tight temporal and spatial regulation in which the fusion of membranes is driven by the formation of the ternary SNARE complex. Syntaxin 1a, a core component of the exocytic SNARE1 complex in neurones and neuroendocrine cells, is regulated directly by munc18-1, its cognate SM (Sec1p/Munc18) protein. SM proteins show remarkable structural conservation throughout evolution indicating a common binding mechanism and function. However, SM proteins possess disparate binding mechanisms and regulatory effects, with munc18-1, the major brain isoform, classed as atypical in both its binding specificity and mode. We now show that munc18-1 interacts with syntaxin 1a through two mechanistically distinct modes of binding, both in vitro and in living cells, in contrast to current models. Furthermore these functionally divergent interactions occur at distinct cellular locations. These findings provide a molecular explanation for the multiple, spatially distinct roles of munc18-1. PMID:17264080

  11. 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.

  12. A functional protein pore with a "retro" transmembrane domain.

    PubMed Central

    Cheley, S.; Braha, O.; Lu, X.; Conlan, S.; Bayley, H.

    1999-01-01

    Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane beta-barrel of alpha-hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif. PMID:10386875

  13. 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.

  14. How a single residue in individual β-thymosin/WH2 domains controls their functions in actin assembly

    PubMed Central

    Didry, Dominique; Cantrelle, Francois-Xavier; Husson, Clotilde; Roblin, Pierre; Moorthy, Anna M Eswara; Perez, Javier; Le Clainche, Christophe; Hertzog, Maud; Guittet, Eric; Carlier, Marie-France; van Heijenoort, Carine; Renault, Louis

    2012-01-01

    β-Thymosin (βT) and WH2 domains are widespread, intrinsically disordered actin-binding peptides that display significant sequence variability and different regulations of actin self-assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers like Thymosin-β4, or enhance motility by directing polarized filament assembly like Ciboulot βT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-β4, Ciboulot, TetraThymosinβ and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-β4. Functionally different βT/WH2 domains differ by distinct dynamics of their C-terminal half interactions with G-actin pointed face. These C-terminal interaction dynamics are controlled by the strength of electrostatic interactions with G-actin. At physiological ionic strength, a single salt bridge with actin located next to their central LKKT/V motif induces G-actin sequestration in both isolated long βT and WH2 domains. The results open perspectives for elucidating the functions of βT/WH2 domains in other modular proteins. PMID:22193718

  15. Simple Separation of Functionally Distinct Populations of Lamin-Binding Proteins.

    PubMed

    Berk, Jason M; Wilson, Katherine L

    2016-01-01

    The inner membrane of the nuclear envelope (NE) is home to hundreds of integral membrane proteins (NE transmembrane proteins, "NETs") with conserved or tissue-specific roles in genome organization and nuclear function. Nearly all characterized NETs bind A- or B-type lamins directly. However, hundreds of NETs remain uncharacterized, collectively posing an enormous gap that must be bridged to understand nuclear function and genome biology. We provide technically simple protocols for the separation and recovery of functionally distinct populations of NETs and A-type lamins. This protocol was developed for emerin, an inner nuclear membrane protein that binds lamins and barrier-to-autointegration factor (BANF1) as a component of nuclear lamina structure, and has diverse roles in nuclear assembly, signaling, and gene regulation. This protocol separates easily solubilized ("easy") populations of nuclear lamina proteins (emerin, lamin A, BAF) from "sonication-dependent" populations. Depending on cell type, the "easy" and "sonication-dependent" fractions each contain up to about half the available emerin, A-type lamins, and BAF, whereas B-type lamins and histone H3 are predominantly sonication dependent. The two populations of emerin have distinct posttranslational modifications, and only one population associates with BAF. This method may be useful for functional screening or analysis of other lamin-associated proteins, including novel NETs emerging from proteomic studies.

  16. Feeding characteristics reveal functional distinctions among browsing herbivorous fishes on coral reefs

    NASA Astrophysics Data System (ADS)

    Streit, Robert P.; Hoey, Andrew S.; Bellwood, David R.

    2015-12-01

    The removal of macroalgal biomass by fishes is a key process on coral reefs. Numerous studies have identified the fish species responsible for removing mature macroalgae, and have identified how this varies spatially, temporally, and among different algal types. None, however, have considered the behavioural and morphological traits of the browsing fishes and how this may influence the removal of macroalgal material. Using video observations of fish feeding on the brown macroalga Sargassum polycystum, we quantified the feeding behaviour and morphology of the four dominant browsing species on the Great Barrier Reef ( Kyphosus vaigiensis, Naso unicornis, Siganus canaliculatus, and Siganus doliatus). The greatest distinction between species was the algal material they targeted. K. vaigiensis and N. unicornis bit on the entire macroalgal thallus in approximately 90 % of bites. In contrast, Si. canaliculatus and Si. doliatus avoided biting the stalks, with 80-98 % of bites being on the macroalgal leaves only. This distinctive grouping into `entire thallus-biters' versus `leaf-biters' was not supported by size-standardized measures of biting morphology. Rather, species-specific adult body sizes, tooth shape, and feeding behaviour appear to underpin this functional distinction, with adults of the two larger fish species ( N. unicornis and K. vaigiensis) eating the entire macroalgal thallus, while the two smaller species ( Si. canaliculatus and Si. doliatus) bite only leaves. These findings caution against assumed homogeneity within this, and potentially other, functional groups on coral reefs. As functional redundancy within the macroalgal browsers is limited, the smaller `leaf-biting' species are unlikely to be able to compensate functionally for the loss of larger `entire thallus-biting' species.

  17. Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

    SciTech Connect

    Parent, Kristin N.; Tang, Jinghua; Cardone, Giovanni; Gilcrease, Eddie B.; Janssen, Mandy E.; Olson, Norman H.; Casjens, Sherwood R.; Baker, Timothy S.

    2014-09-15

    CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer.

  18. MASTICATORY FUNCTION OF OBESE CANDIDATES TO BARIATRIC SURGERY FROM DISTINCT SOCIOECONOMIC CLASSES

    PubMed Central

    PASSERI, Celso Roberto; ANDRADE, Jacira Alves Caracik de Camargo; TOMAL, Karla Thaíza; PRACUCHO, Eduardo Marcucci; de CAMPOS, Livia Paschoalino; SALES-PERES, Silvia Helena de Carvalho

    2016-01-01

    ABSTRACT Background: Obesity and metabolic syndrome can be labeled as worldwide outbreak; thus, both have led to serious public health problem. Oral health can be worsened by both, obesity and metabolic syndrome. Tooth loss harms masticatory function, essential status to whom will be submitted to bariatric surgery. Aim: Assess masticatory function of obese candidates to bariatric surgery, who belong to distinct socioeconomic class range, in order to recognize hazard factors and the bias of socioeconomic factor in this context. Methods: Observational cross-section study, with samples comprised by two groups of patients, with distinct socioeconomic class range, one of them belonging to public health system (SUSG) and the other to private clinic (CPG), candidates to bariatric surgery. Were assessed anthropometric data, comorbidities and medicines usage, blood tests, habits and the number of dental functional units. Results: The groups SUSG and CPG were homogeneous taking into account gender (p=0,890) and age range (p=0,170). The number of dental functional units was higher in the private group (p<0.001). The impaired masticatory function was rather present among public group (p<0.001) and female gender (p<0,001). Regarded as blood tests, fasting glucose was higher in female in SUSG (p<0,001). The following hazard factors have corroborated to have patients rated as impaired masticatory function: belong to public service (OR: 8.420, p=0.003), higher age (OR: 1.186, p<0.001), female gender (OR: 0.153, p=0.029), diabetes mellitus (OR: 2.545, p=0.045) and smokers (OR: 2.951, p=0.043). Conclusion: The general health and masticatory function of female SUSG were worse, highlighting the socioeconomic condition as hazard factor. PMID:27683777

  19. Representation, control, or reasoning? Distinct functions for theory of mind within the medial prefrontal cortex.

    PubMed

    Hartwright, Charlotte E; Apperly, Ian A; Hansen, Peter C

    2014-04-01

    The medial pFC (mPFC) is frequently reported to play a central role in Theory of Mind (ToM). However, the contribution of this large cortical region in ToM is not well understood. Combining a novel behavioral task with fMRI, we sought to demonstrate functional divisions between dorsal and rostral mPFC. All conditions of the task required the representation of mental states (beliefs and desires). The level of demands on cognitive control (high vs. low) and the nature of the demands on reasoning (deductive vs. abductive) were varied orthogonally between conditions. Activation in dorsal mPFC was modulated by the need for control, whereas rostral mPFC was modulated by reasoning demands. These findings fit with previously suggested domain-general functions for different parts of mPFC and suggest that these functions are recruited selectively in the service of ToM.

  20. The Structure of Treponema pallidum Tp0624 Reveals a Modular Assembly of Divergently Functionalized and Previously Uncharacterized Domains

    PubMed Central

    Wetherell, Charmaine; Cameron, Caroline E.

    2016-01-01

    Treponema pallidum subspecies pallidum is the causative agent of syphilis, a chronic, multistage, systemic infection that remains a major global health concern. The molecular mechanisms underlying T. pallidum pathogenesis are incompletely understood, partially due to the phylogenetic divergence of T. pallidum. One aspect of T. pallidum that differentiates it from conventional Gram-negative bacteria, and is believed to play an important role in pathogenesis, is its unusual cell envelope ultrastructure; in particular, the T. pallidum peptidoglycan layer is chemically distinct, thinner and more distal to the outer membrane. Established functional roles for peptidoglycan include contributing to the structural integrity of the cell envelope and stabilization of the flagellar motor complex, which are typically mediated by the OmpA domain-containing family of proteins. To gain insight into the molecular mechanisms that govern peptidoglycan binding and cell envelope biogenesis in T. pallidum we report here the structural characterization of the putative OmpA-like domain-containing protein, Tp0624. Analysis of the 1.70 Å resolution Tp0624 crystal structure reveals a multi-modular architecture comprised of three distinct domains including a C-terminal divergent OmpA-like domain, which we show is unable to bind the conventional peptidoglycan component diaminopimelic acid, and a previously uncharacterized tandem domain unit. Intriguingly, bioinformatic analysis indicates that the three domains together are found in all orthologs from pathogenic treponemes, but are not observed together in genera outside Treponema. These findings provide the first structural insight into a multi-modular treponemal protein containing an OmpA-like domain and its potential role in peptidoglycan coordination and stabilization of the T. pallidum cell envelope. PMID:27832149

  1. AINTEGUMENTA-LIKE genes have partly overlapping functions with AINTEGUMENTA but make distinct contributions to Arabidopsis thaliana flower development.

    PubMed

    Krizek, Beth A

    2015-08-01

    AINTEGUMENTA (ANT) is an important regulator of Arabidopsis flower development that has overlapping functions with the related AINTEGUMENTA-LIKE6 (AIL6) gene in floral organ initiation, identity specification, growth, and patterning. Two other AINTEGUMENTA-LIKE (AIL) genes, AIL5 and AIL7, are expressed in developing flowers in spatial domains that partly overlap with those of ANT. Here, it is shown that AIL5 and AIL7 also act in a partially redundant manner with ANT. The results demonstrate that AIL genes exhibit unequal genetic redundancy with roles for AIL5, AIL6, and AIL7 only revealed in the absence of ANT function. ant ail5 and ant ail7 double mutant flowers show alterations in floral organ positioning and growth, sepal fusion, and reductions in petal number. In ant ail5, petals are often replaced by filaments or dramatically reduced in size. ant ail7 double mutants produce increased numbers of carpels, which have defects in valve fusion and a loss of apical tissues. The distinct phenotypes of ant ail5, ant ail7 and the previously characterized ant ail6 indicate that AIL5, AIL6, and AIL7 make unique contributions to flower development. These distinct roles are also supported by genetic analyses of ant ail triple mutants. While ant ail5 ail6 triple mutants closely resemble ant ail6 double mutants, ant ail5 ail7 triple mutants exhibit more severe deviations from the wild type than either ant ail5 or ant ail7 double mutants. Furthermore, it is shown that AIL5, AIL6, and AIL7 act in a dose dependent manners in ant and other mutant backgrounds.

  2. Distinctive Feature of Microbial Communities and Bacterial Functional Profiles in Tricholoma matsutake Dominant Soil

    PubMed Central

    Oh, Seung-Yoon; Fong, Jonathan J.; Park, Myung Soo; Lim, Young Woon

    2016-01-01

    Tricholoma matsutake, the pine mushroom, is a valuable forest product with high economic value in Asia, and plays an important ecological role as an ectomycorrhizal fungus. Around the host tree, T. matsutake hyphae generate a distinctive soil aggregating environment called a fairy ring, where fruiting bodies form. Because T. matsutake hyphae dominate the soil near the fairy ring, this species has the potential to influence the microbial community. To explore the influence of T. matsutake on the microbial communities, we compared the microbial community and predicted bacterial function between two different soil types—T. matsutake dominant and T. matsutake minor. DNA sequence analyses showed that fungal and bacterial diversity were lower in the T. matsutake dominant soil compared to T. matsutake minor soil. Some microbial taxa were significantly more common in the T. matsutake dominant soil across geographic locations, many of which were previously identified as mycophillic or mycorrhiza helper bacteria. Between the two soil types, the predicted bacterial functional profiles (using PICRUSt) had significantly distinct KEGG modules. Modules for amino acid uptake, carbohydrate metabolism, and the type III secretion system were higher in the T. matsutake dominant soil than in the T. matsutake minor soil. Overall, similar microbial diversity, community structure, and bacterial functional profiles of the T. matsutake dominant soil across geographic locations suggest that T. matsutake may generate a dominance effect. PMID:27977803

  3. Three functionally distinct classes of C-fibre nociceptors in primates.

    PubMed

    Wooten, Matthew; Weng, Hao-Jui; Hartke, Timothy V; Borzan, Jasenka; Klein, Amanda H; Turnquist, Brian; Dong, Xinzhong; Meyer, Richard A; Ringkamp, Matthias

    2014-06-20

    In primates, C-fibre polymodal nociceptors are broadly classified into two groups based on mechanosensitivity. Here we demonstrate that mechanically sensitive polymodal nociceptors that respond either quickly (QC) or slowly (SC) to a heat stimulus differ in responses to a mild burn, heat sensitization, conductive properties and chemosensitivity. Superficially applied capsaicin and intradermal injection of β-alanine, an MrgprD agonist, excite vigorously all QCs. Only 40% of SCs respond to β-alanine, and their response is only half that of QCs. Mechanically insensitive C-fibres (C-MIAs) are β-alanine insensitive but vigorously respond to capsaicin and histamine with distinct discharge patterns. Calcium imaging reveals that β-alanine and histamine activate distinct populations of capsaicin-responsive neurons in primate dorsal root ganglion. We suggest that histamine itch and capsaicin pain are peripherally encoded in C-MIAs, and that primate polymodal nociceptive afferents form three functionally distinct subpopulations with β-alanine responsive QC fibres likely corresponding to murine MrgprD-expressing, non-peptidergic nociceptive afferents.

  4. Affinity for self antigen selects regulatory T cells with distinct functional properties

    PubMed Central

    Wyss, Lena; Stadinski, Brian D.; King, Carolyn G.; Schallenberg, Sonja; McCarthy, Nicholas I.; Lee, Jun Young; Kretschmer, Karsten; Terracciano, Luigi M.; Anderson, Graham; Surh, Charles D.; Huseby, Eric S.; Palmer, Ed

    2016-01-01

    How regulatory T cells (Treg cell) control lymphocyte homeostasis is not fully understood. Here we identify two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. Triplehi (GITRhiPD-1hiCD25hi) Treg cell are highly self-reactive and control lympho-proliferation in peripheral lymph nodes. Triplelo (GITRloPD-1loCD25lo) Treg cells are less self-reactive and limit development of colitis by promoting conversion of CD4+ Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (scurfy) mice lack Treg cells, they contain Triplehi-like and Triplelo-like CD4+ T cells with distinct pathological properties. Scurfy TriplehiCD4+T cells infiltrate the skin whereas scurfy TripleloCD4+T cells induce colitis and wasting disease. These findings indicate that T cell receptor affinity for self-antigens drives the differentiation of Tregs into distinct subsets with non-overlapping regulatory activities. PMID:27478940

  5. Genomic and functional characterization of the diverse immunoglobulin domain-containing protein (DICP) family

    PubMed Central

    Haire, Robert N.; Cannon, John P.; O’Driscoll, Marci L.; Ostrov, David A.; Mueller, M. Gail; Turner, Poem M.; Litman, Ronda T.; Litman, Gary W.; Yoder, Jeffrey A.

    2012-01-01

    A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs varies between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and effect parallel patterns of ligand recognition that potentially impact species-specific advantages. PMID:22386706

  6. Single-stranded DNA-binding proteins: multiple domains for multiple functions.

    PubMed

    Dickey, Thayne H; Altschuler, Sarah E; Wuttke, Deborah S

    2013-07-02

    The recognition of single-stranded DNA (ssDNA) is integral to myriad cellular functions. In eukaryotes, ssDNA is present stably at the ends of chromosomes and at some promoter elements. Furthermore, it is formed transiently by several cellular processes including telomere synthesis, transcription, and DNA replication, recombination, and repair. To coordinate these diverse activities, a variety of proteins have evolved to bind ssDNA in a manner specific to their function. Here, we review the recognition of ssDNA through the analysis of high-resolution structures of proteins in complex with ssDNA. This functionally diverse set of proteins arises from a limited set of structural motifs that can be modified and arranged to achieve distinct activities, including a range of ligand specificities. We also investigate the ways in which these domains interact in the context of large multidomain proteins/complexes. These comparisons reveal the structural features that define the range of functions exhibited by these proteins.

  7. Distinct functions of the RNA polymerase σ subunit region 3.2 in RNA priming and promoter escape

    PubMed Central

    Pupov, Danil; Kuzin, Ivan; Bass, Irina; Kulbachinskiy, Andrey

    2014-01-01

    The σ subunit of bacterial RNA polymerase (RNAP) has been implicated in all steps of transcription initiation, including promoter recognition and opening, priming of RNA synthesis, abortive initiation and promoter escape. The post-promoter-recognition σ functions were proposed to depend on its conserved region σ3.2 that directly contacts promoter DNA immediately upstream of the RNAP active centre and occupies the RNA exit path. Analysis of the transcription effects of substitutions and deletions in this region in Escherichia coli σ70 subunit, performed in this work, suggests that (i) individual residues in the σ3.2 finger collectively contribute to RNA priming by RNAP, likely by the positioning of the template DNA strand in the active centre, but are not critical to promoter escape; (ii) the physical presence of σ3.2 in the RNA exit channel is important for promoter escape; (iii) σ3.2 promotes σ dissociation during initiation and suppresses σ-dependent promoter-proximal pausing; (iv) σ3.2 contributes to allosteric inhibition of the initiating NTP binding by rifamycins. Thus, region σ3.2 performs distinct functions in transcription initiation and its inhibition by antibiotics. The B-reader element of eukaryotic factor TFIIB likely plays similar roles in RNAPII transcription, revealing common principles in transcription initiation in various domains of life. PMID:24452800

  8. Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

    PubMed Central

    Soh, Timothy K.

    2015-01-01

    ABSTRACT Vesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) with the large polymerase protein (L). To study the assembly of single viral particles, we tagged M and P with fluorescent proteins. We selected from a library of viruses with insertions in the M gene a replication-competent virus containing a fluorescent M and combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintained the same bullet shape appearance as wild-type VSV but had a modest increase in particle length, reflecting the increased genome size. Imaging of the released particles revealed a variation in the amount of M and P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the late domains in M, which serve to recruit the endosomal sorting complex required for transport (ESCRT) machinery during budding. Mutations in late domains resulted in the accumulation of virions that failed to pinch off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M tagged with enhanced green fluorescent protein and M tagged with mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late-domain mutant into the released virions. In contrast, the intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV. IMPORTANCE Assembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication

  9. Endocytotic routes of cobra cardiotoxins depend on spatial distribution of positively charged and hydrophobic domains to target distinct types of sulfated glycoconjugates on cell surface.

    PubMed

    Lee, Shao-Chen; Lin, Chien-Chu; Wang, Chia-Hui; Wu, Po-Long; Huang, Hsuan-Wei; Chang, Chung-I; Wu, Wen-guey

    2014-07-18

    Cobra cardiotoxins (CTX) are a family of three-fingered basic polypeptides known to interact with diverse targets such as heparan sulfates, sulfatides, and integrins on cell surfaces. After CTX bind to the membrane surface, they are internalized to intracellular space and exert their cytotoxicity via an unknown mechanism. By the combined in vitro kinetic binding, three-dimensional x-ray structure determination, and cell biology studies on the naturally abundant CTX homologues from the Taiwanese cobra, we showed that slight variations on the spatial distribution of positively charged or hydrophobic domains among CTX A2, A3, and A4 could lead to significant changes in their endocytotic pathways and action mechanisms via distinct sulfated glycoconjugate-mediated processes. The intracellular locations of these structurally similar CTX after internalization are shown to vary between the mitochondria and lysosomes via either dynamin2-dependent or -independent processes with distinct membrane cholesterol sensitivity. Evidence is presented to suggest that the shifting between the sulfated glycoconjugates as distinct targets of CTX A2, A3, and A4 might play roles in the co-evolutionary arms race between venomous snake toxins to cope with different membrane repair mechanisms at the cellular levels. The sensitivity of endocytotic routes to the spatial distribution of positively charged or hydrophobic domains may provide an explanation for the diverse endocytosis pathways of other cell-penetrating basic polypeptides.

  10. Function of the ATR N-terminal domain revealed by an ATM/ATR chimera

    SciTech Connect

    Chen Xinping; Zhao Runxiang; Glick, Gloria G.; Cortez, David . E-mail: david.cortez@vanderbilt.edu

    2007-05-01

    The ATM and ATR kinases function at the apex of checkpoint signaling pathways. These kinases share significant sequence similarity, phosphorylate many of the same substrates, and have overlapping roles in initiating cell cycle checkpoints. However, they sense DNA damage through distinct mechanisms. ATR primarily senses single stranded DNA (ssDNA) through its interaction with ATRIP, and ATM senses double strand breaks through its interaction with Nbs1. We determined that the N-terminus of ATR contains a domain that binds ATRIP. Attaching this domain to ATM allowed the fusion protein (ATM*) to bind ATRIP and associate with RPA-coated ssDNA. ATM* also gained the ability to localize efficiently to stalled replication forks as well as double strand breaks. Despite having normal kinase activity when tested in vitro and being phosphorylated on S1981 in vivo, ATM* is defective in checkpoint signaling and does not complement cellular deficiencies in either ATM or ATR. These data indicate that the N-terminus of ATR is sufficient to bind ATRIP and to promote localization to sites of replication stress.

  11. Distinct Molecular Evolutionary Mechanisms Underlie the Functional Diversification of the Wnt and TGFβ Signaling Pathways

    PubMed Central

    Konikoff, Charlotte E.; Wisotzkey, Robert G.; Stinchfield, Michael J.

    2010-01-01

    The canonical Wnt pathway is one of the oldest and most functionally diverse of animal intercellular signaling pathways. Though much is known about loss-of-function phenotypes for Wnt pathway components in several model organisms, the question of how this pathway achieved its current repertoire of functions has not been addressed. Our phylogenetic analyses of 11 multigene families from five species belonging to distinct phyla, as well as additional analyses employing the 12 Drosophila genomes, suggest frequent gene duplications affecting ligands and receptors as well as co-evolution of new ligand–receptor pairs likely facilitated the expansion of this pathway’s capabilities. Further, several examples of recent gene loss are visible in Drosophila when compared to family members in other phyla. By comparison the TGFβ signaling pathway is characterized by ancient gene duplications of ligands, receptors, and signal transducers with recent duplication events restricted to the vertebrate lineage. Overall, the data suggest that two distinct molecular evolutionary mechanisms can create a functionally diverse developmental signaling pathway. These are the recent dynamic generation of new genes and ligand–receptor interactions as seen in the Wnt pathway and the conservative adaptation of ancient pre-existing genes to new roles as seen in the TGFβ pathway. From a practical perspective, the former mechanism limits the investigator’s ability to transfer knowledge of specific pathway functions across species while the latter facilitates knowledge transfer. Electronic supplementary material The online version of this article (doi:10.1007/s00239-010-9337-z) contains supplementary material, which is available to authorized users. PMID:20339843

  12. Molecular basis of the functional distinction between Cln1 and Cln2 cyclins

    PubMed Central

    Quilis, Inma; Igual, Juan Carlos

    2012-01-01

    Cln1 and Cln2 are very similar but not identical cyclins. In this work, we tried to describe the molecular basis of the functional distinction between Cln1 and Cln2. We constructed chimeric cyclins containing different fragments of Cln1 and Cln2 and performed several functional analysis that make it possible to distinguish between Cln1 or Cln2. We identified that region between amino acids 225 and 299 of Cln2 is not only necessary but also sufficient to confer Cln2 specific functionality compared with Cln1. We also studied Cln1 and Cln2 subcellular localization identifying additional differences between them. Both cyclins are distributed between the nucleus and the cytoplasm, but Cln1 shows stronger nuclear accumulation. Nuclear import of both cyclins is mediated by the classical nuclear import pathway and by sequences in the N-terminal end of the proteins. For Cln2, but not for Cln1, a nuclear export mechanism mediated by karyopherin Msn5 has been identified. Strikingly, Cln2 export depends on a Msn5-dependent NES between amino acids 225 and 299. In fact, the introduction of this region confers to Cln1 an export mechanism dependent on Msn5; importantly, this causes the gain of Cln2-specific cytosolic functions and the impairment of nuclear function. In short, a region from Cln2 controlling an Msn5-dependent nuclear export mechanism confers a specific functionality to Cln2 compared with Cln1. PMID:22889732

  13. Distinct domains of the spinal muscular atrophy protein SMN are required for targeting to Cajal bodies in mammalian cells.

    PubMed

    Renvoisé, Benoît; Khoobarry, Kevinee; Gendron, Marie-Claude; Cibert, Christian; Viollet, Louis; Lefebvre, Suzie

    2006-02-15

    Mutations of the survival motor neuron gene SMN1 cause the inherited disease spinal muscular atrophy (SMA). The ubiquitous SMN protein facilitates the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs). The protein is detected in the cytoplasm, nucleoplasm and enriched with snRNPs in nuclear Cajal bodies. It is structurally divided into at least an amino-terminal region rich in basic amino acid residues, a central Tudor domain, a self-association tyrosine-glycine-box and an exon7-encoded C-terminus. To examine the domains required for the intranuclear localization of SMN, we have used fluorescently tagged protein mutants transiently overexpressed in mammalian cells. The basic amino acid residues direct nucleolar localization of SMN mutants. The Tudor domain promotes localization of proteins in the nucleus and it cooperates with the basic amino acid residues and the tyrosine-glycine-box for protein localization in Cajal bodies. Moreover, the most frequent disease-linked mutant SMNDeltaex7 reduces accumulation of snRNPs in Cajal bodies, suggesting that the C-terminus of SMN participates in targeting to Cajal bodies. A reduced number of Cajal bodies in patient fibroblasts associates with the absence of snRNPs in Cajal bodies, revealing that intranuclear snRNA organization is modified in disease. These results indicate that direct and indirect mechanisms regulate localization of SMN in Cajal bodies.

  14. Geographically Distinct and Domain-Specific Sequence Variations in the Alleles of Rice Blast Resistance Gene Pib

    PubMed Central

    Vasudevan, Kumar; Vera Cruz, Casiana M.; Gruissem, Wilhelm; Bhullar, Navreet K.

    2016-01-01

    Rice blast is caused by Magnaporthe oryzae, which is the most destructive fungal pathogen affecting rice growing regions worldwide. The rice blast resistance gene Pib confers broad-spectrum resistance against Southeast Asian M. oryzae races. We investigated the allelic diversity of Pib in rice germplasm originating from 12 major rice growing countries. Twenty-five new Pib alleles were identified that have unique single nucleotide polymorphisms (SNPs), insertions and/or deletions, in addition to the polymorphic nucleotides that are shared between the different alleles. These partially or completely shared polymorphic nucleotides indicate frequent sequence exchange events between the Pib alleles. In some of the new Pib alleles, nucleotide diversity is high in the LRR domain, whereas, in others it is distributed among the NB-ARC and LRR domains. Most of the polymorphic amino acids in LRR and NB-ARC2 domains are predicted as solvent-exposed. Several of the alleles and the unique SNPs are country specific, suggesting a diversifying selection of alleles in various geographical locations in response to the locally prevalent M. oryzae population. Together, the new Pib alleles are an important genetic resource for rice blast resistance breeding programs and provide new information on rice-M. oryzae interactions at the molecular level. PMID:27446145

  15. Geographically Distinct and Domain-Specific Sequence Variations in the Alleles of Rice Blast Resistance Gene Pib.

    PubMed

    Vasudevan, Kumar; Vera Cruz, Casiana M; Gruissem, Wilhelm; Bhullar, Navreet K

    2016-01-01

    Rice blast is caused by Magnaporthe oryzae, which is the most destructive fungal pathogen affecting rice growing regions worldwide. The rice blast resistance gene Pib confers broad-spectrum resistance against Southeast Asian M. oryzae races. We investigated the allelic diversity of Pib in rice germplasm originating from 12 major rice growing countries. Twenty-five new Pib alleles were identified that have unique single nucleotide polymorphisms (SNPs), insertions and/or deletions, in addition to the polymorphic nucleotides that are shared between the different alleles. These partially or completely shared polymorphic nucleotides indicate frequent sequence exchange events between the Pib alleles. In some of the new Pib alleles, nucleotide diversity is high in the LRR domain, whereas, in others it is distributed among the NB-ARC and LRR domains. Most of the polymorphic amino acids in LRR and NB-ARC2 domains are predicted as solvent-exposed. Several of the alleles and the unique SNPs are country specific, suggesting a diversifying selection of alleles in various geographical locations in response to the locally prevalent M. oryzae population. Together, the new Pib alleles are an important genetic resource for rice blast resistance breeding programs and provide new information on rice-M. oryzae interactions at the molecular level.

  16. Two functionally distinct kinetochore pools of BubR1 ensure accurate chromosome segregation

    PubMed Central

    Zhang, Gang; Mendez, Blanca Lopez; Sedgwick, Garry G.; Nilsson, Jakob

    2016-01-01

    The BubR1/Bub3 complex is an important regulator of chromosome segregation as it facilitates proper kinetochore–microtubule interactions and is also an essential component of the spindle assembly checkpoint (SAC). Whether BubR1/Bub3 localization to kinetochores in human cells stimulates SAC signalling or only contributes to kinetochore–microtubule interactions is debated. Here we show that two distinct pools of BubR1/Bub3 exist at kinetochores and we uncouple these with defined BubR1/Bub3 mutants to address their function. The major kinetochore pool of BubR1/Bub3 is dependent on direct Bub1/Bub3 binding and is required for chromosome alignment but not for the SAC. A distinct pool of BubR1/Bub3 localizes by directly binding to phosphorylated MELT repeats on the outer kinetochore protein KNL1. When we prevent the direct binding of BubR1/Bub3 to KNL1 the checkpoint is weakened because BubR1/Bub3 is not incorporated into checkpoint complexes efficiently. In conclusion, kinetochore localization supports both known functions of BubR1/Bub3. PMID:27457023

  17. Hierarchical modularity in ERα transcriptional network is associated with distinct functions and implicates clinical outcomes.

    PubMed

    Tang, Binhua; Hsu, Hang-Kai; Hsu, Pei-Yin; Bonneville, Russell; Chen, Su-Shing; Huang, Tim H-M; Jin, Victor X

    2012-01-01

    Recent genome-wide profiling reveals highly complex regulation networks among ERα and its targets. We integrated estrogen (E2)-stimulated time-series ERα ChIP-seq and gene expression data to identify the ERα-centered transcription factor (TF) hubs and their target genes, and inferred the time-variant hierarchical network structures using a Bayesian multivariate modeling approach. With its recurrent motif patterns, we determined three embedded regulatory modules from the ERα core transcriptional network. The GO analyses revealed the distinct biological function associated with each of three embedded modules. The survival analysis showed the genes in each module were able to render a significant survival correlation in breast cancer patient cohorts. In summary, our Bayesian statistical modeling and modularity analysis not only reveals the dynamic properties of the ERα-centered regulatory network and associated distinct biological functions, but also provides a reliable and effective genomic analytical approach for the analysis of dynamic regulatory network for any given TF.

  18. Regional functional connectivity predicts distinct cognitive impairments in Alzheimer's disease spectrum.

    PubMed

    Ranasinghe, Kamalini G; Hinkley, Leighton B; Beagle, Alexander J; Mizuiri, Danielle; Dowling, Anne F; Honma, Susanne M; Finucane, Mariel M; Scherling, Carole; Miller, Bruce L; Nagarajan, Srikantan S; Vossel, Keith A

    2014-01-01

    Understanding neural network dysfunction in neurodegenerative disease is imperative to effectively develop network-modulating therapies. In Alzheimer's disease (AD), cognitive decline associates with deficits in resting-state functional connectivity of diffuse brain networks. The goal of the current study was to test whether specific cognitive impairments in AD spectrum correlate with reduced functional connectivity of distinct brain regions. We recorded resting-state functional connectivity of alpha-band activity in 27 patients with AD spectrum--22 patients with probable AD (5 logopenic variant primary progressive aphasia, 7 posterior cortical atrophy, and 10 early-onset amnestic/dysexecutive AD) and 5 patients with mild cognitive impairment due to AD. We used magnetoencephalographic imaging (MEGI) to perform an unbiased search for regions where patterns of functional connectivity correlated with disease severity and cognitive performance. Functional connectivity measured the strength of coherence between a given region and the rest of the brain. Decreased neural connectivity of multiple brain regions including the right posterior perisylvian region and left middle frontal cortex correlated with a higher degree of disease severity. Deficits in executive control and episodic memory correlated with reduced functional connectivity of the left frontal cortex, whereas visuospatial impairments correlated with reduced functional connectivity of the left inferior parietal cortex. Our findings indicate that reductions in region-specific alpha-band resting-state functional connectivity are strongly correlated with, and might contribute to, specific cognitive deficits in AD spectrum. In the future, MEGI functional connectivity could be an important biomarker to map and follow defective networks in the early stages of AD.

  19. Distinct patterns of functional brain connectivity correlate with objective performance and subjective beliefs

    PubMed Central

    Barttfeld, Pablo; Wicker, Bruno; McAleer, Phil; Belin, Pascal; Cojan, Yann; Graziano, Martín; Leiguarda, Ramón; Sigman, Mariano

    2013-01-01

    The degree of correspondence between objective performance and subjective beliefs varies widely across individuals. Here we demonstrate that functional brain network connectivity measured before exposure to a perceptual decision task covaries with individual objective (type-I performance) and subjective (type-II performance) accuracy. Increases in connectivity with type-II performance were observed in networks measured while participants directed attention inward (focus on respiration), but not in networks measured during states of neutral (resting state) or exogenous attention. Measures of type-I performance were less sensitive to the subjects’ specific attentional states from which the networks were derived. These results suggest the existence of functional brain networks indexing objective performance and accuracy of subjective beliefs distinctively expressed in a set of stable mental states. PMID:23801762

  20. Functional chromatography reveals three natural products that target the same protein with distinct mechanisms of action

    PubMed Central

    Kang, MinJin; Wu, Tongde; Wijeratne, E. M. Kithsiri; Lau, Eric C.; Mason, Damian J.; Mesa, Celestina; Tillotson, Joseph; Zhang, Donna D.; Gunatilaka, A. A. Leslie; La Clair, James J.

    2014-01-01

    Access to lead compounds with defined molecular targets continues to be a barrier to the translation of natural product resources. As a solution, we have developed a system that uses discreet, recombinant proteins as the vehicles for natural product isolation. Here, we describe the use of this functional chromatographic method to identify natural products that bind to the AAA+ chaperone, p97, a promising cancer target. Application of this method to a panel of fungal and plant extracts identified rheoemodin, 1-hydroxydehydroherbarin and phomapyrrolidone A as distinct p97 modulators. Excitingly, each of these molecules displayed a unique mechanism of p97 modulation. This discovery provides strong support for the application of functional chromatography to the discovery of protein modulators that would likely escape traditional high-throughput or phenotypic screening platforms. PMID:25125376

  1. Generation of a Functionally Distinct Rhizopus oryzae Lipase through Protein Folding Memory.

    PubMed

    Satomura, Atsushi; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2015-01-01

    Rhizopus oryzae lipase (ROL) has a propeptide at its N-terminus that functions as an intramolecular chaperone and facilitates the folding of mature ROL (mROL). In this study, we successfully generated a functionally distinct imprinted mROL (mROLimp) through protein folding memory using a mutated propeptide. The mutated propeptide left its structural memory on mROL and produced mROLimp that exhibited different substrate specificities compared with mROLWT (prepared from the wild type propeptide), although the amino acid sequences of both mROLs were the same. mROLimp showed a preference for substrates with medium chain-length acyl groups and, noticeably, recognized a peptidase-specific substrate. In addition, ROLimp was more stable than mROLWT. These results strongly suggest that proteins with identical amino acid sequences can fold into different conformations and that mutations in intramolecular chaperones can dynamically induce changes in enzymatic activity.

  2. Regularized Laplace-Fourier-Domain Full Waveform Inversion Using a Weighted l 2 Objective Function

    NASA Astrophysics Data System (ADS)

    Jun, Hyunggu; Kwon, Jungmin; Shin, Changsoo; Zhou, Hongbo; Cogan, Mike

    2017-03-01

    Full waveform inversion (FWI) can be applied to obtain an accurate velocity model that contains important geophysical and geological information. FWI suffers from the local minimum problem when the starting model is not sufficiently close to the true model. Therefore, an accurate macroscale velocity model is essential for successful FWI, and Laplace-Fourier-domain FWI is appropriate for obtaining such a velocity model. However, conventional Laplace-Fourier-domain FWI remains an ill-posed and ill-conditioned problem, meaning that small errors in the data can result in large differences in the inverted model. This approach also suffers from certain limitations related to the logarithmic objective function. To overcome the limitations of conventional Laplace-Fourier-domain FWI, we introduce a weighted l 2 objective function, instead of the logarithmic objective function, as the data-domain objective function, and we also introduce two different model-domain regularizations: first-order Tikhonov regularization and prior model regularization. The weighting matrix for the data-domain objective function is constructed to suitably enhance the far-offset information. Tikhonov regularization smoothes the gradient, and prior model regularization allows reliable prior information to be taken into account. Two hyperparameters are obtained through trial and error and used to control the trade-off and achieve an appropriate balance between the data-domain and model-domain gradients. The application of the proposed regularizations facilitates finding a unique solution via FWI, and the weighted l 2 objective function ensures a more reasonable residual, thereby improving the stability of the gradient calculation. Numerical tests performed using the Marmousi synthetic dataset show that the use of the weighted l 2 objective function and the model-domain regularizations significantly improves the Laplace-Fourier-domain FWI. Because the Laplace-Fourier-domain FWI is improved, the

  3. Regularized Laplace-Fourier-Domain Full Waveform Inversion Using a Weighted l 2 Objective Function

    NASA Astrophysics Data System (ADS)

    Jun, Hyunggu; Kwon, Jungmin; Shin, Changsoo; Zhou, Hongbo; Cogan, Mike

    2016-09-01

    Full waveform inversion (FWI) can be applied to obtain an accurate velocity model that contains important geophysical and geological information. FWI suffers from the local minimum problem when the starting model is not sufficiently close to the true model. Therefore, an accurate macroscale velocity model is essential for successful FWI, and Laplace-Fourier-domain FWI is appropriate for obtaining such a velocity model. However, conventional Laplace-Fourier-domain FWI remains an ill-posed and ill-conditioned problem, meaning that small errors in the data can result in large differences in the inverted model. This approach also suffers from certain limitations related to the logarithmic objective function. To overcome the limitations of conventional Laplace-Fourier-domain FWI, we introduce a weighted l 2 objective function, instead of the logarithmic objective function, as the data-domain objective function, and we also introduce two different model-domain regularizations: first-order Tikhonov regularization and prior model regularization. The weighting matrix for the data-domain objective function is constructed to suitably enhance the far-offset information. Tikhonov regularization smoothes the gradient, and prior model regularization allows reliable prior information to be taken into account. Two hyperparameters are obtained through trial and error and used to control the trade-off and achieve an appropriate balance between the data-domain and model-domain gradients. The application of the proposed regularizations facilitates finding a unique solution via FWI, and the weighted l 2 objective function ensures a more reasonable residual, thereby improving the stability of the gradient calculation. Numerical tests performed using the Marmousi synthetic dataset show that the use of the weighted l 2 objective function and the model-domain regularizations significantly improves the Laplace-Fourier-domain FWI. Because the Laplace-Fourier-domain FWI is improved, the

  4. Understory plant communities and the functional distinction between savanna trees, forest trees, and pines

    SciTech Connect

    Veldman, Joseph W.; Mattingly, W. Brett; Brudvig, Lars A.

    2013-02-01

    Although savanna trees and forest trees are thought to represent distinct functional groups with different effects on ecosystem processes, few empirical studies have examined these effects. In particular, it remains unclear if savanna and forest trees differ in their ability to coexist with understory plants, which comprise the majority of plant diversity in most savannas. We used structural equation modeling (SEM) and data from 157 sites across three locations in the southeastern United States to understand the effects of broadleaf savanna trees, broadleaf forest trees, and pine trees on savanna understory plant communities. After accounting for underlying gradients in fire frequency and soil moisture, abundances (i.e., basal area and stem density) of forest trees and pines, but not savanna trees, were negatively correlated with the cover and density (i.e., local-scale species richness) of C4 graminoid species, a defining savanna understory functional group that is linked to ecosystem flammability. In analyses of the full understory community, abundances of trees from all functional groups were negatively correlated with species density and cover. For both the C4 and full communities, fire frequency promoted understory plants directly, and indirectly by limiting forest tree abundance. There was little indirect influence of fire on the understory mediated through savanna trees and pines, which are more fire tolerant than forest trees. We conclude that tree functional identity is an important factor that influences overstory tree relationships with savanna understory plant communities. In particular, distinct relationships between trees and C4 graminoids have implications for grass-tree coexistence and vegetation-fire feedbacks that maintain savanna environments and their associated understory plant diversity.

  5. Common and Distinct Amygdala-Function Perturbations in Depressed vs Anxious Adolescents

    PubMed Central

    Beesdo, Katja; Lau, Jennifer Y. F.; Guyer, Amanda E.; McClure-Tone, Erin B.; Monk, Christopher S.; Nelson, Eric E.; Fromm, Stephen J.; Goldwin, Michelle A.; Wittchen, Hans-Ulrich; Leibenluft, Ellen; Ernst, Monique; Pine, Daniel S.

    2010-01-01

    Context Few studies directly compare amygdala function in depressive and anxiety disorders. Data from longitudinal research emphasize the need for such studies in adolescents. Objective To compare amygdala response to varying attention and emotion conditions among adolescents with major depressive disorder (MDD) or anxiety disorders, relative to adolescents with no psychopathology. Design Case-control study. Setting Government clinical research institute. Participants Eighty-seven adolescents matched on age, sex, intelligence, and social class: 26 with MDD (14 with and 12 without anxiety disorders), 16 with anxiety disorders but no depression, and 45 without psychopathology. Main Outcome Measures Blood oxygen level–dependent signal in the amygdala, measured by means of event-related functional magnetic resonance imaging. During imaging, participants viewed facial expressions (neutral, fearful, angry, and happy) while attention was constrained (afraid, hostility, and nose-width ratings) or unconstrained (passive viewing). Results Left and right amygdala activation differed as a function of diagnosis, facial expression, and attention condition both when patients with comorbid MDD and anxiety were included and when they were excluded (group × emotion × attention interactions, P≤.03). Focusing on fearful face–viewing events, patients with anxiety and those with MDD both differed in amygdala responses from healthy participants and from each other during passive viewing. However, both MDD and anxiety groups, relative to healthy participants, exhibited similar signs of amygdala hyperactivation to fearful faces when subjectively experienced fear was rated. Conclusions Adolescent MDD and anxiety disorders exhibit common and distinct functional neural correlates during face processing. Attention modulates the degree to which common or distinct amygdala perturbations manifest in these patient groups, relative to healthy peers. PMID:19255377

  6. Understory plant communities and the functional distinction between savanna trees, forest trees, and pines.

    PubMed

    Veldman, Joseph W; Mattingly, W Brett; Brudvig, Lars A

    2013-02-01

    Although savanna trees and forest trees are thought to represent distinct functional groups with different effects on ecosystem processes, few empirical studies have examined these effects. In particular, it remains unclear if savanna and forest trees differ in their ability to coexist with understory plants, which comprise the majority of plant diversity in most savannas. We used structural equation modeling (SEM) and data from 157 sites across three locations in the southeastern United States to understand the effects of broadleaf savanna trees, broadleaf forest trees, and pine trees on savanna understory plant communities. After accounting for underlying gradients in fire frequency and soil moisture, abundances (i.e., basal area and stem density) of forest trees and pines, but not savanna trees, were negatively correlated with the cover and density (i.e., local-scale species richness) of C4 graminoid species, a defining savanna understory functional group that is linked to ecosystem flammability. In analyses of the full understory community, abundances of trees from all functional groups were negatively correlated with species density and cover. For both the C4 and full communities, fire frequency promoted understory plants directly, and indirectly by limiting forest tree abundance. There was little indirect influence of fire on the understory mediated through savanna trees and pines, which are morefire tolerant than forest trees. We conclude that tree functional identity is an important factor that influences overstory tree relationships with savanna understory plant communities. In particular, distinct relationships between trees and C4 graminoids have implications for grass-tree coexistence and vegetation-fire feedbacks that maintain savanna environments and their associated understory plant diversity.

  7. Structural and functional analysis of the pro-domain of human cathelicidin, LL-37

    PubMed Central

    Pazgier, Marzena; Ericksen, Bryan; Ling, Minhua; Toth, Eric; Shi, Jishu; Li, Xiangdong; Galliher-Beckley, Amy; Lan, Liqiong; Zou, Guozhang; Zhan, Changyou; Yuan, Weirong; Pozharski, Edwin; Lu, Wuyuan

    2013-01-01

    Cathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large precursor molecules with a highly conserved pro-domain known as the cathelin-like domain (CLD). CLDs have high degrees of sequence homology to cathelin, a protein isolated from pig leukocytes and belonging to the cystatin family of cysteine protease inhibitors. In this report, we describe for the first time the X-ray crystal structure of the human CLD (hCLD) of the sole human cathelicidin, LL-37. The structure of hCLD, determined at 1.93 Å resolution, shows the cystatin-like fold and is highly similar to the structure of the CLD of the pig cathelicidin, protegrin-3. We assayed the in vitro antibacterial activities of hCLD, LL-37 and the precursor form, pro-cathelicidin (also known as hCAP18), and we found that the unprocessed protein inhibited the growth of Gram-negative bacteria with efficiencies comparable to the mature peptide, LL-37. In addition, the antibacterial activity of LL-37 was not inhibited by hCLD intermolecularly, since exogenously added hCLD had no effect on the bactericidal activity of the mature peptide. hCLD itself lacked antimicrobial function and did not inhibit the cysteine protease, cathepsin L. Our results contrast with previous reports of hCLD activity. A comparative structural analysis between hCLD and the cysteine protease inhibitor stefin A showed why hCLD is unable to function as an inhibitor of cysteine proteases. In this respect, the cystatin scaffold represents an ancestral structural platform from which proteins evolved divergently, with some losing inhibitory functions. PMID:23406372

  8. Three acetylcholinesterases of the pinewood nematode, Bursaphelenchus xylophilus: insights into distinct physiological functions.

    PubMed

    Kang, Jae Soon; Lee, Dae-Weon; Choi, Jae Young; Je, Yeon Ho; Koh, Young Ho; Lee, Si Hyeock

    2011-02-01

    Acetylcholinesterase (AChE) plays a key role in postsynaptic transmission in most animals. Nematodes encode multiple AChEs, implying its functional diversity. To explore physiological functions of multiple AChEs, three distinct AChEs (BxACE-1, BxACE-2, and BxACE-3) were identified and characterized from the pinewood nematode. Sequencing comparison with Torpedo AChE and Caenorhabditis elegans ACEs identified choline-binding site, catalytic triad functional site, three internal disulfide bonds and aromatic residues for the catalytic gorge. Transcriptional profiling by quantitative real-time PCR revealed that BxACE-3 is more actively transcribed than BxACE-1 (2-3 times) and BxACE-2 (9-18 times) in both propagative and dispersal stages. The three BxACEs were functionally expressed using baculovirus system. Kinetic analysis of in vitro-expressed BxACEs revealed that the substrate specificity was highest in BxACE-1 whereas the catalytic efficiency was highest in BxACE-2. In inhibition assay, BxACE-3 showed the lowest inhibition rate. Taken together, it appears that both BxACE-1 and BxACE-2 play common but non-overlapping roles in synaptic transmission, whereas BxACE-3 may have non-neuronal functions. The current findings should provide valuable insights into the evolutionary process and various physiological roles of AChE.

  9. Computational Prediction of Protein Function Based on Weighted Mapping of Domains and GO Terms

    PubMed Central

    Teng, Zhixia; Guo, Maozu; Dai, Qiguo; Wang, Chunyu; Li, Jin; Liu, Xiaoyan

    2014-01-01

    In this paper, we propose a novel method, SeekFun, to predict protein function based on weighted mapping of domains and GO terms. Firstly, a weighted mapping of domains and GO terms is constructed according to GO annotations and domain composition of the proteins. The association strength between domain and GO term is weighted by symmetrical conditional probability. Secondly, the mapping is extended along the true paths of the terms based on GO hierarchy. Finally, the terms associated with resident domains are transferred to host protein and real annotations of the host protein are determined by association strengths. Our careful comparisons demonstrate that SeekFun outperforms the concerned methods on most occasions. SeekFun provides a flexible and effective way for protein function prediction. It benefits from the well-constructed mapping of domains and GO terms, as well as the reasonable strategy for inferring annotations of protein from those of its domains. PMID:24868539

  10. Functional domains of the Xenopus replication licensing factor Cdt1.

    PubMed

    Ferenbach, Andrew; Li, Anatoliy; Brito-Martins, Marta; Blow, J Julian

    2005-01-01

    During late mitosis and early G1, replication origins are licensed for subsequent replication by loading heterohexamers of the mini-chromosome maintenance proteins (Mcm2-7). To prevent re-replication of DNA, the licensing system is down-regulated at other cell cycle stages. A small protein called geminin plays an important role in this down-regulation by binding and inhibiting the Cdt1 component of the licensing system. We examine here the organization of Xenopus Cdt1, delimiting regions of Cdt1 required for licensing and regions required for geminin interaction. The C-terminal 377 residues of Cdt1 are required for licensing and the extreme C-terminus contains a domain that interacts with an Mcm(2,4,6,7) complex. Two regions of Cdt1 interact with geminin: one at the N-terminus, and one in the centre of the protein. Only the central region binds geminin tightly enough to successfully compete with full-length Cdt1 for geminin binding. This interaction requires a predicted coiled-coil domain that is conserved amongst metazoan Cdt1 homologues. Geminin forms a homodimer, with each dimer binding one molecule of Cdt1. Separation of the domains necessary for licensing activity from domains required for a strong interaction with geminin generated a construct, whose licensing activity was partially insensitive to geminin inhibition.

  11. Blood pressure control has distinct effects on executive function, attention, memory and markers of cerebrovascular damage.

    PubMed

    Semplicini, A; Inverso, G; Realdi, A; Macchini, L; Maraffon, M; Puato, M; Zanardo, M; Tirrito, C; Amodio, P; Schiff, S; Mapelli, D; Manara, R

    2011-02-01

    Hypertension causes cognitive impairment, involving mainly executive functions, but the effect of blood pressure (BP) control on the different cognitive domains is still debated. We correlated executive function, attention and memory with BP control and cerebrovascular damage in 60 undemented middle-aged hypertensives at baseline and after 6-year follow-up. At first evaluation, the patients with poor BP control had higher score of white matter lesions, reduced cerebrovascular reserve capacity and greater carotid intima-media thickness (IMT) than those with good BP control. Performance on executive tests correlated with IMT and with performance on attention tests, which was impaired by low diastolic BP. At long-term follow-up, performance in attention and executive tests improved in spite of the minor improvement of BP control, increased IMT and worse memory. Low diastolic BP has a negative effect on attention, which affects executive performance at first cross-sectional examination. This confounding effect has to be taken into consideration when planning studies on cognitive function. Longitudinal studies are required to unravel the effect of BP control on cognitive function, as only long-term antihypertensive treatment improves both attention and executive performance.

  12. Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability

    PubMed Central

    Yoshida, Keisuke; Hisabori, Toru

    2016-01-01

    The thiol-based redox regulation system is believed to adjust chloroplast functions in response to changes in light environments. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been traditionally considered to serve as a transmitter of light signals to target enzymes. However, emerging data indicate that chloroplasts have a complex redox network composed of diverse redox-mediator proteins and target enzymes. Despite extensive research addressing this system, two fundamental questions are still unresolved: How are redox pathways orchestrated within chloroplasts, and why are chloroplasts endowed with a complicated redox network? In this report, we show that NADPH-Trx reductase C (NTRC) is a key redox-mediator protein responsible for regulatory functions distinct from those of the classically known FTR/Trx system. Target screening and subsequent biochemical assays indicated that NTRC and the Trx family differentially recognize their target proteins. In addition, we found that NTRC is an electron donor to Trx-z, which is a key regulator of gene expression in chloroplasts. We further demonstrate that cooperative control of chloroplast functions via the FTR/Trx and NTRC pathways is essential for plant viability. Arabidopsis double mutants impaired in FTR and NTRC expression displayed lethal phenotypes under autotrophic growth conditions. This severe growth phenotype was related to a drastic loss of photosynthetic performance. These combined results provide an expanded map of the chloroplast redox network and its biological functions. PMID:27335455

  13. Functional analyses of two cellular binding domains of bovine lactadherin.

    PubMed

    Andersen, M H; Graversen, H; Fedosov, S N; Petersen, T E; Rasmussen, J T

    2000-05-23

    The glycoprotein bovine lactadherin (formerly known as PAS-6/7) comprises two EGF-like domains and two C-like domains found in blood clotting factors V and VIII. Bovine lactadherin binds to alpha(v)beta(5) integrin in an RGD-dependent manner and also to phospholipids, especially phosphatidyl serine. To define and characterize these bindings the interactions between lactadherin and different mammalian cell types were investigated. Using recombinant forms of bovine lactadherin, the human breast carcinomas MCF-7 cells expressing the alpha(v)beta(5) integrin receptor were shown to bind specifically to RGD containing lactadherin but not to a mutated RGE lactadherin. A monoclonal antibody against the alpha(v)beta(5) integrin receptor and a synthetic RGD-containing peptide inhibited the adhesion of MCF-7 cells to lactadherin. Green monkey kidney MA-104 cells, also expressing the alpha(v)beta(3) together with the alpha(v)beta(5) integrin, showed binding to bovine lactadherin via both integrins. To investigate the interaction of lipid with lactadherin two fragments were expressed corresponding to the C1C2 domains and the C2 domain. Both fragments bound to phosphatidyl serine in a concentration-dependent manner with an affinity similar to native lactadherin (K(d) = 1.8 nM). A peptide corresponding to the C-terminal part of the C2 domain inhibited the binding of lactadherin to phospholipid in a concentration-dependent manner, and finally it was shown that lactadherin mediates binding between artificial phosphatidyl serine membranes and MCF-7 cells. Taken together these results show that lactadherin can act as link between two surfaces by binding to integrin receptors through its N-terminus and to phospholipids through its C-terminus.

  14. Affinity for self antigen selects Treg cells with distinct functional properties.

    PubMed

    Wyss, Lena; Stadinski, Brian D; King, Carolyn G; Schallenberg, Sonja; McCarthy, Nicholas I; Lee, Jun Young; Kretschmer, Karsten; Terracciano, Luigi M; Anderson, Graham; Surh, Charles D; Huseby, Eric S; Palmer, Ed

    2016-09-01

    The manner in which regulatory T cells (Treg cells) control lymphocyte homeostasis is not fully understood. We identified two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. We found that GITR(hi)PD-1(hi)CD25(hi) (Triple(hi)) Treg cells were highly self-reactive and controlled lympho-proliferation in peripheral lymph nodes. GITR(lo)PD-1(lo)CD25(lo) (Triple(lo)) Treg cells were less self-reactive and limited the development of colitis by promoting the conversion of CD4(+) Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (Scurfy) mice lacked Treg cells, they contained Triple(hi)-like and Triple(lo)-like CD4(+) T cells zsuper> T cells infiltrated the skin, whereas Scurfy Triple(lo)CD4(+) T cells induced colitis and wasting disease. These findings indicate that the affinity of the T cell antigen receptor for self antigen drives the differentiation of Treg cells into distinct subsets with non-overlapping regulatory activities.

  15. Distinct Roles of the Repeat-Containing Regions and Effector Domains of the Vibrio vulnificus Multifunctional-Autoprocessing Repeats-in-Toxin (MARTX) Toxin

    PubMed Central

    Kim, Byoung Sik; Gavin, Hannah E.

    2015-01-01

    ABSTRACT Vibrio vulnificus is a seafood-borne pathogen that destroys the intestinal epithelium, leading to rapid bacterial dissemination and death. The most important virulence factor is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin comprised of effector domains in the center region flanked by long repeat-containing regions which are well conserved among MARTX toxins and predicted to translocate effector domains. Here, we examined the role of the repeat-containing regions using a modified V. vulnificus MARTX (MARTXVv) toxin generated by replacing all the internal effector domains with β-lactamase (Bla). Bla activity was detected in secretions from the bacterium and also in the cytosol of intoxicated epithelial cells. The modified MARTXVv toxin without effector domains retained its necrotic activity but lost its cell-rounding activity. Further, deletion of the carboxyl-terminal repeat-containing region blocked toxin secretion from the bacterium. Deletion of the amino-terminal repeat-containing region had no effect on secretion but completely abolished translocation and necrosis. Neither secretion nor translocation was affected by enzymatically inactivating the cysteine protease domain of the toxin. These data demonstrate that the amino-terminal and carboxyl-terminal repeat-containing regions of the MARTXVv toxin are necessary and sufficient for the delivery of effector domains and epithelial cell lysis in vitro but that effector domains are required for other cytopathic functions. Furthermore, Ca2+-dependent secretion of the modified MARTXVv toxin suggests that nonclassical RTX-like repeats found in the carboxyl-terminal repeat-containing region are functionally similar to classical RTX repeats found in other RTX proteins. PMID:25827415

  16. Functional analysis of ligand-binding and signal transduction domains of CD69 and CD23 C-type lectin leukocyte receptors.

    PubMed

    Sancho, D; Santis, A G; Alonso-Lebrero, J L; Viedma, F; Tejedor, R; Sánchez-Madrid, F

    2000-10-01

    CD69 and CD23 are leukocyte receptors with distinctive pattern of cell expression and functional features that belong to different C-type lectin receptor subfamilies. To assess the functional equivalence of different domains of these structurally related proteins, a series of CD69/CD23 chimeras exchanging the carbohydrate recognition domain, the neck region, and the transmembrane and cytoplasmic domains were generated. Biochemical analysis revealed the importance of the neck region (Cys68) in the dimerization of CD69. Functional analysis of these chimeras in RBL-2H3 mast cells and Jurkat T cell lines showed the interchangeability of structural domains of both proteins regarding Ca2+ fluxes, serotonin release, and TNF-alpha synthesis. The type of the signal transduced mainly relied on the cytoplasmic domain and was independent of receptor oligomerization. The cytoplasmic domain of CD69 transduced a Ca2+-mediated signaling that was dependent on the extracellular uptake of Ca2+. Furthermore, a significant production of TNF-alpha was induced through the cytoplasmic domain of CD69 in RBL-2H3 cells, which was additive to that promoted via FcepsilonRI, thus suggesting a role for CD69 in the late phase of reactions mediated by mast cells. Our results provide new important data on the functional equivalence of homologous domains of these two leukocyte receptors.

  17. 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

  18. 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.

  19. Alternate transcripts of a floral developmental regulator have both distinct and redundant functions in opium poppy

    PubMed Central

    Hands, Philip; Vosnakis, Nikolaos; Betts, Donna; Irish, Vivian F.; Drea, Sinéad

    2011-01-01

    Background and Aims The MADS-box transcription factor AGAMOUS (AG) is an important regulator of stamen and fruit identity as well as floral meristem determinacy in a number of core eudicots and monocots. However, its role outside of these groups has not been assessed explicitly. Examining its role in opium poppy, a basal eudicot, could uncover much about the evolution and development of flower and fruit development in the angiosperms. Methods AG orthologues were isolated by degenerate RT-PCR and the gene sequence and structure examined; gene expression was characterized using in situ hybridization and the function assessed using virus-induced gene silencing. Key Results In opium poppy, a basal eudicot, the AGAMOUS orthologue is alternatively spliced to produce encoded products that vary at the C-terminus, termed PapsAG-1 and PapsAG-2. Both transcripts are expressed at high levels in stamens and carpels. The functional implications of this alternative transcription were examined using virus-induced gene silencing and the results show that PapsAG-1 has roles in stamen and carpel identity, reflecting those found for Arabidopsis AG. In contrast, PapsAG-2, while displaying redundancy in these functions, has a distinctive role in aspects of carpel development reflected in septae, ovule and stigma defects seen in the loss-of-function line generated. Conclusions These results describe the first explicit functional analysis of an AG-clade gene in a basal eudicot; illustrate one of the few examples of the functional consequences of alternative splicing in transcription factors and reveal the importance of alternative transcription, as well as gene duplication, as a driving force in evolution. PMID:21385783

  20. The N- and C-Terminal Domains Differentially Contribute to the Structure and Function of Dystrophin and Utrophin Tandem Calponin-Homology Domains.

    PubMed

    Singh, Surinder M; Bandi, Swati; Mallela, Krishna M G

    2015-11-24

    Dystrophin and utrophin are two muscle proteins involved in Duchenne/Becker muscular dystrophy. Both proteins use tandem calponin-homology (CH) domains to bind to F-actin. We probed the role of N-terminal CH1 and C-terminal CH2 domains in the structure and function of dystrophin tandem CH domain and compared with our earlier results on utrophin to understand the unifying principles of how tandem CH domains work. Actin cosedimentation assays indicate that the isolated CH2 domain of dystrophin weakly binds to F-actin compared to the full-length tandem CH domain. In contrast, the isolated CH1 domain binds to F-actin with an affinity similar to that of the full-length tandem CH domain. Thus, the obvious question is why the dystrophin tandem CH domain requires CH2, when its actin binding is determined primarily by CH1. To answer, we probed the structural stabilities of CH domains. The isolated CH1 domain is very unstable and is prone to serious aggregation. The isolated CH2 domain is very stable, similar to the full-length tandem CH domain. These results indicate that the main role of CH2 is to stabilize the tandem CH domain structure. These conclusions from dystrophin agree with our earlier results on utrophin, indicating that this phenomenon of differential contribution of CH domains to the structure and function of tandem CH domains may be quite general. The N-terminal CH1 domains primarily determine the actin binding function whereas the C-terminal CH2 domains primarily determine the structural stability of tandem CH domains, and the extent of stabilization depends on the strength of inter-CH domain interactions.

  1. Gain-of-function SOS1 mutations cause a distinctive form of noonansyndrome

    SciTech Connect

    Tartaglia, Marco; Pennacchio, Len A.; Zhao, Chen; Yadav, KamleshK.; Fodale, Valentina; Sarkozy, Anna; Pandit, Bhaswati; Oishi, Kimihiko; Martinelli, Simone; Schackwitz, Wendy; Ustaszewska, Anna; Martin, Joes; Bristow, James; Carta, Claudio; Lepri, Francesca; Neri, Cinzia; Vasta,Isabella; Gibson, Kate; Curry, Cynthia J.; Lopez Siguero, Juan Pedro; Digilio, Maria Cristina; Zampino, Giuseppe; Dallapiccola, Bruno; Bar-Sagi, Dafna; Gelb, Brude D.

    2006-09-01

    Noonan syndrome (NS) is a developmental disordercharacterized by short stature, facial dysmorphia, congenital heartdefects and skeletal anomalies1. Increased RAS-mitogenactivated proteinkinase (MAPK) signaling due to PTPN11 and KRAS mutations cause 50 percentof NS2-6. Here, we report that 22 of 129 NS patients without PTPN11 orKRAS mutation (17 percent) have missense mutations in SOS1, which encodesa RAS-specific guanine nucleotide exchange factor (GEF). SOS1 mutationscluster at residues implicated in the maintenance of SOS1 in itsautoinhibited form and ectopic expression of two NS-associated mutantsinduced enhanced RAS activation. The phenotype associated with SOS1defects is distinctive, although within NS spectrum, with a highprevalence of ectodermal abnormalities but generally normal developmentand linear growth. Our findings implicate for the first timegain-of-function mutations in a RAS GEF in inherited disease and define anew mechanism by which upregulation of the RAS pathway can profoundlychange human development.

  2. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms

    SciTech Connect

    Kober, Daniel L.; Alexander-Brett, Jennifer M.; Karch, Celeste M.; Cruchaga, Carlos; Colonna, Marco; Holtzman, Michael J.; Brett, Thomas J.

    2016-12-20

    Genetic variations in the myeloid immune receptor TREM2 are linked to several neurodegenerative diseases. To determine how TREM2 variants contribute to these diseases, we performed structural and functional studies of wild-type and variant proteins. Our 3.1 Å TREM2 crystal structure revealed that mutations found in Nasu-Hakola disease are buried whereas Alzheimer’s disease risk variants are found on the surface, suggesting that these mutations have distinct effects on TREM2 function. Biophysical and cellular methods indicate that Nasu-Hakola mutations impact protein stability and decrease folded TREM2 surface expression, whereas Alzheimer’s risk variants impact binding to a TREM2 ligand. Additionally, the Alzheimer’s risk variants appear to epitope map a functional surface on TREM2 that is unique within the larger TREM family. These findings provide a guide to structural and functional differences among genetic variants of TREM2, indicating that therapies targeting the TREM2 pathway should be tailored to these genetic and functional differences with patient-specific medicine approaches for neurodegenerative disorders.

  3. Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms

    PubMed Central

    Kober, Daniel L; Alexander-Brett, Jennifer M; Karch, Celeste M; Cruchaga, Carlos; Colonna, Marco; Holtzman, Michael J; Brett, Thomas J

    2016-01-01

    Genetic variations in the myeloid immune receptor TREM2 are linked to several neurodegenerative diseases. To determine how TREM2 variants contribute to these diseases, we performed structural and functional studies of wild-type and variant proteins. Our 3.1 Å TREM2 crystal structure revealed that mutations found in Nasu-Hakola disease are buried whereas Alzheimer’s disease risk variants are found on the surface, suggesting that these mutations have distinct effects on TREM2 function. Biophysical and cellular methods indicate that Nasu-Hakola mutations impact protein stability and decrease folded TREM2 surface expression, whereas Alzheimer’s risk variants impact binding to a TREM2 ligand. Additionally, the Alzheimer’s risk variants appear to epitope map a functional surface on TREM2 that is unique within the larger TREM family. These findings provide a guide to structural and functional differences among genetic variants of TREM2, indicating that therapies targeting the TREM2 pathway should be tailored to these genetic and functional differences with patient-specific medicine approaches for neurodegenerative disorders. DOI: http://dx.doi.org/10.7554/eLife.20391.001 PMID:27995897

  4. Generation of functionally distinct isoforms of PTBP3 by alternative splicing and translation initiation

    PubMed Central

    Tan, Lit-Yeen; Whitfield, Peter; Llorian, Miriam; Monzon-Casanova, Elisa; Diaz-Munoz, Manuel D.; Turner, Martin; Smith, Christopher W.J.

    2015-01-01

    Polypyrimidine tract binding protein (PTBP1) is a widely expressed RNA binding protein that acts as a regulator of alternative splicing and of cytoplasmic mRNA functions. Vertebrates contain two closely-related paralogs with >75% amino acid sequence identity. Early replacement of PTBP1 by PTBP2 during neuronal differentiation causes a concerted set of splicing changes. By comparison, very little is known about the molecular functions or physiological roles of PTBP3, although its expression and conservation throughout the vertebrates suggest a role in haematopoietic cells. To begin to understand its functions we have characterized the mRNA and protein isoform repertoire of PTBP3. Combinatorial alternative splicing events at the 5′ end of the gene allow for the generation of eight mRNA and three major protein isoforms. Individual mRNAs generate up to three protein isoforms via alternative translation initiation by re-initiation and leaky scanning using downstream AUG codons. The N-terminally truncated PTBP3 isoforms lack nuclear localization signals and/or most of the RRM1 domain and vary in their RNA binding properties and nuclear/cytoplasmic distribution, suggesting that PTBP3 may have major post-transcriptional cytoplasmic roles. Our findings set the stage for understanding the non-redundant physiological roles of PTBP3. PMID:25940628

  5. CD6-ligand interactions: a paradigm for SRCR domain function?

    PubMed

    Aruffo, A; Bowen, M A; Patel, D D; Haynes, B F; Starling, G C; Gebe, J A; Bajorath, J

    1997-10-01

    The scavenger receptor cysteine-rich (SRCR) superfamily, which includes proteins expressed by leukocytes, can be subdivided into groups A and B. Group B contains the lymphocyte cell-surface receptor CD6. This article reviews recent progress in understanding the interaction between CD6 and its ligand, activated leukocyte cell adhesion molecule (ALCAM). Analysis of the CD6-ALCAM interaction may help to understand how other SRCR domains bind to their ligands.

  6. Mineral and organic growing media have distinct community structure, stability and functionality in soilless culture systems

    PubMed Central

    Grunert, Oliver; Hernandez-Sanabria, Emma; Vilchez-Vargas, Ramiro; Jauregui, Ruy; Pieper, Dietmar H.; Perneel, Maaike; Van Labeke, Marie-Christine; Reheul, Dirk; Boon, Nico

    2016-01-01

    The choice of soilless growing medium for plant nutrition, growth and support is crucial for improving the eco-sustainability of the production in horticultural systems. As our current understanding of the functional microbial communities inhabiting this ecosystem is still limited, we examined the microbial community development of the two most important growing media (organic and mineral) used in open soilless horticultural systems. We aimed to identify factors that influence community composition over time, and to compare the distribution of individual taxa across growing media, and their potential functionality. High throughput sequencing analysis revealed a distinctive and stable microbial community in the organic growing medium. Humidity, pH, nitrate-N, ammonium-N and conductivity were uncovered as the main factors associated with the resident bacterial communities. Ammonium-N was correlated with Rhizobiaceae abundance, while potential competitive interactions among both Methylophilaceae and Actinobacteridae with Rhizobiaceae were suggested. Our results revealed that soilless growing media are unique niches for diverse bacterial communities with temporal functional stability, which may possibly impact the resistance to external forces. These differences in communities can be used to develop strategies to move towards a sustainable horticulture with increased productivity and quality. PMID:26728128

  7. Analysis of regulatory network topology reveals functionally distinct classes of microRNAs

    PubMed Central

    Yu, Xueping; Lin, Jimmy; Zack, Donald J.; Mendell, Joshua T.; Qian, Jiang

    2008-01-01

    MicroRNAs (miRNAs) negatively regulate the expression of target genes at the post-transcriptional level. Little is known about the crosstalk between miRNAs and transcription factors (TFs). Here we provide data suggesting that the interaction patterns between TFs and miRNAs can influence the biological functions of miRNAs. From this global survey, we find that a regulated feedback loop, in which two TFs regulate each other and one miRNA regulates both of the factors, is the most significantly overrepresented network motif. Mathematical modeling shows that the miRNA in this motif stabilizes the feedback loop to resist environmental perturbation, providing one mechanism to explain the robustness of developmental programs that is contributed by miRNAs. Furthermore, on the basis of a network motif profile analysis, we demonstrate the existence of two classes of miRNAs with distinct network topological properties. The first class of miRNAs is regulated by a large number of TFs, whereas the second is regulated by only a few TFs. The differential expression level of the two classes of miRNAs in embryonic developmental stages versus adult tissues suggests that the two classes may have fundamentally different biological functions. Our results demonstrate that the TFs and miRNAs extensively interact with each other and the biological functions of miRNAs may be wired in the regulatory network topology. PMID:18927108

  8. The PRE and PQ box are functionally distinct yeast pheromone response elements.

    PubMed Central

    Sengupta, P; Cochran, B H

    1990-01-01

    Saccharomyces cerevisiae mating pheromones function by binding to cell surface receptors and activating signal transduction processes which regulate gene expression. In this report, we have analyzed the minimum sequence requirements for conferring both a and alpha mating pheromone inducibilities onto a heterologous promoter. Here we show that the repetitive pheromone response element (PRE) which binds to STE12 protein is sufficient to confer pheromone responsiveness only when present in multiple copies. Moreover, by itself, it is preferentially responsive to alpha factor in a cells. In contrast, a single copy of the PQ box of the STE3 upstream activation sequence (UAS) is sufficient to confer a-factor responsiveness in alpha cells. The PQ box binds both MCM1 and MAT alpha 1 in a cooperative manner, and neither the P nor Q site alone is sufficient to confer a-factor responsiveness. In a cells, however, even multiple copies of the PQ box fail to confer alpha-factor responsiveness. Therefore, the PRE and the PQ box are functionally distinct pheromone-responsive elements with opposite cell type specificities. Moreover, these results indicate that the MCM1 protein functions in a signal transduction pathway in a manner analogous to that of its mammalian homolog, the serum response factor, which regulates the expression of the c-fos proto-oncogene in mammals. PMID:2247085

  9. Linking structure and function in food webs: maximization of different ecological functions generates distinct food web structures.

    PubMed

    Yen, Jian D L; Cabral, Reniel B; Cantor, Mauricio; Hatton, Ian; Kortsch, Susanne; Patrício, Joana; Yamamichi, Masato

    2016-03-01

    Trophic interactions are central to ecosystem functioning, but the link between food web structure and ecosystem functioning remains obscure. Regularities (i.e. consistent patterns) in food web structure suggest the possibility of regularities in ecosystem functioning, which might be used to relate structure to function. We introduce a novel, genetic algorithm approach to simulate food webs with maximized throughput (a proxy for ecosystem functioning) and compare the structure of these simulated food webs to real empirical food webs using common metrics of food web structure. We repeat this analysis using robustness to secondary extinctions (a proxy for ecosystem resilience) instead of throughput to determine the relative contributions of ecosystem functioning and ecosystem resilience to food web structure. Simulated food webs that maximized robustness were similar to real food webs when connectance (i.e. levels of interaction across the food web) was high, but this result did not extend to food webs with low connectance. Simulated food webs that maximized throughput or a combination of throughput and robustness were not similar to any real food webs. Simulated maximum-throughput food webs differed markedly from maximum-robustness food webs, which suggests that maximizing different ecological functions can generate distinct food web structures. Based on our results, food web structure would appear to have a stronger relationship with ecosystem resilience than with ecosystem throughput. Our genetic algorithm approach is general and is well suited to large, realistically complex food webs. Genetic algorithms can incorporate constraints on structure and can generate outputs that can be compared directly to empirical data. Our method can be used to explore a range of maximization or minimization hypotheses, providing new perspectives on the links between structure and function in ecological systems.

  10. 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

  11. Molecular and functional characterization of two distinct IGF binding protein-6 genes in zebrafish.

    PubMed

    Wang, Xianlei; Lu, Ling; Li, Yun; Li, Mingyu; Chen, Chen; Feng, Qiang; Zhang, Chunyang; Duan, Cunming

    2009-05-01

    Insulin-like growth factor binding proteins (IGFBPs) are high-affinity binding partners for IGFs and play important roles in modulating IGF activities. In this study, we have identified and characterized two functional IGFBP-6 genes in zebrafish. Structural, phylogenetic, and comparative genomic analyses indicate that they are co-orthologs of the human IGFBP-6 gene. To gain insight into how the duplicated genes may have evolved through partitioning of ancestral functions, gene expression and functional studies were carried out. In adult fish, IGFBP-6a mRNA was most abundantly expressed in the muscle. The levels of IGFBP-6a mRNA in nonmuscle tissues were very low or barely detectable. In comparison, the levels of IGFBP-6b mRNA were high in the brain, heart, and muscle, but very low or undetectable in other adult tissues. During embryogenesis, the IGFBP-6a mRNA levels were relatively low. The IGFBP-6b mRNA levels were low during the initial 48 h. They became significantly higher at 72 and 96 h postfertilization. Overexpression of zebrafish IGFBP-6a and IGFBP-6b caused a similar degree of reduction in body size and developmental rate. No notable effects were observed on cell fate or patterning in these transgenic fish. These data suggest that the duplicated igfbp-6 genes encode two functionally similar proteins, but they have evolved distinct spatial and temporal expression patterns. These findings are consistent with the notion of an additional gene duplication event in teleost fish and have provided novel insight into the structural and functional evolution of the IGFBP gene family.

  12. Nitrification rates in Arctic soils are associated with functionally distinct populations of ammonia-oxidizing archaea

    NASA Astrophysics Data System (ADS)

    Alves, Ricardo J. E.; Wanek, Wolfgang; Zappe, Anna; Richter, Andreas; Svenning, Mette M.; Schleper, Christa; Urich, Tim

    2014-05-01

    The functioning of Arctic soil ecosystems is crucially important for global climate, although basic knowledge regarding their biogeochemical processes is lacking. Nitrogen (N) is the major limiting nutrient in these environments, and therefore it is particularly important to gain a better understanding of the microbial populations catalyzing transformations that influence N bioavailability. However, microbial communities driving this process remain largely uncharacterized in Arctic soils, namely those catalyzing the rate-limiting step of ammonia (NH3) oxidation. Eleven Arctic soils from Svalbard were analyzed through a polyphasic approach, including determination of gross nitrification rates through a 15N pool dilution method, qualitative and quantitative analyses of ammonia-oxidizing archaea (AOA) and bacteria (AOB) populations based on the functional marker gene amoA (encoding the ammonia monooxygenase subunit A), and enrichment of AOA in laboratory cultures. AOA were the only NH3 oxidizers detected in five out of 11 soils, and outnumbered AOB by 1 to 3 orders of magnitude in most others. AOA showed a great overall phylogenetic diversity that was differentially distributed across soil ecosystems, and exhibited an uneven population composition that reflected the dominance of a single AOA phylotype in each population. Moreover, AOA populations showed a multifactorial association with the soil properties, which reflected an overall distribution associated with tundra type and with several physico-chemical parameters combined, namely pH and soil moisture and N contents (i.e., NO3- and dissolved organic N). Remarkably, the different gross in situ and potential nitrification rates between soils were associated with distinct AOA phylogenetic clades, suggesting differences in their nitrifying potential, both under the native NH3 conditions and as a response to higher NH3 availability. This was further supported by the selective enrichment of two AOA clades that exhibited

  13. cDNA cloning reveals two mouse beta5 integrin transcripts distinct in cytoplasmic domains as a result of alternative splicing.

    PubMed Central

    Zhang, H; Tan, S M; Lu, J

    1998-01-01

    The integrin beta5 subunit has only been found to form a heterodimer with subunit alphav which acts as a vitronectin receptor. Integrin alphavbeta5 has been implicated in cell migration and growth factor-induced angiogenesis. In the present study, a mouse liver cDNA library was screened using a human beta5 cDNA fragment obtained by reverse transcriptase PCR (RT-PCR). Three of the clones (MB5, MB15 and MB17) overlapped to give an open reading frame, called beta5A, which is homologous to the human beta5 subunit. The sequence of another clone (MB26), called beta5B, was identical with beta5A, except for a deletion of 29 bp near the 3' end of the open reading frame. The 29 bp deletion resulted in an open-reading-frame shift and a completely different C-terminal sequence in beta5B. beta5A and beta5B were shown, by RT-PCR, to be co-expressed in most mouse tissues tested, although beta5B mRNA was detected at much lower levels than beta5A. beta5A and beta5B mRNAs were also detected in the mouse monocytic cell line, J774, and in isolated mouse peritoneal macrophages. Adhesion of peritoneal macrophages has been shown to up-regulate the expression of both beta5A and beta5B mRNAs. The 29 bp sequence begins with a putative intron-splicing donor site (GTGAT...). A 3' fragment of the mouse integrin beta5 gene was cloned by PCR and sequenced showing that the 29 bp sequence was also immediately followed by an intron. Therefore, the 29 bp sequence was apparently expressed as part of the beta5A mRNA but was spliced out as part of the downstream intron in beta5B. Since the cytoplasmic domains of the integrin beta subunits are important in cytoskeleton attachment and signalling, the two alternatively spliced beta5 isoforms may have distinct roles in cell adhesion and other cellular functions. PMID:9531507

  14. Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability

    PubMed Central

    Klebba, Joseph E.; Galletta, Brian J.; Nye, Jonathan; Plevock, Karen M.; Buster, Daniel W.; Hollingsworth, Natalie A.; Slep, Kevin C.

    2015-01-01

    Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4’s tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl–Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification. PMID:25688134

  15. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families.

    PubMed Central

    Gilkes, N R; Henrissat, B; Kilburn, D G; Miller, R C; Warren, R A

    1991-01-01

    Several types of domain occur in beta-1, 4-glycanases. The best characterized of these are the catalytic domains and the cellulose-binding domains. The domains may be joined by linker sequences rich in proline or hydroxyamino acids or both. Some of the enzymes contain repeated sequences up to 150 amino acids in length. The enzymes can be grouped into families on the basis of sequence similarities between the catalytic domains. There are sequence similarities between the cellulose-binding domains, of which two types have been identified, and also between some domains of unknown function. The beta-1, 4-glycanases appear to have arisen by the shuffling of a relatively small number of progenitor sequences. PMID:1886523

  16. 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

  17. Cell-to-cell movement of potato virus X involves distinct functions of the coat protein.

    PubMed

    Fedorkin, O; Solovyev, A; Yelina, N; Zamyatnin, A; Zinovkin, R; Mäkinen, K; Schiemann, J; Yu Morozov, S

    2001-02-01

    Complementation of movement-deficient potato virus X (PVX) coat protein (CP) mutants, namely PVX.CP-Xho lacking the 18 C-terminal amino acid residues and PVX.DeltaCP lacking the entire CP gene, was studied by transient co-expression with heterologous proteins. These data demonstrated that the potyvirus CPs and both the major and minor CPs of beet yellows closterovirus could complement cell-to-cell movement of PVX.CP-Xho but not PVX.DeltaCP. These data also indicated that the C-terminally truncated PVX CP lacked a movement function which could be provided in trans by the CPs of other filamentous viruses, whereas another movement determinant specified by some region outside the most C-terminal part of the PVX CP could not be complemented either by potyvirus or closterovirus CPs. Surprisingly, the CP of spherical cocksfoot mottle sobemovirus rescued all of the PVX CP movement functions, complementing the spread of PVX.CP-Xho and, to a lesser extent, PVX.DeltaCP. Both these mutants were also rescued by the tobacco mosaic virus (TMV) movement protein (MP). To shed light on the movement function of PVX CP, attempts were made to complement PVX.CP-Xho by a series of TMV MP mutants. An internal deletion abolished complementation, suggesting that the internal region of TMV MP, which includes a number of overlapping functional domains important for cell-to-cell transport, provides an activity complementing movement determinant(s) specified by the C-terminal region of PVX CP.

  18. Distinct functional connectivity of the hippocampus during semantic and phonemic fluency.

    PubMed

    Glikmann-Johnston, Yifat; Oren, Noga; Hendler, Talma; Shapira-Lichter, Irit

    2015-03-01

    Verbal fluency tasks are typically used in neuropsychological practice for assessment of language function in a variety of neurological disorders. Recently, it has been shown that the hippocampus, a region thought to be exclusive to the domain of memory, is also involved in tests of semantic fluency. The present study further explores hippocampal contribution to verbal fluency using functional Magnetic Resonance Imaging (fMRI) and examining mean activity and inter-regional functional connectivity with known task-related brain regions. Given the clear lateralization of brain areas involved in language, lateralization of hippocampal involvement in semantic and phonemic word fluency was also investigated. Different hippocampal recruitment during semantic and phonemic fluency was found: greater change in activity was seen during semantic fluency, as compared with phonemic fluency. This pattern was obtained in the right and the left hippocampus, with no lateralization effects. Functional connectivity analyses corroborate the notion of selective contribution of the hippocampus to semantic fluency. During the semantic fluency task, connectivity levels between the hippocampi and components of the semantic network did not differ from connectivity levels within the semantic network. In contrast, during the phonemic fluency task, the hippocampi were less correlated with components of the phonemic network, as compared to the within phonemic network connectivity. Importantly, hippocampal connectivity with the semantic network was task-dependent and restricted to periods of semantic fluency performance. Altogether, results suggest that the right and the left hippocampus are integral components of the brain network that selectively supports verbal semantic fluency, but not phonemic fluency.

  19. Processes of fungal proteome evolution and gain of function: gene duplication and domain rearrangement

    NASA Astrophysics Data System (ADS)

    Cohen-Gihon, Inbar; Sharan, Roded; Nussinov, Ruth

    2011-06-01

    During evolution, organisms have gained functional complexity mainly by modifying and improving existing functioning systems rather than creating new ones ab initio. Here we explore the interplay between two processes which during evolution have had major roles in the acquisition of new functions: gene duplication and protein domain rearrangements. We consider four possible evolutionary scenarios: gene families that have undergone none of these event types; only gene duplication; only domain rearrangement, or both events. We characterize each of the four evolutionary scenarios by functional attributes. Our analysis of ten fungal genomes indicates that at least for the fungi clade, species significantly appear to gain complexity by gene duplication accompanied by the expansion of existing domain architectures via rearrangements. We show that paralogs gaining new domain architectures via duplication tend to adopt new functions compared to paralogs that preserve their domain architectures. We conclude that evolution of protein families through gene duplication and domain rearrangement is correlated with their functional properties. We suggest that in general, new functions are acquired via the integration of gene duplication and domain rearrangements rather than each process acting independently.

  20. Impact of Distinct Poxvirus Infections on the Specificities and Functionalities of CD4+ T Cell Responses

    PubMed Central

    Siciliano, Nicholas A.; Hersperger, Adam R.; Lacuanan, Aimee M.; Xu, Ren-Huan; Sidney, John; Sette, Alessandro; Sigal, Luis J.

    2014-01-01

    ABSTRACT The factors that determine CD4+ T cell (TCD4+) specificities, functional capacity, and memory persistence in response to complex pathogens remain unclear. We explored these parameters in the C57BL/6 mouse through comparison of two highly related (>92% homology) poxviruses: ectromelia virus (ECTV), a natural mouse pathogen, and vaccinia virus (VACV), a heterologous virus that nevertheless elicits potent immune responses. In addition to elucidating several previously unidentified major histocompatibility complex class II (MHC-II)-restricted epitopes, we observed many qualitative and quantitative differences between the TCD4+ repertoires, including responses not elicited by VACV despite complete sequence conservation. In addition, we observed functional heterogeneity between ECTV- and VACV-specific TCD4+ at both a global and individual epitope level, particularly greater expression of the cytolytic marker CD107a from TCD4+ following ECTV infection. Most striking were differences during the late memory phase where, in contrast to ECTV, VACV infection failed to elicit measurable epitope-specific TCD4+ as determined by intracellular cytokine staining. These findings illustrate the strong influence of epitope-extrinsic factors on TCD4+ responses and memory. IMPORTANCE Much of our understanding concerning host-pathogen relationships in the context of poxvirus infections stems from studies of VACV in mice. However, VACV is not a natural mouse pathogen, and therefore, the relevance of results obtained using this model may be limited. Here, we explored the MHC class II-restricted TCD4+ repertoire induced by mousepox (ECTV) infection and the functional profile of the responding epitope-specific TCD4+, comparing these results to those induced by VACV infection under matched conditions. Despite a high degree of homology between the two viruses, we observed distinct specificity and functional profiles of TCD4+ responses at both acute and memory time points, with VACV

  1. The expression pattern of genes involved in early neurogenesis suggests distinct and conserved functions in the diplopod Glomeris marginata.

    PubMed

    Pioro, Hilary L; Stollewerk, Angelika

    2006-01-01

    We have shown recently that the expression and function of proneural genes is conserved in chelicerates and myriapods, although groups of neural precursors are specified in the ventral neuroectoderm of these arthropod groups, rather than single cells as in insects and crustaceans. We present additional evidence that the pattern of neurogenesis seen in chelicerates and in previously analyzed myriapod species is representative of both arthropod groups, by analysing the formation of neural precursors in the diplopod Archispirostreptus sp. This raises the question as to what extent the genetic network has been modified to result in different modes of neurogenesis in the arthropod group. To find out which components of the neural genetic network might account for the different mode of neural precursor formation in chelicerates and myriapods, we identified genes in the diplopod Glomeris marginata that are known to be involved in early neurogenesis in Drosophila and studied their expression pattern. In Drosophila, early neurogenesis is controlled by proneural genes that encode HLH transcription factors. These genes belong to two major subfamilies, the achaete-scute group and the atonal group. Different proneural proteins activate both a common neural programme and distinct neuronal subtype-specific target genes. We show that the expression pattern of homologs of the Drosophila proneural genes daughterless, atonal, and Sox B1 are partially conserved in Glomeris mariginata. While the expression of the pan-neural gene snail is conserved in the ventral neuroectoderm of G. marginata, we found an additional expression domain in the ventral midline. We conclude that, although the components of the genetic network involved in specification of neural precursors seem to be conserved in chelicerates, myriapods, and Drosophila, the function of some of the genes might have changed during evolution.

  2. The RST and PARP-like domain containing SRO protein family: analysis of protein structure, function and conservation in land plants

    PubMed Central

    2010-01-01

    Background The SROs (SIMILAR TO RCD-ONE) are a group of plant-specific proteins which have important functions in stress adaptation and development. They contain the catalytic core of the poly(ADP-ribose) polymerase (PARP) domain and a C-terminal RST (RCD-SRO-TAF4) domain. In addition to these domains, several, but not all, SROs contain an N-terminal WWE domain. Results SROs are present in all analyzed land plants and sequence analysis differentiates between two structurally distinct groups; cryptogams and monocots possess only group I SROs whereas eudicots also contain group II. Group I SROs possess an N-terminal WWE domain (PS50918) but the WWE domain is lacking in group II SROs. Group I domain structure is widely represented in organisms as distant as humans (for example, HsPARP11). We propose a unified nomenclature for the SRO family. The SROs are able to interact with transcription factors through the C-terminal RST domain but themselves are generally not regulated at the transcriptional level. The most conserved feature of the SROs is the catalytic core of the poly(ADP-ribose) polymerase (PS51059) domain. However, bioinformatic analysis of the SRO PARP domain fold-structure and biochemical assays of AtRCD1 suggested that SROs do not possess ADP-ribosyl transferase activity. Conclusions The SROs are a highly conserved family of plant specific proteins. Sequence analysis of the RST domain implicates a highly preserved protein structure in that region. This might have implications for functional conservation. We suggest that, despite the presence of the catalytic core of the PARP domain, the SROs do not possess ADP-ribosyl transferase activity. Nevertheless, the function of SROs is critical for plants and might be related to transcription factor regulation and complex formation. PMID:20226034

  3. The novel finding of four distinct prepro-IGF-I E domains in a perciform fish, Sciaenops ocellatus, during ontogeny.

    PubMed

    Faulk, Cynthia K; Pérez-Domínguez, Rafael; Webb, Kenneth A; Holt, G Joan

    2010-10-01

    In fishes, insulin-like growth factor-I (IGF-I) stimulates growth and differentiation but also plays a role in a number of other processes including osmoregulation, metabolism, immune response and reproduction. This study presents the cDNA encoding multiple prepro-IGF-I transcripts obtained from red drum, Sciaenopsocellatus, and examines differential expression in select adult tissues and during ontogeny. Four distinct transcripts were sequenced which were identical in the coding region for the signal (132 bp) and mature (204 bp) peptides but differed in the coding region of the E peptide by the exclusion of 117 (Ea-1), 81 (Ea-2) or 36 (Ea-3) bp compared to the 222 bp present in Ea-4. Analysis of the pertinent portion of the genomic sequence of this gene suggests that the transcripts are a result of alternative splicing. This is the first report of the expression of all four known prepro-IGF-I transcripts in a teleost other than a salmonid. The deduced amino acid sequences exhibited 70-95% identity with teleosts and somewhat lower identity to other vertebrates (60-75%). Three of the 4 transcripts (Ea-2, Ea-3, Ea-4) were expressed in the liver, ovary, spleen, gall bladder, brain, red muscle, pancreas and spinal cord of adults. Only the Ea-4 transcript was expressed in adult stomach tissue while no signal was detected in pituitary, retina, intestine, adipose or white muscle. In contrast, all 4 transcripts were expressed throughout ontogeny. The apparent expression of the Ea-1 transcript only during the larval stage may indicate a developmental role for this E peptide in red drum.

  4. Phosphorylation of Synaptojanin Differentially Regulates Endocytosis of Functionally Distinct Synaptic Vesicle Pools

    PubMed Central

    Geng, Junhua; Wang, Liping; Lee, Joo Yeun; Chen, Chun-Kan

    2016-01-01

    The rapid replenishment of synaptic vesicles through endocytosis is crucial for sustaining synaptic transmission during intense neuronal activity. Synaptojanin (Synj), a phosphoinositide phosphatase, is known to play an important role in vesicle recycling by promoting the uncoating of clathrin following synaptic vesicle uptake. Synj has been shown to be a substrate of the minibrain (Mnb) kinase, a fly homolog of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A); however, the functional impacts of Synj phosphorylation by Mnb are not well understood. Here we identify that Mnb phosphorylates Synj at S1029 in Drosophila. We find that phosphorylation of Synj at S1029 enhances Synj phosphatase activity, alters interaction between Synj and endophilin, and promotes efficient endocytosis of the active cycling vesicle pool (also referred to as exo-endo cycling pool) at the expense of reserve pool vesicle endocytosis. Dephosphorylated Synj, on the other hand, is deficient in the endocytosis of the active recycling pool vesicles but maintains reserve pool vesicle endocytosis to restore total vesicle pool size and sustain synaptic transmission. Together, our findings reveal a novel role for Synj in modulating reserve pool vesicle endocytosis and further indicate that dynamic phosphorylation and dephosphorylation of Synj differentially maintain endocytosis of distinct functional synaptic vesicle pools. SIGNIFICANCE STATEMENT Synaptic vesicle endocytosis sustains communication between neurons during a wide range of neuronal activities by recycling used vesicle membrane and protein components. Here we identify that Synaptojanin, a protein with a known role in synaptic vesicle endocytosis, is phosphorylated at S1029 in vivo by the Minibrain kinase. We further demonstrate that the phosphorylation status of Synaptojanin at S1029 differentially regulates its participation in the recycling of distinct synaptic vesicle pools. Our results reveal a new role for

  5. Functional characterization of myrcene hydroxylases from two geographically distinct Ips pini populations.

    PubMed

    Song, Minmin; Kim, Amy C; Gorzalski, Andrew J; MacLean, Marina; Young, Sharon; Ginzel, Matthew D; Blomquist, Gary J; Tittiger, Claus

    2013-04-01

    Ips pini bark beetles use myrcene hydroxylases to produce the aggregation pheromone component, ipsdienol, from myrcene. The enantiomeric ratio of pheromonal ipsdienol is an important prezygotic mating isolation mechanism of I. pini and differs among geographically distinct populations. We explored the substrate and product ranges of myrcene hydroxylases (CYP9T2 and CYP9T3) from reproductively-isolated western and eastern I. pini. The two cytochromes P450 share 94% amino acid identity. CYP9T2 mRNA levels were not induced in adults exposed to myrcene-saturated atmosphere. Functional assays of recombinant enzymes showed both hydroxylated myrcene, (+)- and (-)-α-pinene, 3-carene, and R-(+)-limonene, but not α-phellandrene, (-)-β-pinene, γ-terpinene, or terpinolene, with evidence that CYP9T2 strongly preferred myrcene over other substrates. They differed in the enantiomeric ratios of ipsdienol produced from myrcene, and in the products resulting from different α-pinene enantiomers. These data provide new information regarding bark beetle pheromone evolution and factors affecting cytochrome P450 structure-function relationships.

  6. Two distinct functional high affinity receptors for mouse interleukin-3 (IL-3).

    PubMed Central

    Hara, T; Miyajima, A

    1992-01-01

    The human interleukin-3 receptor (IL-3R) is composed of an IL-3 specific alpha subunit (IL-3R alpha) and a common beta subunit (beta c) that is shared by IL-3, granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-5 receptors. In contrast to the human, the mouse has two distinct but related genes, AIC2A and AIC2B, both of which are homologous to the human beta c gene. AIC2B has proved to encode a common beta subunit between mouse GM-CSF and IL-5 receptors. AIC2A is unique to the mouse and encodes a low affinity IL-3 binding protein. Based on the observation that the AIC2A protein is a component of a high affinity IL-3R, we searched for a cDNA encoding a protein which conferred high affinity IL-3 binding when coexpressed with the AIC2A protein in COS7 cells. We obtained such a cDNA (SUT-1) encoding a mature protein of 70 kDa that has weak homology to the human IL-3R alpha. The SUT-1 protein bound IL-3 with low affinity and formed high affinity receptors not only with the AIC2A protein but also with the AIC2B protein. Both high affinity IL-3Rs expressed on a mouse T cell line, CTLL-2, showed similar IL-3 binding properties and transmitted a growth signal in response to IL-3. Thus, the mouse has two distinct functional high affinity IL-3Rs, providing a molecular explanation for the differences observed between mouse and human IL-3Rs. Images PMID:1582416

  7. Two distinct phosphorylation events govern the function of muscle FHOD3.

    PubMed

    Iskratsch, Thomas; Reijntjes, Susan; Dwyer, Joseph; Toselli, Paul; Dégano, Irene R; Dominguez, Isabel; Ehler, Elisabeth

    2013-03-01

    Posttranslational modifications such as phosphorylation are universally acknowledged regulators of protein function. Recently we characterised a striated muscle-specific isoform of the formin FHOD3 that displays distinct subcellular targeting and protein half-life compared to its non-muscle counterpart and which is dependent on phosphorylation by CK2 (formerly casein kinase 2). We now show that the two isoforms of FHOD3 are already expressed in the vertebrate embryonic heart. Analysis of CK2 alpha knockout mice showed that phosphorylation by CK2 is also required for proper targeting of muscle FHOD3 to the myofibrils in embryonic cardiomyocytes in situ. The localisation of muscle FHOD3 in the sarcomere varies depending on the maturation state, being either broader or restricted to the Z-disc proper in the adult heart. Following myofibril disassembly, such as that in dedifferentiating adult rat cardiomyocytes in culture, the expression of non-muscle FHOD3 is up-regulated, which is reversed once the myofibrils are reassembled. The shift in expression levels of different isoforms is accompanied by an increased co-localisation with p62, which is involved in autophagy, and affects the half-life of FHOD3. Phosphorylation of three amino acids in the C-terminus of FHOD3 by ROCK1 is sufficient for activation, which results in increased actin filament synthesis in cardiomyocytes and also a broader localisation pattern of FHOD3 in the myofibrils. ROCK1 can directly phosphorylate FHOD3, and FHOD3 seems to be the downstream mediator of the exaggerated actin filament formation phenotype that is induced in cardiomyocytes upon the overexpression of constitutively active ROCK1. We conclude that the expression of the muscle FHOD3 isoform is characteristic of the healthy mature heart and that two distinct phosphorylation events are crucial to regulate the activity of this isoform in thin filament assembly and maintenance.

  8. Microbial Pattern Recognition Causes Distinct Functional Micro-RNA Signatures in Primary Human Monocytes

    PubMed Central

    Häsler, Robert; Jacobs, Gunnar; Till, Andreas; Grabe, Nils; Cordes, Christian; Nikolaus, Susanna; Lao, Kaiqin

    2012-01-01

    Micro-RNAs (miRNAs) are short, non-coding RNAs that regulate gene expression post transcriptionally. Several studies have demonstrated the relevance of miRNAs for a wide range of cellular mechanisms, however, the current knowledge on how miRNAs respond to relevant external stimuli, e.g. in disease scenarios is very limited. To generate a descriptive picture of the miRNA network associated to inflammatory responses, we quantified the levels of 330 miRNAs upon stimulation with a panel of pro-inflammatory components such as microbial pattern molecules (flagellin, diacylated lipopeptide lipopolysaccharide, muramyl dipeptide), infection with Listeria monocytogenes and TNF-α as pro-inflammatory control in primary human monocytes using real time PCR. As a result, we found distinct miRNA response clusters for each stimulus used. Additionally, we identified potential target genes of three selected miRNAs miR-129-5p, miR-146a and miR-378 which were part of PAMP-specific response clusters by transfecting THP1 monocytes with the corresponding pre- or anti-miRNAs and microfluidic PCR arrays. The miRNAs induced distinct transcriptomal signatures, e.g. overexpression of miRNA129-5p, which was selectively upregulated by the NOD2-elicitor MDP, led to an upregulation of DEFB1, IRAK1, FBXW7 and IKK γ (Nemo). Our findings on highly co-regulated clusters of miRNAs support the hypothesis that miRNAs act in functional groups. This study indicates that miRNAs play an important role in fine-tuning inflammatory mechanisms. Further investigation in the field of miRNA responses will help to understand their effects on gene expression and may close the regulatory gap between mRNA and protein expression in inflammatory diseases. PMID:22363568

  9. Two distinct phosphorylation events govern the function of muscle FHOD3

    PubMed Central

    Iskratsch, Thomas; Reijntjes, Susan; Dwyer, Joseph; Toselli, Paul; Dégano, Irene R.; Dominguez, Isabel; Ehler, Elisabeth

    2013-01-01

    Posttranslational modifications such as phosphorylation are universally acknowledged regulators of protein function. Recently we characterised a striated muscle-specific isoform of the formin FHOD3 that displays distinct subcellular targeting and protein half-life compared to its non-muscle counterpart, which is dependent on phosphorylation by CK2 (formerly casein kinase 2). We now show that the two isoforms of FHOD3 are already expressed in the vertebrate embryonic heart. Analysis of CK2alpha knockout mice showed that phosphorylation by CK2 is required for proper targeting of muscle FHOD3 to the myofibrils also in embryonic cardiomyocytes in situ. The localisation of muscle FHOD3 in the sarcomere varies depending on the maturation state, being either broader or restricted to the Z-disc proper in adult heart. Following myofibril disassembly such as in dedifferentiating adult rat cardiomyocytes in culture, the expression of non-muscle FHOD3 is up-regulated, which is reversed once the myofibrils are reassembled. The shift in expression levels of different isoforms is accompanied by an increased co-localisation with p62, which is involved in autophagy, and affects the half-life of FHOD3. Phosphorylation of three amino acids in the C-terminus of FHOD3 by ROCK1 is sufficient for activation, which results in increased actin filament synthesis in cardiomyocytes and also a broader localisation pattern of FHOD3 in the myofibrils. ROCK1 can directly phosphorylate FHOD3 and FHOD3 seems to be the downstream mediator of the exaggerated actin filament formation phenotype that is induced in cardiomyocytes upon the overexpression of constitutively active ROCK1. We conclude that the expression of the muscle FHOD3 isoform is characteristic for the healthy mature heart and that two distinct phosphorylation events are crucial to regulate its activity in thin filament assembly and maintenance. PMID:23052206

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

    PubMed

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

    2015-06-01

    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.

  11. It takes two to tango: the structure and function of LIM, RING, PHD and MYND domains.

    PubMed

    Matthews, J M; Bhati, M; Lehtomaki, E; Mansfield, R E; Cubeddu, L; Mackay, J P

    2009-01-01

    LIM (Lin-11, Isl-1, Mec-3), RING (Really interesting new gene), PHD (Plant homology domain) and MYND (myeloid, Nervy, DEAF-1) domains are all zinc-binding domains that ligate two zinc ions. Unlike the better known classical zinc fingers, these domains do not bind DNA, but instead mediate interactions with other proteins. LIM-domain containing proteins have diverse functions as regulators of gene expression, cell adhesion and motility and signal transduction. RING finger proteins are generally associated with ubiquitination; the presence of such a domain is the defining feature of a class of E3 ubiquitin protein ligases. PHD proteins have been associated with SUMOylation but most recently have emerged as a chromatin recognition motif that reads the methylation state of histones. The function of the MYND domain is less clear, but MYND domains are also found in proteins that have ubiquitin ligase and/or histone methyltransferase activity. Here we review the structure-function relationships for these domains and discuss strategies to modulate their activity.

  12. Is membrane occupation and recognition nexus domain functional in plant phosphatidylinositol phosphate kinases?

    PubMed

    Mikami, Koji; Saavedra, Laura; Sommarin, Marianne

    2010-10-01

    Phosphatidylinositol phosphate kinase (PIPK) catalyzes a key step controlling cellular contents of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], a critical intracellular messenger involved in vesicle trafficking and modulation of actin cytoskeleton and also a substrate of phospholipase C to produce the two intracellular messengers, diacylglycerol and inositol-1,4,5-trisphosphate. In addition to the conserved C-terminal PIPK catalytic domain, plant PIPKs contain a unique structural feature consisting of a repeat of membrane occupation and recognition nexus (MORN) motifs, called the MORN domain, in the N-terminal half. The MORN domain has previously been proposed to regulate plasma membrane localization and phosphatidic acid (PA)-inducible activation. Recently, the importance of the catalytic domain, but not the MORN domain, in these aspects was demonstrated. These conflicting data raise the question about the function of the MORN domain in plant PIPKs. We therefore performed analyses of PpPIPK1 from the moss Physcomitrella patens to elucidate the importance of the MORN domain in the control of enzymatic activity; however, we found no effect on either enzymatic activity or activation by PA. Taken together with our previous findings of lack of function in plasma membrane localization, there is no positive evidence indicating roles of the MORN domain in enzymatic and functional regulations of PpPIPK1. Therefore, further biochemical and reverse genetic analyses are necessary to understand the biological significance of the MORN domain in plant PIPKs.

  13. 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.

  14. Genetic, structural, and molecular insights into the function of ras of complex proteins domains.

    PubMed

    Civiero, Laura; Dihanich, Sybille; Lewis, Patrick A; Greggio, Elisa

    2014-07-17

    Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson's disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role that ROC domains play in regulating protein function and signaling pathways. In this review, recent advances in the structural information available for proteins containing ROC domains, along with insights into enzymatic function and the integration of ROC domains as molecular switches in a cellular and organismal context, are explored.

  15. 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

    2000-05-09

    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.

  16. Further characterization of functional domains of PerA, role of amino and carboxy terminal domains in DNA binding.

    PubMed

    Ibarra, J Antonio; García-Zacarias, Claudia M; Lara-Ochoa, Cristina; Carabarin-Lima, Alejandro; Tecpanecatl-Xihuitl, J Sergio; Perez-Rueda, Ernesto; Martínez-Laguna, Ygnacio; Puente, José L

    2013-01-01

    PerA is a key regulator of virulence genes in enteropathogenic E. coli. PerA is a member of the AraC/XylS family of transcriptional regulators that directly regulates the expression of the bfp and per operons in response to different environmental cues. Here, we characterized mutants in both the amino (NTD) and carboxy (CTD) terminal domains of PerA that affect its ability to activate the expression of the bfp and per promoters. Mutants at residues predicted to be important for DNA binding within the CTD had a significant defect in their ability to bind to the regulatory regions of the bfp and per operons and, consequently, in transcriptional activation. Notably, mutants in specific NTD residues were also impaired to bind to DNA suggesting that this domain is involved in structuring the protein for correct DNA recognition. Mutations in residues E116 and D168, located in the vicinity of the putative linker region, significantly affected the activation of the perA promoter, without affecting PerA binding to the per or bfp regulatory sequences. Overall these results provide additional evidence of the importance of the N-terminal domain in PerA activity and suggest that the activation of these promoters involves differential interactions with the transcriptional machinery. This study further contributes to the characterization of the functional domains of PerA by identifying critical residues involved in DNA binding, differential promoter activation and, potentially, in the possible response to environmental cues.

  17. Recombinant human IgG antibodies recognizing distinct extracellular domains of EGF receptor exhibit different degrees of growth inhibitory effects on human A431 cancer cells.

    PubMed

    Chang, Chialun; Takayanagi, Atsushi; Yoshida, Tetsuhiko; Shimizu, Nobuyoshi

    2013-05-01

    Recently, we isolated 4 distinct kinds of single chain antibody against human EGF receptor (EGFR) after screening the Keio phage display scFv library by using two methods of target-guided proximity labeling. In the current study, these monovalent scFv antibodies were converted to bivalent IgGs of humanized forms (hIgGs) by recombinant technology using the specially designed expression vectors followed by protein production in CHO cells. The resulting recombinant hIgGs were examined for their binding specificity using several different transformed human BJ cell lines that express deletion mutants of EGFR, each lacking one of 4 distinct extracellular domains (L1, L2, C1 and C2). Immuno-fluorescent microscopy and immuno-precipitation assay on these cells indicated that 4 distinct kinds of hIgGs bind to one of 3 different domains (L1, C1 and C2). Then, these hIgGs were further examined for biological effects on human A431 cancer cells, which overexpress EGFR. The results indicated that hIgG38 binding to L1 and hIgG45 binding to C2 substantially suppressed the EGF-induced phosphorylation of EGFR, resulting in the growth inhibition of A431 cancer cells. On the contrary, hIgG40 binding to C1 and hIgG42 binding to another site (epitope) of C2 exhibited no such inhibitory effects. Thus, the newly produced four recombinant hIgG antibodies recognize 4 different sites (epitopes) in 3 different extracellular domains of EGFR and exhibit different biological effects on cancer cells. These characteristics are somewhat different from the currently utilized therapeutic anti-EGFR antibodies. Hence, these hIgG antibodies will be invaluable as a research tool for the detailed molecular analysis of the EGFR-mediated signal transduction mechanism and more importantly a possible application as new therapeutic agents to treat certain types of cancers.

  18. Distinct effects of childhood ADHD and cannabis use on brain functional architecture in young adults.

    PubMed

    Kelly, Clare; Castellanos, F Xavier; Tomaselli, Olivia; Lisdahl, Krista; Tamm, Leanne; Jernigan, Terry; Newman, Erik; Epstein, Jeffery N; Molina, Brooke S G; Greenhill, Laurence L; Potkin, Steven G; Hinshaw, Stephen; Swanson, James M

    2017-01-01

    One of the most salient long-term implications of a childhood diagnosis of ADHD is an increased risk for substance use, abuse, or dependence in adolescence and adulthood. The extent to which cannabis use affects ADHD-related alterations in brain functional organization is unknown, however. To address this research gap, we recruited a sample of 75 individuals aged 21-25 years with and without a childhood diagnosis of ADHD Combined Type, who were either frequent users or non-users of cannabis. These participants have been followed longitudinally since age 7-9.9 years as part of a large multi-site longitudinal study of ADHD, the Multimodal Treatment Study of Children with ADHD (MTA). We examined task-independent intrinsic functional connectivity (iFC) within 9 functional networks using a 2 × 2 design, which compared four groups of participants: (1) individuals with a childhood diagnosis of ADHD who currently use cannabis (n = 23); (2) individuals with ADHD who do not currently use cannabis (n = 22); (3) comparisons who currently use cannabis (n = 15); and (4) comparisons who do not currently use cannabis (n = 15). The main effects of childhood ADHD were primarily weakened iFC in networks supporting executive function and somatomotor control. Contrary to expectations, effects of cannabis use were distinct from those of diagnostic group and no interactions were observed. Exploratory brain-behavior analyses suggested that ADHD-related effects were primarily linked with poorer neurocognitive performance. Deficits in the integrity of functional networks supporting executive function and somatomotor control are consistent with the phenotypic and neurocognitive features of ADHD. Our data suggest that cannabis use does not exacerbate ADHD-related alterations, but this finding awaits replication in a larger sample. Longitudinal neuroimaging studies are urgently required to delineate the neurodevelopmental cascade that culminates in positive and negative outcomes

  19. Structure and function of Toll/interleukin-1 receptor/resistance protein (TIR) domains.

    PubMed

    Ve, Thomas; Williams, Simon J; Kobe, Bostjan

    2015-02-01

    The Toll/interleukin-1 receptor/resistance protein (TIR) domain is a protein-protein interaction domain consisting of 125-200 residues, widely distributed in animals, plants and bacteria but absent from fungi, archea and viruses. In plants and animals, these domains are found in proteins with functions in innate immune pathways, while in bacteria, some TIR domain-containing proteins interfere with the innate immune pathways in the host. TIR domains function as protein scaffolds, mostly involving self-association and homotypic interactions with other TIR domains. In the last 15 years, the three-dimensional structures of TIR domains from several mammalian, plant and bacterial proteins have been reported. These structures, jointly with functional data including the identification of interacting proteins, have started to provide insight into the molecular basis of the assembly of animal and plant immune signaling complexes, and for host immunosuppression by bacterial pathogens. This review focuses on the current knowledge of the structures of the TIR domains and how the structure relates to function.

  20. 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…

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

    PubMed

    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.

  2. 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

  3. Zebrafish mesonephric renin cells are functionally conserved and comprise two distinct morphological populations.

    PubMed

    Rider, Sebastien A; Christian, Helen C; Mullins, Linda J; Howarth, Amelia R; MacRae, Calum A; Mullins, John J

    2017-04-01

    Zebrafish provide an excellent model in which to assess the role of the renin-angiotensin system in renal development, injury, and repair. In contrast to mammals, zebrafish kidney organogenesis terminates with the mesonephros. Despite this, the basic functional structure of the nephron is conserved across vertebrates. The relevance of teleosts for studies relating to the regulation of the renin-angiotensin system was established by assessing the phenotype and functional regulation of renin-expressing cells in zebrafish. Transgenic fluorescent reporters for renin (ren), smooth muscle actin (acta2), and platelet-derived growth factor receptor-beta (pdgfrb) were studied to determine the phenotype and secretory ultrastructure of perivascular renin-expressing cells. Whole kidney ren transcription responded to altered salinity, pharmacological renin-angiotensin system inhibition, and renal injury. Mesonephric ren-expressing cells occupied niches at the preglomerular arteries and afferent arterioles, forming intermittent epithelioid-like multicellular clusters exhibiting a granular secretory ultrastructure. In contrast, renin cells of the efferent arterioles were thin bodied and lacked secretory granules. Renin cells expressed the perivascular cell markers acta2 and pdgfrb Transcriptional responses of ren to physiological challenge support the presence of a functional renin-angiotensin system and are consistent with the production of active renin. The reparative capability of the zebrafish kidney was harnessed to demonstrate that ren transcription is a marker for renal injury and repair. Our studies demonstrate substantive conservation of renin regulation across vertebrates, and ultrastructural studies of renin cells reveal at least two distinct morphologies of mesonephric perivascular ren-expressing cells.

  4. Differential expression of two distinct functional isoforms of melanopsin (Opn4) in the mammalian retina.

    PubMed

    Pires, Susana S; Hughes, Steven; Turton, Michael; Melyan, Zare; Peirson, Stuart N; Zheng, Lei; Kosmaoglou, Maria; Bellingham, James; Cheetham, Michael E; Lucas, Robert J; Foster, Russell G; Hankins, Mark W; Halford, Stephanie

    2009-09-30

    Melanopsin is the photopigment that confers photosensitivity to a subset of retinal ganglion cells (pRGCs) that regulate many non-image-forming tasks such as the detection of light for circadian entrainment. Recent studies have begun to subdivide the pRGCs on the basis of morphology and function, but the origin of these differences is not yet fully understood. Here we report the identification of two isoforms of melanopsin from the mouse Opn4 locus, a previously described long isoform (Opn4L) and a novel short isoform (Opn4S) that more closely resembles the sequence and structure of rat and human melanopsins. Both isoforms, Opn4L and Opn4S, are expressed in the ganglion cell layer of the retina, traffic to the plasma membrane and form a functional photopigment in vitro. Quantitative PCR revealed that Opn4S is 40 times more abundant than Opn4L. The two variants encode predicted proteins of 521 and 466 aa and only differ in the length of their C-terminal tails. Antibodies raised to isoform-specific epitopes identified two discrete populations of melanopsin-expressing RGCs, those that coexpress Opn4L and Opn4S and those that express Opn4L only. Recent evidence suggests that pRGCs show a range of anatomical subtypes, which may reflect the functional diversity reported for mouse Opn4-mediated light responses. The distinct isoforms of Opn4 described in this study provide a potential molecular basis for generating this diversity, and it seems likely that their differential expression plays a role in generating the variety of pRGC light responses found in the mammalian retina.

  5. A Comprehensive Analysis of RALF Proteins in Green Plants Suggests There Are Two Distinct Functional Groups

    PubMed Central

    Campbell, Liam; Turner, Simon R.

    2017-01-01

    Rapid Alkalinization Factors (RALFs) are small, cysteine-rich peptides known to be involved in various aspects of plant development and growth. Although RALF peptides have been identified within many species, a single wide-ranging phylogenetic analysis of the family across the plant kingdom has not yet been undertaken. Here, we identified RALF proteins from 51 plant species that represent a variety of land plant lineages. The inferred evolutionary history of the 795 identified RALFs suggests that the family has diverged into four major clades. We found that much of the variation across the family exists within the mature peptide region, suggesting clade-specific functional diversification. Clades I, II, and III contain the features that have been identified as important for RALF activity, including the RRXL cleavage site and the YISY motif required for receptor binding. In contrast, members of clades IV that represent a third of the total dataset, is highly diverged and lacks these features that are typical of RALFs. Members of clade IV also exhibit distinct expression patterns and physico-chemical properties. These differences suggest a functional divergence of clades and consequently, we propose that the peptides within clade IV are not true RALFs, but are more accurately described as RALF-related peptides. Expansion of this RALF–related clade in the Brassicaceae is responsible for the large number of RALF genes that have been previously described in Arabidopsis thaliana. Future experimental work will help to establish the nature of the relationship between the true RALFs and the RALF-related peptides, and whether they function in a similar manner. PMID:28174582

  6. Functional Analysis of CP2-Like Domain and SAM-Like Domain in TFCP2L1, Novel Pluripotency Factor of Embryonic Stem Cells.

    PubMed

    Kim, Chang Min; Jang, Tae-Ho; Park, Hyun Ho

    2016-06-01

    TFCP2L1 is a transcription factor that facilitates establishment and maintenance of pluripotency in embryonic stem cells by forming a complex transcriptional network with other transcription factors (OCT4, SOX2, and NANOG). TFCP2L1 contains two distinct domains, the CP2-like domain at the N-terminus and the SAM-like domain at the C-terminus. In this study, we found that TFCP2L1 is hexamerized in solution via the C-terminal SAM-like domain. We also found that homo-oligomerization of SAM-like domain is dependent on the concentration of the proteins. Finally, we found that TFCP2L1 binds directly to DNA via the N-terminal CP2-like domain.

  7. Mapping Viral Functional Domains for Genetic Diversity in Plants

    PubMed Central

    Pita, Justin S.

    2013-01-01

    Cucumber mosaic virus (CMV) comprises numerous isolates with various levels of in-host diversity. Subgroup-distinctive features of the Fny and LS strains provided us with a platform to genetically map the viral control elements for genetic variation in planta. We found that both RNAs 1 and 2 controlled levels of genetic diversity, and further fine mapping revealed that the control elements of mutation frequency reside within the first 596 amino acids (aa) of RNA 1. The 2a/2b overlapping region of the 2a protein also contributed to control of viral genetic variation. Furthermore, the 3′ nontranslated region (NTR) of RNA 3 constituted a hot spot of polymorphism, where the majority of fixed mutations found in the population were clustered. The 2b gene of CMV, a viral suppressor of gene silencing, controls the abundance of the fixed mutants in the viral population via a host-dependent mechanism. PMID:23115283

  8. Structure of the Mtb CarD/RNAP β-lobes complex reveals the molecular basis of interaction and presents a distinct DNA-binding domain for Mtb CarD.

    PubMed

    Gulten, Gulcin; Sacchettini, James C

    2013-10-08

    CarD from Mycobacterium tuberculosis (Mtb) is an essential protein shown to be involved in stringent response through downregulation of rRNA and ribosomal protein genes. CarD interacts with the β-subunit of RNAP and this interaction is vital for Mtb's survival during the persistent infection state. We have determined the crystal structure of CarD in complex with the RNAP β-subunit β1 and β2 domains at 2.1 Å resolution. The structure reveals the molecular basis of CarD/RNAP interaction, providing a basis to further our understanding of RNAP regulation by CarD. The structural fold of the CarD N-terminal domain is conserved in RNAP interacting proteins such as TRCF-RID and CdnL, and displays similar interactions to the predicted homology model based on the TRCF/RNAP β1 structure. Interestingly, the structure of the C-terminal domain, which is required for complete CarD function in vivo, represents a distinct DNA-binding fold.

  9. Similar and Distinct Properties of MUPP1 and Patj, Two Homologous PDZ Domain-Containing Tight-Junction Proteins ▿ †

    PubMed Central

    Adachi, Makoto; Hamazaki, Yoko; Kobayashi, Yuka; Itoh, Masahiko; Tsukita, Sachiko; Furuse, Mikio; Tsukita, Shoichiro

    2009-01-01

    MUPP1 and Patj are both composed of an L27 domain and multiple PDZ domains (13 and 10 domains, respectively) and are localized to tight junctions (TJs) in epithelial cells. Although Patj is known to be responsible for the organization of TJs and epithelial polarity, characterization of MUPP1 is lacking. In this study, we found that MUPP1 and Patj share several binding partners, including JAM1, ZO-3, Pals1, Par6, and nectins (cell-cell adhesion molecules at adherens junctions). MUPP1 and Patj exhibited similar subcellular distributions, and the mechanisms with which they localize to TJs also appear to overlap. Despite these similarities, functional studies have revealed that Patj is indispensable for the establishment of TJs and epithelial polarization, whereas MUPP1 is not. Thus, although MUPP1 and Patj share several molecular properties, their functions are entirely different. We present evidence that the signaling mediated by Pals1, which has a higher affinity for Patj than for MUPP1 and is involved in the activation of the Par6-aPKC complex, is of principal importance for the function of Patj in epithelial cells. PMID:19255144

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

    PubMed Central

    2015-01-01

    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

  11. 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.

  12. C-terminal domains of bacterial proteases: structure, function and the biotechnological applications.

    PubMed

    Huang, J; Wu, C; Liu, D; Yang, X; Wu, R; Zhang, J; Ma, C; He, H

    2017-01-01

    C-terminal domains widely exist in the C-terminal region of multidomain proteases. As a β-sandwich domain in multidomain protease, the C-terminal domain plays an important role in proteolysis including regulation of the secretory process, anchoring and swelling the substrate molecule, presenting as an inhibitor for the preprotease and adapting the protein structural flexibility and stability. In this review, the diversity, structural characteristics and biological function of C-terminal protease domains are described. Furthermore, the application prospects of C-terminal domains, including polycystic kidney disease, prepeptidase C-terminal and collagen-binding domain, in the area of medicine and biological artificial materials are also discussed.

  13. Protein Domain of Unknown Function 3233 is a Translocation Domain of Autotransporter Secretory Mechanism in Gamma proteobacteria

    PubMed Central

    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 1/3rd 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

  14. The functionally distinct hemoglobins of the Arctic spotted wolffish Anarhichas minor.

    PubMed

    Verde, Cinzia; Carratore, Vito; Riccio, Antonio; Tamburrini, Maurizio; Parisi, Elio; Di Prisco, Guido

    2002-09-27

    The Arctic fish Anarhichas minor, a benthic sedentary species, displays high hemoglobin multiplicity. The three major hemoglobins (Hb 1, Hb 2, and Hb 3) show important functional differences in pH and organophosphate regulation, subunit cooperativity, and response of oxygen binding to temperature. Hb 1 and Hb 2 display a low, effector-enhanced Bohr effect and no Root effect. In contrast, Hb 3 displays pronounced Bohr and Root effects, accompanied by strong organophosphate regulation. Hb 1 has the beta (beta(1)) chain in common with Hb 2; Hb 3 and Hb 2 share the alpha (alpha(2)) chain. The amino acid sequences have been established. Several substitutions in crucial positions were observed, such as Cys in place of C-terminal His in the beta(1) chain of Hb 1 and Hb 2. In Hb 3, Val E11 of the beta(2) chain is replaced by Ile. Homology modeling revealed an unusual structure of the Hb 3 binding site of inositol hexakisphoshate. Phylogenetic analysis indicated that only Hb 2 displays higher overall similarity with the major Antarctic hemoglobins. The oxygen transport system of A. minor differs remarkably from those of Antarctic Notothenioidei, indicating distinct evolutionary pathways in the regulatory mechanisms of the fish respiratory system in the two polar environments.

  15. Ly-1 B cells: functionally distinct lymphocytes that secrete IgM autoantibodies.

    PubMed Central

    Hayakawa, K; Hardy, R R; Honda, M; Herzenberg, L A; Steinberg, A D; Herzenberg, L A

    1984-01-01

    Studies presented here introduce another perspective on the mechanisms responsible for IgM autoantibody production. A unique subpopulation of B lymphocytes (Ly-1 B) that concomitantly expresses IgM, IgD, Ia, and Ly-1 membrane glycoproteins is present at higher frequencies in NZB and NZB-related mice. The Ly-1 B subpopulation in these autoimmune animals is responsible for the "spontaneous" IgM secretion demonstrated with cultured NZB spleen cells and contains the cells that secrete typical NZB IgM autoantibodies to single-stranded DNA and to thymocytes. In addition, the Ly-1 B population in normal mouse strains (and in NZB) contains virtually all of the spleen cells that secrete IgM autoantibodies reactive with bromelain-treated mouse erythrocytes. Since a different B-cell subpopulation (IgM+, IgD-, Ly-1) secretes most of the IgM antibodies produced in responses to exogenous antigens, we conclude that Ly-1 B cells constitute a functionally distinct B-cell population important in certain kinds of autoimmunity. PMID:6609363

  16. Beyond polarity: functional membrane domains in astrocytes and Müller cells.

    PubMed

    Derouiche, Amin; Pannicke, Thomas; Haseleu, Julia; Blaess, Sandra; Grosche, Jens; Reichenbach, Andreas

    2012-11-01

    Various ependymoglial cells display varying degrees of process specialization, in particular processes contacting mesenchymal borders (pia, blood vessels, vitreous body), or those lining the ventricular surface. Within the neuropil, glial morphology, cellular contacts, and interaction partners are complex. It appears that glial processes contacting neurons, specific parts of neurons, or mesenchymal or ventricular borders are characterized by specialized membranes. We propose a concept of membrane domains in addition to the existing concept of ependymoglial polarity. Such membrane domains are equipped with certain membrane-bound proteins, enabling them to function in their specific environment. This review focuses on Müller cells and astrocytes and discusses exemplary the localization of established glial markers in membrane domains. We distinguish three functional glial membrane domains based on their typical molecular arrangement. The domain of the endfoot specifically displays the complex of dystrophin-associated proteins, aquaporin 4 and the potassium channel Kir4.1. We show that the domain of microvilli and the peripheral glial process in the Müller cell share the presence of ezrin, as do peripheral astrocyte processes. As a third domain, the Müller cell has peripheral glial processes related to a specific subtype of synapse. Although many details remain to be studied, the idea of glial membrane domains may permit new insights into glial function and pathology.

  17. Functional connectivity with distinct neural networks tracks fluctuations in gain/loss framing susceptibility.

    PubMed

    Smith, David V; Sip, Kamila E; Delgado, Mauricio R

    2015-07-01

    Multiple large-scale neural networks orchestrate a wide range of cognitive processes. For example, interoceptive processes related to self-referential thinking have been linked to the default-mode network (DMN); whereas exteroceptive processes related to cognitive control have been linked to the executive-control network (ECN). Although the DMN and ECN have been postulated to exert opposing effects on cognition, it remains unclear how connectivity with these spatially overlapping networks contribute to fluctuations in behavior. While previous work has suggested the medial-prefrontal cortex (MPFC) is involved in behavioral change following feedback, these observations could be linked to interoceptive processes tied to DMN or exteroceptive processes tied to ECN because MPFC is positioned in both networks. To address this problem, we employed independent component analysis combined with dual-regression functional connectivity analysis. Participants made a series of financial decisions framed as monetary gains or losses. In some sessions, participants received feedback from a peer observing their choices; in other sessions, feedback was not provided. Following feedback, framing susceptibility-indexed as the increase in gambling behavior in loss frames compared to gain frames-was heightened in some participants and diminished in others. We examined whether these individual differences were linked to differences in connectivity by contrasting sessions containing feedback against those that did not contain feedback. We found two key results. As framing susceptibility increased, the MPFC increased connectivity with DMN; in contrast, temporal-parietal junction decreased connectivity with the ECN. Our results highlight how functional connectivity patterns with distinct neural networks contribute to idiosyncratic behavioral changes.

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

    PubMed

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

    2015-02-27

    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.

  19. Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

    PubMed Central

    Lila, T; Drubin, D G

    1997-01-01

    In a variety of organisms, a number of proteins associated with the cortical actin cytoskeleton contain SH3 domains, suggesting that these domains may provide the physical basis for functional interactions among structural and regulatory proteins in the actin cytoskeleton. We present evidence that SH3 domains mediate at least two independent functions of the Saccharomyces cerevisiae actin-binding protein Abp1p in vivo. Abp1p contains a single SH3 domain that has recently been shown to bind in vitro to the adenylyl cyclase-associated protein Srv2p. Immunofluorescence analysis of Srv2p subcellular localization in strains carrying mutations in either ABP1 or SRV2 reveals that the Abp1p SH3 domain mediates the normal association of Srv2p with the cortical actin cytoskeleton. We also show that a site in Abp1p itself is specifically bound by the SH3 domain of the actin-associated protein Rvs167p. Genetic analysis provides evidence that Abp1p and Rvs167p have functions that are closely interrelated. Abp1 null mutations, like rvs167 mutations, result in defects in sporulation and reduced viability under certain suboptimal growth conditions. In addition, mutations in ABP1 and RVS167 yield similar profiles of genetic "synthetic lethal" interactions when combined with mutations in genes encoding other cytoskeletal components. Mutations which specifically disrupt the SH3 domain-mediated interaction between Abp1p and Srv2p, however, show none of the shared phenotypes of abp1 and rvs167 mutations. We conclude that the Abp1p SH3 domain mediates the association of Srv2p with the cortical actin cytoskeleton, and that Abp1p performs a distinct function that is likely to involve binding by the Rvs167p SH3 domain. Overall, work presented here illustrates how SH3 domains can integrate the activities of multiple actin cytoskeleton proteins in response to varying environmental conditions. Images PMID:9190214

  20. Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

    PubMed

    Lila, T; Drubin, D G

    1997-02-01

    In a variety of organisms, a number of proteins associated with the cortical actin cytoskeleton contain SH3 domains, suggesting that these domains may provide the physical basis for functional interactions among structural and regulatory proteins in the actin cytoskeleton. We present evidence that SH3 domains mediate at least two independent functions of the Saccharomyces cerevisiae actin-binding protein Abp1p in vivo. Abp1p contains a single SH3 domain that has recently been shown to bind in vitro to the adenylyl cyclase-associated protein Srv2p. Immunofluorescence analysis of Srv2p subcellular localization in strains carrying mutations in either ABP1 or SRV2 reveals that the Abp1p SH3 domain mediates the normal association of Srv2p with the cortical actin cytoskeleton. We also show that a site in Abp1p itself is specifically bound by the SH3 domain of the actin-associated protein Rvs167p. Genetic analysis provides evidence that Abp1p and Rvs167p have functions that are closely interrelated. Abp1 null mutations, like rvs167 mutations, result in defects in sporulation and reduced viability under certain suboptimal growth conditions. In addition, mutations in ABP1 and RVS167 yield similar profiles of genetic "synthetic lethal" interactions when combined with mutations in genes encoding other cytoskeletal components. Mutations which specifically disrupt the SH3 domain-mediated interaction between Abp1p and Srv2p, however, show none of the shared phenotypes of abp1 and rvs167 mutations. We conclude that the Abp1p SH3 domain mediates the association of Srv2p with the cortical actin cytoskeleton, and that Abp1p performs a distinct function that is likely to involve binding by the Rvs167p SH3 domain. Overall, work presented here illustrates how SH3 domains can integrate the activities of multiple actin cytoskeleton proteins in response to varying environmental conditions.

  1. Reader domain specificity and lysine demethylase-4 family function

    PubMed Central

    Su, Zhangli; Wang, Fengbin; Lee, Jin-Hee; Stephens, Kimberly E.; Papazyan, Romeo; Voronina, Ekaterina; Krautkramer, Kimberly A.; Raman, Ana; Thorpe, Jeremy J.; Boersma, Melissa D.; Kuznetsov, Vyacheslav I.; Miller, Mitchell D.; Taverna, Sean D.; Phillips, George N.; Denu, John M.

    2016-01-01

    The KDM4 histone demethylases are conserved epigenetic regulators linked to development, spermatogenesis and tumorigenesis. However, how the KDM4 family targets specific chromatin regions is largely unknown. Here, an extensive histone peptide microarray analysis uncovers trimethyl-lysine histone-binding preferences among the closely related KDM4 double tudor domains (DTDs). KDM4A/B DTDs bind strongly to H3K23me3, a poorly understood histone modification recently shown to be enriched in meiotic chromatin of ciliates and nematodes. The 2.28 Å co-crystal structure of KDM4A-DTD in complex with H3K23me3 peptide reveals key intermolecular interactions for H3K23me3 recognition. Furthermore, analysis of the 2.56 Å KDM4B-DTD crystal structure pinpoints the underlying residues required for exclusive H3K23me3 specificity, an interaction supported by in vivo co-localization of KDM4B and H3K23me3 at heterochromatin in mammalian meiotic and newly postmeiotic spermatocytes. In vitro demethylation assays suggest H3K23me3 binding by KDM4B stimulates H3K36 demethylation. Together, these results provide a possible mechanism whereby H3K23me3-binding by KDM4B directs localized H3K36 demethylation during meiosis and spermatogenesis. PMID:27841353

  2. A Distinct Mechanism for Coactivator versus Corepressor Function by Histone Methyltransferase G9a in Transcriptional Regulation*

    PubMed Central

    Purcell, Daniel J.; Jeong, Kwang Won; Bittencourt, Danielle; Gerke, Daniel S.; Stallcup, Michael R.

    2011-01-01

    Histone methyltransferase G9a has been understood primarily as a corepressor of gene expression, but we showed previously that G9a positively regulates nuclear receptor-mediated transcription in reporter gene assays. Here, we show that endogenous G9a contributes to the estradiol (E2)-dependent induction of some endogenous target genes of estrogen receptor (ER)α in MCF-7 breast cancer cells while simultaneously limiting the E2-induced expression of other ERα target genes. Thus, G9a has a dual and selective role as a coregulator for ERα target genes. The ERα binding regions associated with the pS2 gene, which requires G9a for E2-induced expression, are transiently occupied by G9a at 15 min after beginning E2 treatment, suggesting that G9a coactivator function is by direct interaction with ERα target genes. Transient reporter gene assays with deletion mutants of G9a demonstrated that domains previously associated with the corepressor functions of G9a (C-terminal methyltransferase domain, ankyrin repeat domain, and cysteine-rich domain) were unnecessary for G9a coactivator function in ERα-mediated transcription. In contrast, the N-terminal domain of G9a was necessary and sufficient for enhancement of ERα-mediated transcription and for E2-induced occupancy of G9a on ERα binding sites associated with endogenous target genes of ERα. In addition to a previously identified activation domain, this region contains a previously uncharacterized ligand-dependent ERα binding function, indicating how G9a is recruited to the target genes. Therefore, the coactivator and corepressor functions of G9a involve different G9a domains and different molecular mechanisms. PMID:21984853

  3. Small Molecule-Induced Domain Swapping as a Mechanism for Controlling Protein Function and Assembly

    PubMed Central

    Karchin, Joshua M.; Ha, Jeung-Hoi; Namitz, Kevin E.; Cosgrove, Michael S.; Loh, Stewart N.

    2017-01-01

    Domain swapping is the process by which identical proteins exchange reciprocal segments to generate dimers. Here we introduce induced domain swapping (INDOS) as a mechanism for regulating protein function. INDOS employs a modular design consisting of the fusion of two proteins: a recognition protein that binds a triggering molecule, and a target protein that undergoes a domain swap in response to binding of the triggering ligand. The recognition protein (FK506 binding protein) is inserted into functionally-inactivated point mutants of two target proteins (staphylococcal nuclease and ribose binding protein). Binding of FK506 to the FKBP domain causes the target domain to first unfold, then refold via domain swap. The inactivating mutations become ‘swapped out’ in the dimer, increasing nuclease and ribose binding activities by 100-fold and 15-fold, respectively, restoring them to near wild-type values. INDOS is intended to convert an arbitrary protein into a functional switch, and is the first example of rational design in which a small molecule is used to trigger protein domain swapping and subsequent activation of biological function. PMID:28287617

  4. The role of chromosome domains in shaping the functional genome.

    PubMed

    Sexton, Tom; Cavalli, Giacomo

    2015-03-12

    The genome must be highly compacted to fit within eukaryotic nuclei but must be accessible to the transcriptional machinery to allow appropriate expression of genes in different cell types and throughout developmental pathways. A growing body of work has shown that the genome, analogously to proteins, forms an ordered, hierarchical structure that closely correlates and may even be causally linked with regulation of functions such as transcription. This review describes our current understanding of how these functional genomic "secondary and tertiary structures" form a blueprint for global nuclear architecture and the potential they hold for understanding and manipulating genomic regulation.

  5. 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

  6. A Family of Salmonella Virulence Factors Functions as a Distinct Class of Autoregulated E3 Ubiquitin Ligases

    SciTech Connect

    Quezada, C.; Hicks, S; Galan, J; Stebbins, C

    2009-01-01

    Processes as diverse as receptor binding and signaling, cytoskeletal dynamics, and programmed cell death are manipulated by mimics of host proteins encoded by pathogenic bacteria. We show here that the Salmonella virulence factor SspH2 belongs to a growing class of bacterial effector proteins that harness and subvert the eukaryotic ubiquitination pathway. This virulence protein possesses ubiquitination activity that depends on a conserved cysteine residue. A crystal structure of SspH2 reveals a canonical leucine-rich repeat (LRR) domain that interacts with a unique E{sub 3} ligase [which we have termed NEL for Novel E{sub 3} Ligase] C-terminal fold unrelated to previously observed HECT or RING-finger E{sub 3} ligases. Moreover, the LRR domain sequesters the catalytic cysteine residue contained in the NEL domain, and we suggest a mechanism for activation of the ligase requiring a substantial conformational change to release the catalytic domain for function. We also show that the N-terminal domain targets SspH2 to the apical plasma membrane of polarized epithelial cells and propose a model whereby binding of the LRR to proteins at the target site releases the ligase domain for site-specific function.

  7. 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

  8. 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.

  9. Visual Input to the Drosophila Central Complex by Developmentally and Functionally Distinct Neuronal Populations.

    PubMed

    Omoto, Jaison Jiro; Keleş, Mehmet Fatih; Nguyen, Bao-Chau Minh; Bolanos, Cheyenne; Lovick, Jennifer Kelly; Frye, Mark Arthur; Hartenstein, Volker

    2017-03-23

    The Drosophila central brain consists of stereotyped neural lineages, developmental-structural units of macrocircuitry formed by the sibling neurons of single progenitors called neuroblasts. We demonstrate that the lineage principle guides the connectivity and function of neurons, providing input to the central complex, a collection of neuropil compartments important for visually guided behaviors. One of these compartments is the ellipsoid body (EB), a structure formed largely by the axons of ring (R) neurons, all of which are generated by a single lineage, DALv2. Two further lineages, DALcl1 and DALcl2, produce neurons that connect the anterior optic tubercle, a central brain visual center, with R neurons. Finally, DALcl1/2 receive input from visual projection neurons of the optic lobe medulla, completing a three-legged circuit that we call the anterior visual pathway (AVP). The AVP bears a fundamental resemblance to the sky-compass pathway, a visual navigation circuit described in other insects. Neuroanatomical analysis and two-photon calcium imaging demonstrate that DALcl1 and DALcl2 form two parallel channels, establishing connections with R neurons located in the peripheral and central domains of the EB, respectively. Although neurons of both lineages preferentially respond to bright objects, DALcl1 neurons have small ipsilateral, retinotopically ordered receptive fields, whereas DALcl2 neurons share a large excitatory receptive field in the contralateral hemifield. DALcl2 neurons become inhibited when the object enters the ipsilateral hemifield and display an additional excitation after the object leaves the field of view. Thus, the spatial position of a bright feature, such as a celestial body, may be encoded within this pathway.

  10. Expression and functional analysis of a cytoplasmic domain variant of the beta 1 integrin subunit.

    PubMed

    Balzac, F; Belkin, A M; Koteliansky, V E; Balabanov, Y V; Altruda, F; Silengo, L; Tarone, G

    1993-04-01

    We have previously described a variant form of the integrin beta 1 subunit (beta 1B)1 characterized by an altered sequence at the cytoplasmic domain. Using polyclonal antibodies to a synthetic peptide corresponding to the unique sequence of the beta 1B, we analyzed the expression of this molecule in human tissues and cultured cells. Western blot analysis showed that the beta 1B is expressed in skin and liver and, in lower amounts, in skeletal and cardiac muscles. The protein was not detectable in brain, kidney, and smooth muscle. In vitro cultured keratinocytes and hepatoma cells are positive, but fibroblasts, endothelial cells, and smooth muscle cells are negative. An astrocytoma cell line derived from immortalized fetal astrocytes was found to express beta 1B. In these cells beta 1B represent integral of 30% of the beta 1 and form heterodimers with alpha 1 and alpha 5 subunits. To investigate the functional properties of beta 1B, the full-length cDNA coding for this molecule was transfected into CHO cells. Stable transfectants were selected and the beta 1B was identified by a mAb that discriminate between the transfected human protein and the endogenous hamster beta 1A. Immunoprecipitation experiments indicated that the beta 1B was exported at the cell surface in association with the endogenous hamster alpha subunits. The alpha 5/beta 1B complex bound to a fibronectin-affinity matrix and was specifically released by RGD-containing peptides. Thus beta 1B and beta 1A are similar as far as the alpha/beta association and fibronectin binding are concerned. The two proteins differ, however, in their subcellular localization. Immunofluorescence studies indicated, in fact, that beta 1B, in contrast to beta 1A, does not localize in focal adhesions. The restricted tissue distribution and the distinct subcellular localization, suggest that beta 1B has unique functional properties.

  11. Expression and functional analysis of a cytoplasmic domain variant of the beta 1 integrin subunit

    PubMed Central

    1993-01-01

    We have previously described a variant form of the integrin beta 1 subunit (beta 1B)1 characterized by an altered sequence at the cytoplasmic domain. Using polyclonal antibodies to a synthetic peptide corresponding to the unique sequence of the beta 1B, we analyzed the expression of this molecule in human tissues and cultured cells. Western blot analysis showed that the beta 1B is expressed in skin and liver and, in lower amounts, in skeletal and cardiac muscles. The protein was not detectable in brain, kidney, and smooth muscle. In vitro cultured keratinocytes and hepatoma cells are positive, but fibroblasts, endothelial cells, and smooth muscle cells are negative. An astrocytoma cell line derived from immortalized fetal astrocytes was found to express beta 1B. In these cells beta 1B represent integral of 30% of the beta 1 and form heterodimers with alpha 1 and alpha 5 subunits. To investigate the functional properties of beta 1B, the full- length cDNA coding for this molecule was transfected into CHO cells. Stable transfectants were selected and the beta 1B was identified by a mAb that discriminate between the transfected human protein and the endogenous hamster beta 1A. Immunoprecipitation experiments indicated that the beta 1B was exported at the cell surface in association with the endogenous hamster alpha subunits. The alpha 5/beta 1B complex bound to a fibronectin-affinity matrix and was specifically released by RGD-containing peptides. Thus beta 1B and beta 1A are similar as far as the alpha/beta association and fibronectin binding are concerned. The two proteins differ, however, in their subcellular localization. Immunofluorescence studies indicated, in fact, that beta 1B, in contrast to beta 1A, does not localize in focal adhesions. The restricted tissue distribution and the distinct subcellular localization, suggest that beta 1B has unique functional properties. PMID:7681433

  12. Cerebellar on-beam and lateral inhibition: two functionally distinct circuits.

    PubMed

    Cohen, D; Yarom, Y

    2000-04-01

    Optical imaging of voltage-sensitive dyes in an isolated cerebellum preparation was used to study the spatiotemporal functional organization of the inhibitory systems in the cerebellar cortex. Responses to surface stimulation of the cortex reveal two physiologically distinct inhibitory systems, which we refer to as lateral and on-beam inhibition following classical terminology. Lateral inhibition occurs throughout the area responding to a stimulus, whereas on-beam inhibition is confined to the area directly excited by parallel fibers. The time course of the lateral inhibition is twice as long as that of the on-beam inhibition. Both inhibitory responses increase with stimulus intensity, but the lateral inhibition has a lower threshold, and it saturates at lower stimulus intensity. The amplitude of the on-beam inhibition is linearly related to the excitation at the same location, whereas that of the lateral inhibition is linearly related to the excitation at the center of the beam. Repetitive stimulation is required to activate on-beam inhibition, whereas the same stimulus paradigm reveals prolonged depression of the lateral inhibition. We conclude that lateral inhibition reflects the activation of molecular layer interneurons, and its major role is to increase the excitability of the activated area by disinhibition. The on-beam inhibition most likely reflects Golgi cell inhibition of granule cells. However, Purkinje cell collateral inhibition of Golgi cells cannot be excluded. Both possibilities suggest that the role of the on-beam inhibition is to efficiently modulate, in time and space, the mossy fiber input to the cerebellar cortex.

  13. Phragmoplast of the green alga Spirogyra is functionally distinct from the higher plant phragmoplast

    PubMed Central

    1995-01-01

    Cytokinesis in the green alga Spirogyra (Zygnemataceae) is characterized by centripetal growth of a septum, which impinges on a persistent, centrifugally expanding telophase spindle, leading to a phragmoplast-like structure of potential phylogenetic significance (Fowke, L. C., and J. D. Pickett-Heaps. 1969. J. Phycol. 5:273-281). Combining fluorescent tagging of the cytoskeleton in situ and video- enhanced differential interference contrast microscopy of live cells, the process of cytokinesis was investigated with emphasis on cytoskeletal reorganization and concomitant redistribution of organelles. Based on a sequence of cytoskeletal arrangements and the effects of cytoskeletal inhibitors thereon, cytokinetic progression could be divided into three functional stages with respect to the contribution of microfilaments (MFs) and microtubules (MTs): (1) Initiation: in early prophase, a cross wall initial was formed independently of MFs and MTs at the presumptive site of wall growth. (2) Septum ingrowth: numerous organelles accumulated at the cross wall initial concomitant with reorganization of the extensive peripheral interphase MF array into a distinct circumferential MF array. This array guided the ingrowing septum until it contacted the expanding interzonal MT array. (3) Cross wall closure: MFs at the growing edge of the septum coaligned with and extended along the interzonal MTs toward the daughter nuclei. Thus, actin-based transportation of small organelles during this third stage occurred, in part, along a scaffold previously deployed in space by MTs. Displacement of the nuclei- associated interzonal MT array by centrifugation and depolymerization of the phragmoplast-like structure showed that the success of cytokinesis at the third stage depends on the interaction of both MF and MT cytoskeletons. Important features of the phragmoplast-like structure in Spirogyra were different from the higher plant phragmoplast: in particular, MFs were responsible for the

  14. The macro domain protein family: structure, functions, and their potential therapeutic implications.

    PubMed

    Han, Weidong; Li, Xiaolei; Fu, Xiaobing

    2011-01-01

    Macro domains are ancient, highly evolutionarily conserved domains that are widely distributed throughout all kingdoms of life. The 'macro fold' is roughly 25kDa in size and is composed of a mixed α-β fold with similarity to the P loop-containing nucleotide triphosphate hydrolases. They function as binding modules for metabolites of NAD(+), including poly(ADP-ribose) (PAR), which is synthesized by PAR polymerases (PARPs). Although there is a high degree of sequence similarity within this family, particularly for residues that might be involved in catalysis or substrates binding, it is likely that the sequence variation that does exist among macro domains is responsible for the specificity of function of individual proteins. Recent findings have indicated that macro domain proteins are functionally promiscuous and are implicated in the regulation of diverse biological functions, such as DNA repair, chromatin remodeling and transcriptional regulation. Significant advances in the field of macro domain have occurred in the past few years, including biological insights and the discovery of novel signaling pathways. To provide a framework for understanding these recent findings, this review will provide a comprehensive overview of the known and proposed biochemical, cellular and physiological roles of the macro domain family. Recent data that indicate a critical role of macro domain regulation for the proper progression of cellular differentiation programs will be discussed. In addition, the effect of dysregulated expression of macro domain proteins will be considered in the processes of tumorigenesis and bacterial pathogenesis. Finally, a series of observations will be highlighted that should be addressed in future efforts to develop macro domains as effective therapeutic targets.

  15. Modulation and Functional Role of the Orientations of the N- and P-Domains of Cu+ -Transporting ATPase along the Ion Transport Cycle.

    PubMed

    Meng, Dan; Bruschweiler-Li, Lei; Zhang, Fengli; Brüschweiler, Rafael

    2015-08-18

    Ion transport of different P-type ATPases is regulated similarly through the interplay of multiple protein domains. In the presence of ATP, binding of a cation to the ion binding site in the transmembrane helices leads to the phosphorylation of the P-domain, allowing ion transfer across the membrane. The details of the mechanism, however, are not clear. Here, we report the modulation of the orientation between the N- and P-domains of Cu(+)-transporting ATPase along the ion transport cycle using high-resolution nuclear magnetic resonance spectroscopy in solution. On the basis of residual dipolar coupling measurements, it is found that the interdomain orientation (relative openness) of the N- and P-domains is distinctly modulated depending on the specific state of the N- and P-domains along the ion translocation cycle. The two domains' relative position in the apo state is semiopen, whereas it becomes closed upon binding of ATP to the N-domain. After phosphorylation of the P-domain and the release of ADP, the opening, however, becomes the widest among all the states. We reason such wide opening resulting from the departure of ADP prepares the N- and P-domains to accommodate the A-domain for interaction and, hence, promote ion transport and allow dephosphorylation of the P-domain. Such wide interdomain opening is abolished when an Asn to Asp mutation is introduced into the conserved DXXK motif located in the hinge region of the N- and P-domains of Cu(+)-ATPase, suggesting the indispensible role of the N- and P-interdomain orientation during ion transportation. Our results shed new light on the structural and mechanistic details of P-type ATPase function at large.

  16. 55 Amino acid linker between helicase and carboxyl terminal domains of RIG-I functions as a critical repression domain and determines inter-domain conformation.

    PubMed

    Kageyama, Maiko; Takahasi, Kiyohiro; Narita, Ryo; Hirai, Reiko; Yoneyama, Mitsutoshi; Kato, Hiroki; Fujita, Takashi

    2011-11-11

    In virus-infected cells, viral RNA with non-self structural pattern is recognized by DExD/Hbox RNA helicase, RIG-I. Once RIG-I senses viral RNA, it triggers a signaling cascade, resulting in the activation of genes including type I interferon, which activates antiviral responses. Overexpression of N-terminal caspase activation and recruitment domain (CARD) is sufficient to activate signaling; however basal activity of full-length RIG-I is undetectable. The repressor domain (RD), initially identified as a.a. 735-925, is responsible for diminished basal activity; therefore, it is suggested that RIG-I is under auto-repression in uninfected cells and the repression is reversed upon its encounter with viral RNA. In this report, we further delimited RD to a.a. 747-801, which corresponds to a linker connecting the helicase and the C-terminal domain (CTD). Alanine substitutions of the conserved residues in the linker conferred constitutive activity to full-length RIG-I. We found that the constitutive active mutants do not exhibit ATPase activity, suggesting that ATPase is required for de-repression but not signaling itself. Furthermore, trypsin digestion of recombinant RIG-I revealed that the wild-type, but not linker mutant conforms to the trypsin-resistant structure, containing CARD and helicase domain. The result strongly suggests that the linker is responsible for maintaining RIG-I in a "closed" structure to minimize unwanted production of interferon in uninfected cells. These findings shed light on the structural regulation of RIG-I function.

  17. Implications of 3D domain swapping for protein folding, misfolding and function.

    PubMed

    Rousseau, Frederic; Schymkowitz, Joost; Itzhaki, Laura S

    2012-01-01

    Three-dimensional domain swapping is the process by which two identical protein chains exchange a part of their structure to form an intertwined dimer or higher-order oligomer. The phenomenon has been observed in the crystal structures of a range of different proteins. In this chapter we review the experiments that have been performed in order to understand the sequence and structural determinants of domain-swapping and these show how the general principles obtained can be used to engineer proteins to domain swap. We discuss the role of domain swapping in regulating protein function and as one possible mechanism of protein misfolding that can lead to aggregation and disease. We also review a number of interesting pathways of macromolecular assembly involving β-strand insertion or complementation that are related to the domain-swapping phenomenon.

  18. Characterization of the Functional Domains of a Mammalian Voltage-Sensitive Phosphatase.

    PubMed

    Rosasco, Mario G; Gordon, Sharona E; Bajjalieh, Sandra M

    2015-12-15

    Voltage-sensitive phosphatases (VSPs) are proteins that directly couple changes in membrane electrical potential to inositol lipid phosphatase activity. VSPs thus couple two signaling pathways that are critical for cellular functioning. Although a number of nonmammalian VSPs have been characterized biophysically, mammalian VSPs are less well understood at both the physiological and biophysical levels. In this study, we aimed to address this gap in knowledge by determining whether the VSP from mouse, Mm-VSP, is expressed in the brain and contains a functional voltage-sensing domain (VSD) and a phosphatase domain. We report that Mm-VSP is expressed in neurons and is developmentally regulated. To address whether the functions of the VSD and phosphatase domain are retained in Mm-VSP, we took advantage of the modular nature of these domains and expressed each independently as a chimeric protein in a heterologous expression system. We found that the Mm-VSP VSD, fused to a viral potassium channel, was able to drive voltage-dependent gating of the channel pore. The Mm-VSP phosphatase domain, fused to the VSD of a nonmammalian VSP, was also functional: activation resulted in PI(4,5)P2 depletion that was sufficient to inhibit the PI(4,5)P2-regulated KCNQ2/3 channels. While testing the functionality of the VSD and phosphatase domain, we observed slight differences between the activities of Mm-VSP-based chimeras and those of nonmammalian VSPs. Although the properties of VSP chimeras may not completely reflect the properties of native VSPs, the differences we observed in voltage-sensing and phosphatase activity provide a starting point for future experiments to investigate the function of Mm-VSP and other mammalian VSPs. In conclusion, our data reveal that both the VSD and the lipid phosphatase domain of Mm-VSP are functional, indicating that Mm-VSP likely plays an important role in mouse neurophysiology.

  19. Functional Organization of the Yeast SAGA Complex: Distinct Components Involved in Structural Integrity, Nucleosome Acetylation, and TATA-Binding Protein Interaction

    PubMed Central

    Sterner, David E.; Grant, Patrick A.; Roberts, Shannon M.; Duggan, Laura J.; Belotserkovskaya, Rimma; Pacella, Lisa A.; Winston, Fred; Workman, Jerry L.; Berger, Shelley L.

    1999-01-01

    SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function. Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex. We demonstrate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity. Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes. Interestingly, double mutants (gcn5Δ spt3Δ and gcn5Δ spt8Δ) causing loss of a member of each of the moderate classes have severe phenotypes, similar to spt7Δ, spt20Δ, or ada1Δ mutants. In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain. Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo. Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro. Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation. Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription. PMID:9858534

  20. Domain structure and function of matrix metalloprotease 23 (MMP23): role in potassium channel trafficking.

    PubMed

    Galea, Charles A; Nguyen, Hai M; George Chandy, K; Smith, Brian J; Norton, Raymond S

    2014-04-01

    MMP23 is a member of the matrix metalloprotease family of zinc- and calcium-dependent endopeptidases, which are involved in a wide variety of cellular functions. Its catalytic domain displays a high degree of structural homology with those of other metalloproteases, but its atypical domain architecture suggests that it may possess unique functional properties. The N-terminal MMP23 pro-domain contains a type-II transmembrane domain that anchors the protein to the plasma membrane and lacks the cysteine-switch motif that is required to maintain other MMPs in a latent state during passage to the cell surface. Instead of the C-terminal hemopexin domain common to other MMPs, MMP23 contains a small toxin-like domain (TxD) and an immunoglobulin-like cell adhesion molecule (IgCAM) domain. The MMP23 pro-domain can trap Kv1.3 but not closely-related Kv1.2 channels in the endoplasmic reticulum, preventing their passage to the cell surface, while the TxD can bind to the channel pore and block the passage of potassium ions. The MMP23 C-terminal IgCAM domain displays some similarity to Ig-like C2-type domains found in IgCAMs of the immunoglobulin superfamily, which are known to mediate protein-protein and protein-lipid interactions. MMP23 and Kv1.3 are co-expressed in a variety of tissues and together are implicated in diseases including cancer and inflammatory disorders. Further studies are required to elucidate the mechanism of action of this unique member of the MMP family.

  1. Disparate evolution of prion protein domains and the distinct origin of Doppel- and prion-related loci revealed by fish-to-mammal comparisons.

    PubMed

    Rivera-Milla, Eric; Oidtmann, Birgit; Panagiotidis, Cynthia H; Baier, Michael; Sklaviadis, Theodoros; Hoffmann, Rudolf; Zhou, Yi; Solis, Gonzalo P; Stuermer, Claudia A O; Málaga-Trillo, Edward

    2006-02-01

    Prions result from the misfolding and selective accumulation of the host-encoded prion protein (PrP) in the brain. Despite intensive research on mammalian models, basic questions about the biological role of PrP and the evolutionary origin of prion disease remain unanswered. Following our previous identification of novel fish PrP homologues, here we generated new fish PrP sequences and performed genomic analysis to demonstrate the existence of two homologous PrP loci in bony fish, which display extensive molecular variation and are highly expressed in adult and developing fish brains. The fish PrP genomic regions contain PrP-related loci directly downstream of each PrP locus, suggesting an independent origin of prion-related proteins in fish and mammals. Our structural prediction analysis uncovers a conserved molecular "bauplan" for all vertebrate PrPs. The C- and N-terminal protein domains have evolved independently from one another, the former having retained its basic globular structure despite high sequence divergence and the latter having undergone differential expansion-degeneration cycles in its repetitive domains. Our evolutionary analysis redefines fundamental concepts on the functional significance of PrP domains and opens up new possibilities for the experimental analysis of prion misfolding and neurodegeneration in a non-mammalian model like the zebrafish.

  2. Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction.

    PubMed Central

    Nicholson, S E; Willson, T A; Farley, A; Starr, R; Zhang, J G; Baca, M; Alexander, W S; Metcalf, D; Hilton, D J; Nicola, N A

    1999-01-01

    SOCS-1 (suppressor of cytokine signaling-1) is a representative of a family of negative regulators of cytokine signaling (SOCS-1 to SOCS-7 and CIS) characterized by a highly conserved C-terminal SOCS box preceded by an SH2 domain. This study comprehensively examined the ability of several SOCS family members to negatively regulate the gp130 signaling pathway. SOCS-1 and SOCS-3 inhibited both interleukin-6 (IL-6)- and leukemia inhibitory factor (LIF)-induced macrophage differentiation of murine monocytic leukemic M1 cells and LIF induction of a Stat3-responsive reporter construct in 293T fibroblasts. Deletion of amino acids 51-78 in the N-terminal region of SOCS-1 prevented inhibition of LIF signaling. The SOCS-1 and SOCS-3 N-terminal regions were functionally interchangeable, but this did not extend to other SOCS family members. Mutation of SH2 domains abrogated the ability of both SOCS-1 and SOCS-3 to inhibit LIF signal transduction. Unlike SOCS-1, SOCS-3 was unable to inhibit JAK kinase activity in vitro, suggesting that SOCS-1 and SOCS-3 act on the JAK-STAT pathway in different ways. Thus, although inhibition of signaling by SOCS-1 and SOCS-3 requires both the SH2 and N-terminal domains, their mechanisms of action appear to be biochemically different. PMID:9889194

  3. Bile acids modulate signaling by functional perturbation of plasma membrane domains.

    PubMed

    Zhou, Yong; Maxwell, Kelsey N; Sezgin, Erdinc; Lu, Maryia; Liang, Hong; Hancock, John F; Dial, Elizabeth J; Lichtenberger, Lenard M; Levental, Ilya

    2013-12-13

    Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

  4. Domain B protruding at the third beta strand of the alpha/beta barrel in barley alpha-amylase confers distinct isozyme-specific properties.

    PubMed

    Rodenburg, K W; Juge, N; Guo, X J; Søgaard, M; Chaix, J C; Svensson, B

    1994-04-01

    alpha-Amylases belong to the alpha/beta-barrel protein family in which the active site is created by residues located at the C-terminus of the beta strands and in the helix-connecting loops extending from these ends. In the alpha-amylase family, a small separate domain B protrudes at the C-terminus of the third beta strand of the (beta/alpha)8-barrel framework. The 80% identical barley alpha-amylase isozymes 1 and 2 (AMY1 and AMY2, respectively) differ in substrate affinity and turnover rate, CaCl2 stimulation of activity, sensitivity to the endogenous 21-kDa alpha-amylase/subtilisin inhibitor, and stability at low pH. To identify regions that confer these isozyme-specific variations, AMY1-AMY2 hybrid cDNAs were generated by in vivo homologous recombination in yeast. The hybrids AMY1-(1-90)-AMY2-(90-403) and AMY1-(1-161)-AMY2-(161-403) characterized in this study contain the 90-residue and 161-residue N-terminal sequences, respectively, of AMY1 and complementary C-terminal regions of AMY2. AMY1-(1-90)-AMY2-(90-403) comprises the 60-amino-acid domain B of AMY2 and resembles this isozyme in sensitivity to alpha-amylase/subtilisin inhibitor and its low affinity for the substrates p-nitrophenyl alpha-D-maltoheptaoside, amylose and the inhibitor acarbose. Only AMY1-(1-161)-AMY2-(161-403) and AMY1, which both share domain B, are stable at low pH. However, AMY2 and both hybrid AMY species, but not AMY1, show maximum enzyme activity on insoluble blue starch at approximately 10 mM CaCl2. Domain B thus determines several functional and stability properties that distinguish the barley alpha-amylase isozymes.

  5. 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

  6. Optimization of the functional domain of flat plate collectors

    NASA Astrophysics Data System (ADS)

    Ritoux, G.; Irigaray, J.-L.

    1981-12-01

    The variations of the extracted heat flux as function of the temperature of the heat transfer fluid in black and selective surface solar collectors are examined. The heat flux is calculated based on the difference of the initial to the stage of thermal equilibrium of the fluid. A nonlinear system of equations is developed and solved by a fast, iterative method to obtain the equilibrium temperatures. It is found that more flux can be extracted from the solar heat by a collector with only one glass cover than with more than one cover. The captured flux is proportional to the coefficient of transmission of the glass coverings, to the coefficient of absorption of the collector, and to the incident flux. Black painted surfaces were more absorbent than selective surfaces, and highest collection efficiencies were displayed by low temperature collectors. Charts of effective uses of the respective types of collectors for heating swimming pools, hot water, home heat, and for refrigeration and air-conditioning are provided.

  7. Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection

    PubMed Central

    Diner, Benjamin A.; Lum, Krystal K.; Toettcher, Jared E.

    2016-01-01

    ABSTRACT The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses. PMID:27935834

  8. Predicting protein N-glycosylation by combining functional domain and secretion information.

    PubMed

    Li, Sujun; Liu, Boshu; Cai, Yudong; Li, Yixue

    2007-08-01

    Protein N-glycosylation plays an important role in protein function. Yet, at present, few computational methods are available for the prediction of this protein modification. This prompted our development of a support vector machine (SVM)-based method for this task, as well as a partial least squares (PLS) regression based prediction method for comparison. A functional domain feature space was used to create SVM and PLS models, which achieved accuracies of 83.91% and 79.89%, respectively, as evaluated by a leave-one-out cross-validation. Subsequently, SVM and PLS models were developed based on functional domain and protein secretion information, which yielded accuracies of 89.13% and 86%, respectively. This analysis demonstrates that the protein functional domain and secretion information are both efficient predictors of N-glycosylation.

  9. GTP binding to the ROC domain of DAP-kinase regulates its function through intramolecular signalling.

    PubMed

    Carlessi, Rodrigo; Levin-Salomon, Vered; Ciprut, Sara; Bialik, Shani; Berissi, Hanna; Albeck, Shira; Peleg, Yoav; Kimchi, Adi

    2011-09-01

    Death-associated protein kinase (DAPk) was recently suggested by sequence homology to be a member of the ROCO family of proteins. Here, we show that DAPk has a functional ROC (Ras of complex proteins) domain that mediates homo-oligomerization and GTP binding through a defined P-loop motif. Upon binding to GTP, the ROC domain negatively regulates the catalytic activity of DAPk and its cellular effects. Mechanistically, GTP binding enhances an inhibitory autophosphorylation at a distal site that suppresses kinase activity. This study presents a new mechanism of intramolecular signal transduction, by which GTP binding operates in cis to affect the catalytic activity of a distal domain in the protein.

  10. Sensory Processing in Low-Functioning Adults with Autism Spectrum Disorder: Distinct Sensory Profiles and Their Relationships with Behavioral Dysfunction

    ERIC Educational Resources Information Center

    Gonthier, Corentin; Longuépée, Lucie; Bouvard, Martine

    2016-01-01

    Sensory processing abnormalities are relatively universal in individuals with autism spectrum disorder, and can be very disabling. Surprisingly, very few studies have investigated these abnormalities in low-functioning adults with autism. The goals of the present study were (a) to characterize distinct profiles of sensory dysfunction, and (b) to…

  11. 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.

  12. 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.

  13. 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.

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

    PubMed

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

    2016-07-05

    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.

  15. The Rad50 coiled-coil domain is indispensable for Mre11 complex functions.

    PubMed

    Hohl, Marcel; Kwon, Youngho; Galván, Sandra Muñoz; Xue, Xiaoyu; Tous, Cristina; Aguilera, Andrés; Sung, Patrick; Petrini, John H J

    2011-09-04

    The Mre11 complex (Mre11, Rad50 and Xrs2 in Saccharomyces cerevisiae) influences diverse functions in the DNA damage response. The complex comprises the globular DNA-binding domain and the Rad50 hook domain, which are linked by a long and extended Rad50 coiled-coil domain. In this study, we constructed rad50 alleles encoding truncations of the coiled-coil domain to determine which Mre11 complex functions required the full length of the coils. These mutations abolished telomere maintenance and meiotic double-strand break (DSB) formation, and severely impaired homologous recombination, indicating a requirement for long-range action. Nonhomologous end joining, which is probably mediated by the globular domain of the Mre11 complex, was also severely impaired by alteration of the coiled-coil and hook domains, providing the first evidence of their influence on this process. These data show that functions of Mre11 complex are integrated by the coiled coils of Rad50.

  16. Structure and function of the SWIRM domain, a conserved protein module found in chromatin regulatory complexes.

    PubMed

    Da, Guoping; Lenkart, Jeffrey; Zhao, Kehao; Shiekhattar, Ramin; Cairns, Bradley R; Marmorstein, Ronen

    2006-02-14

    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. Specific anchoring modes of two distinct dystrophin rod sub-domains interacting in phospholipid Langmuir films studied by atomic force microscopy and PM-IRRAS.

    PubMed

    Vié, V; Legardinier, S; Chieze, L; Le Bihan, O; Qin, Y; Sarkis, J; Hubert, J-F; Renault, A; Desbat, B; Le Rumeur, E

    2010-08-01

    Dystrophin rod repeats 1-3 sub-domain binds to acidic phosphatidylserine in a small vesicle binding assay, while the repeats 20-24 sub-domain does not. In the present work, we studied the adsorption behaviour of both sub-domains at the air/liquid interface and at the air/lipid interface in a Langmuir trough in order to highlight differences in interfacial properties. The adsorption behaviour of the two proteins at the air/liquid interface shows that they display surface activity while maintaining their alpha-helical secondary structure as shown by PM-IRRAS. Strikingly, R20-24 needs to be highly hydrated even at the interface, while this is not the case for R1-3, indicating that the surface activity is dramatically higher for R1-3 than R20-24. Surface-pressure measurements, atomic force microscopy and PM-IRRAS are used in a Langmuir experiment with DOPC-DOPS monolayers at two different surface pressures, 20 mN/m and 30 mN/m. At the lower surface pressure, the proteins are adsorbed at the lipid film interface while maintaining its alpha-helical structure. After an increase of the surface pressure, R1-3 subsequently produces a stable film, while R20-24 induces a reorganization of the lipid film with a subsequent decrease of the surface pressure close to the initial value. AFM and PM-IRRAS show that R1-3 is present in high amounts at the interface, being arranged in clusters representing 3.3% of the surface at low pressure. By contrast, R20-24 is present at the interface in small amounts bound only by a few electrostatic residues to the lipid film while the major part of the molecule remains floating in the sub-phase. Then for R1-3, the electrostatic interaction between the proteins and the film is enhanced by hydrophobic interactions. At higher surface pressure, the number of protein clusters increases and becomes closer in both cases implying the electrostatic character of the binding. These results indicate that even if the repeats exhibit large structural

  18. The Ω-loop lid domain of phosphoenolpyruvate carboxykinase is essential for catalytic function.

    PubMed

    Johnson, Troy A; Holyoak, Todd

    2012-11-27

    Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Recent studies have demonstrated that the enzyme contains a mobile active site lid domain that undergoes a transition between an open, disorded conformation and a closed, ordered conformation as the enzyme progresses through the catalytic cycle. The understanding of how this mobile domain functions in catalysis is incomplete. Previous studies showed that the closure of the lid domain stabilizes the reaction intermediate and protects the reactive intermediate from spurious protonation and thus contributes to the fidelity of the enzyme. To more fully investigate the roles of the lid domain in PEPCK function, we introduced three mutations that replaced the 11-residue lid domain with one, two, and three glycine residues. Kinetic analysis of the mutant enzymes demonstrates that none of the enzyme constructs exhibit any measurable kinetic activity, resulting in a decrease in the catalytic parameters of at least 10(6). Structural characterization of the mutants in complexes representing the catalytic cycle suggests that the inactivity is due to a role for the lid domain in the formation of the fully closed state of the enzyme that is required for catalytic function. In the absence of the lid domain, the enzyme is unable to achieve the fully closed state and is rendered inactive despite possessing all of the residues and substrates required for catalytic function. This work demonstrates how enzyme catalytic function can be abolished through the alteration of conformational equilibria despite all the elements required for chemical conversion of substrates to products remaining intact.

  19. Hyperandrogenism in female athletes with functional hypothalamic amenorrhea: a distinct phenotype

    PubMed Central

    Javed, Asma; Kashyap, Rahul; Lteif, Aida N

    2015-01-01

    Objective To compare the reproductive, metabolic, and skeletal profiles of young athletic women with functional hypothalamic amenorrhea (FHA) as well as clinical or biochemical hyperandrogenism (FHA-EX+HA) with body mass index matched women with FHA due to exercise (FHA-EX) or anorexia nervosa (FHA-AN) alone. Design Retrospective cohort study. Setting Tertiary care teaching hospital. Population Adolescents and young women, 15–30 years of age, diagnosed with FHA along with concurrent signs of hyperandrogenism (n=22) and body mass index matched control groups consisting of 22 women in each group of FHA-EX and FHA-AN. Main outcomes 1) Reproductive hormone profile: luteinizing hormone (LH), follicle stimulating hormone (FSH), total testosterone, pelvic ultrasound features. 2) Metabolic function and skeletal health markers: fasting glucose, cholesterol, number of stress fractures and bone mineral density as assessed by spine dual-energy X-ray absorptiometry z scores. Results FHA-EX+HA group was older at diagnosis compared to the other groups with a median (interquartile range [IQR]) age of 22 (18.75–25.25) years versus (vs) 17.5 (15.75–19) for FHA-EX; (P<0.01) and 18 (16–22.25) years for FHA-AN (P=0.01). There were no differences among the groups based on number of hours of exercise per week, type of physical activity or duration of amenorrhea. Median (IQR) LH/FSH ratio was higher in FHA-EX+HA than both other groups, 1.44 (1.03–1.77) vs 0.50 (0.20–0.94) for FHA-EX and 0.67 (0.51–0.87) for FHA-AN (P<0.01 for both). Total testosterone concentrations were not different among the groups. Median (IQR) fasting serum glucose concentration was higher in FHA-EX+HA vs FHA-EX, 88.5 mg/dL (82.8–90 mg/dL) vs 83.5 mg/dL (78.8–86.3 mg/dL) (P=0.01) but not different from FHA-AN (P=0.31). Percentage of women with stress fractures was lower in FHA-EX+HA (4.5%) as compared to both FHA-EX (27.3%) and FHA-AN (50%); P=0.04 and 0.01 respectively. The LH/FSH ratio was weakly

  20. The carboxyl terminus of the chemokine receptor CCR3 contains distinct domains which regulate chemotactic signaling and receptor down-regulation in a ligand-dependent manner.

    PubMed

    Sabroe, Ian; Jorritsma, Annelies; Stubbs, Victoria E L; Xanthou, Georgina; Jopling, Louise A; Ponath, Paul D; Williams, Timothy J; Murphy, Philip M; Pease, James E

    2005-04-01

    The chemokine receptor CCR3 regulates the chemotaxis of leukocytes implicated in allergic disease, such as eosinophils. Incubation of eosinophils with CCL11, CCL13 or CCL5 resulted in a rapid decrease of cell-surface CCR3 which was replicated using CCR3 transfectants. Progressive truncation of the CCR3 C terminus by 15 amino acids produced three constructs, Delta340, Delta325 and Delta310. Delta340 and Delta325 were able to bind CCL11 with affinities similar to wild-type CCR3. Delta340 transfectants exhibited enhanced migration and reduced receptor down-regulation in response to CCL11 and CCL13. Delta325 transfectants displayed chemotactic responses to CCL11 and CCL13 similar to wild-type CCR3, and had impaired down-regulation when stimulated with CCL13 but not CCL11. In contrast, neither the Delta325 nor Delta340 truncation affected chemotaxis or receptor down-regulation induced by CCL5. Delta310 transfectants bound CCL11 poorly and were biologically inactive. Inhibitors of p38 mitogen-activated protein kinase and PI3-kinase antagonized eosinophil shape change responses and chemotaxis of transfectants to CCL11 and CCL13. In contrast, shape change but not chemotaxis was sensitive to inhibition of the extracellular signal-regulated kinase kinase pathway suggesting differential regulation of the two responses. Thus, the CCR3 C terminus contains distinct domains responsible for the regulation of receptor desensitization and for coupling to chemotactic responses.

  1. Janus Model of The Na,K-ATPase β-subunit Transmembrane Domain: Distinct Faces Mediate α /β Assembly and β-β Homo-Oligomerization

    PubMed Central

    Barwe, Sonali P.; Kim, Sanguk; Rajasekaran, Sigrid A.; Bowie, James U.; Rajasekaran, Ayyappan K.

    2007-01-01

    Summary Na,K-ATPase is a hetero-oligomer of α- and β-subunits. The Na,K-ATPase β-subunit (Na,K-β ) is involved in both the regulation of ion transport activity, and in cell-cell adhesion. By structure prediction and evolutionary analysis, we identified two distinct faces on the Na,K-β transmembrane domain (TMD) that could mediate protein-protein interactions: a glycine zipper motif and a conserved heptad repeat. Here, we show that the heptad repeat face is involved in the hetero-oligomeric interaction of Na,K-β with Na,K-α , and the glycine zipper face is involved in the homo-oligomerization of Na,K-β . Point mutations in the heptad repeat motif reduced Na,K-β binding to Na,K-α , and Na,K-ATPase activity. Na,K-β TMD homo-oligomerized in biological membranes, and mutation of the glycine zipper motif affected oligomerization and cell-cell adhesion. These results provide a structural basis for understanding how Na,K-β links ion transport and cell-cell adhesion. PMID:17078968

  2. The Novel Plant Protein INAPERTURATE POLLEN1 Marks Distinct Cellular Domains and Controls Formation of Apertures in the Arabidopsis Pollen Exine[C][W

    PubMed Central

    Dobritsa, Anna A.; Coerper, Daniel

    2012-01-01

    Pollen grains protect the sperm cells inside them with the help of the unique cell wall, the exine, which exhibits enormous morphological variation across plant taxa, assembling into intricate and diverse species-specific patterns. How this complex extracellular structure is faithfully deposited at precise sites and acquires precise shape within a species is not understood. Here, we describe the isolation and characterization of the novel Arabidopsis thaliana gene INAPERTURATE POLLEN1 (INP1), which is specifically involved in formation of the pollen surface apertures, which arise by restriction of exine deposition at specific sites. Loss of INP1 leads to the loss of all three apertures in Arabidopsis pollen, and INP1 protein exhibits a unique tripartite localization in developing pollen, indicative of its direct involvement in specification of aperture positions. We also show that aperture length appears to be sensitive to INP1 dosage and INP1 misexpression can affect global exine patterning. Phenotypes of some inp1 mutants indicate that Arabidopsis apertures are initiated at three nonrandom positions around the pollen equator. The identification of INP1 opens up new avenues for studies of how formation of distinct cellular domains results in the production of different extracellular morphologies. PMID:23136373

  3. 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.

  4. Structural and functional definition of the human chitinase chitin-binding domain.

    PubMed

    Tjoelker, L W; Gosting, L; Frey, S; Hunter, C L; Trong, H L; Steiner, B; Brammer, H; Gray, P W

    2000-01-07

    Mammalian chitinase, a chitinolytic enzyme expressed by macrophages, has been detected in atherosclerotic plaques and is elevated in blood and tissues of guinea pigs infected with Aspergillus. Its normal physiological function is unknown. To understand how the enzyme interacts with its substrate, we have characterized the chitin-binding domain. The C-terminal 49 amino acids make up the minimal sequence required for chitin binding activity. The absence of this domain does not affect the ability of the enzyme to hydrolyze the soluble substrate, triacetylchitotriose, but abolishes hydrolysis of insoluble chitin. Within the minimal chitin-binding domain are six cysteines; mutation of any one of these to serine results in complete loss of chitin binding activity. Analysis of purified recombinant chitin-binding domain revealed the presence of three disulfide linkages. The recombinant domain binds specifically to chitin but does not bind chitosan, cellulose, xylan, beta-1, 3-glucan, beta-1,3-1,4-glucan, or mannan. Fluorescently tagged chitin-binding domain was used to demonstrate chitin-specific binding to Saccharomyces cerevisiae, Candida albicans, Mucor rouxii, and Neurospora crassa. These experiments define structural features of the minimal domain of human chitinase required for both specifically binding to and hydrolyzing insoluble chitin and demonstrate relevant binding within the context of the fungal cell wall.

  5. 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

  6. Localization and Function of Pals1-associated Tight Junction Protein in Drosophila Is Regulated by Two Distinct Apical Complexes.

    PubMed

    Sen, Arnab; Sun, Rui; Krahn, Michael P

    2015-05-22

    The transmembrane protein Crumbs (Crb) and its intracellular adaptor protein Pals1 (Stardust, Sdt in Drosophila) play a crucial role in the establishment and maintenance of apical-basal polarity in epithelial cells in various organisms. In contrast, the multiple PDZ domain-containing protein Pals1-associated tight junction protein (PATJ), which has been described to form a complex with Crb/Sdt, is not essential for apical basal polarity or for the stability of the Crb/Sdt complex in the Drosophila epidermis. Here we show that, in the embryonic epidermis, Sdt is essential for the correct subcellular localization of PATJ in differentiated epithelial cells but not during cellularization. Consistently, the L27 domain of PATJ is crucial for the correct localization and function of the protein. Our data further indicate that the four PDZ domains of PATJ function, to a large extent, in redundancy, regulating the function of the protein. Interestingly, the PATJ-Sdt heterodimer is not only recruited to the apical cell-cell contacts by binding to Crb but depends on functional Bazooka (Baz). However, biochemical experiments show that PATJ associates with both complexes, the Baz-Sdt and the Crb-Sdt complex, in the mature epithelium of the embryonic epidermis, suggesting a role of these two complexes for the function of PATJ during the development of Drosophila.

  7. Structural and functional characterization of a cold adapted TPM-domain with ATPase/ADPase activity.

    PubMed

    Cerutti, María L; Otero, Lisandro H; Smal, Clara; Pellizza, Leonardo; Goldbaum, Fernando A; Klinke, Sebastián; Aran, Martín

    2016-11-01

    The Pfam PF04536 TPM_phosphatase family is a broadly conserved family of domains found across prokaryotes, plants and invertebrates. Despite having a similar protein fold, members of this family have been implicated in diverse cellular processes and found in varied subcellular localizations. Very recently, the biochemical characterization of two evolutionary divergent TPM domains has shown that they are able to hydrolyze phosphate groups from different substrates. However, there are still incorrect functional annotations and uncertain relationships between the structure and function of this family of domains. BA41 is an uncharacterized single-pass transmembrane protein from the Antarctic psychrotolerant bacterium Bizionia argentinensis with a predicted compact extracytoplasmic TPM domain and a C-terminal cytoplasmic low complexity region. To shed light on the structural properties that enable TPM domains to adopt divergent roles, we here accomplish a comprehensive structural and functional characterization of the central TPM domain of BA41 (BA41-TPM). Contrary to its predicted function as a beta-propeller methanol dehydrogenase, light scattering and crystallographic studies showed that BA41-TPM behaves as a globular monomeric protein and adopts a conserved Rossmann fold, typically observed in other TPM domain structures. Although the crystal structure reveals the conservation of residues involved in substrate binding, no putative catalytic or intramolecular metal ions were detected. Most important, however, extensive biochemical studies demonstrated that BA41-TPM has hydrolase activity against ADP, ATP, and other di- and triphosphate nucleotides and shares properties of cold-adapted enzymes. The role of BA41 in extracellular ATP-mediated signaling pathways and its occurrence in environmental and pathogenic microorganisms is discussed.

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

    PubMed

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

    2011-11-08

    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

  9. 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

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

    PubMed Central

    Tian, Wei; Yu, Jianzhong; Tomchick, Diana R.; Pan, Duojia; Luo, Xuelian

    2010-01-01

    The Hippo pathway controls organ size and suppresses tumorigenesis in metazoans by blocking cell proliferation and promoting apoptosis. The TEAD1-4 proteins (which contain a DNA-binding domain but lack an activation domain) interact with YAP (which lacks a DNA-binding domain but contains an activation domain) to form functional heterodimeric transcription factors that activate proliferative and prosurvival gene expression programs. The Hippo pathway inhibits the YAP-TEAD hybrid transcription factors by phosphorylating and promoting cytoplasmic retention of YAP. Here we report the crystal structure of the YAP-binding domain (YBD) of human TEAD2. TEAD2 YBD adopts an immunoglobulin-like β-sandwich fold with two extra helix-turn-helix inserts. NMR studies reveal that the TEAD-binding domain of YAP is natively unfolded and that TEAD binding causes localized conformational changes in YAP. In vitro binding and in vivo functional assays define an extensive conserved surface of TEAD2 YBD as the YAP-binding site. Therefore, our studies suggest that a short segment of YAP adopts an extended conformation and forms extensive contacts with a rigid surface of TEAD. Targeting a surface-exposed pocket of TEAD might be an effective strategy to disrupt the YAP-TEAD interaction and to reduce the oncogenic potential of YAP. PMID:20368466

  11. FAH Domain Containing Protein 1 (FAHD-1) Is Required for Mitochondrial Function and Locomotion Activity in C. elegans

    PubMed Central

    Taferner, Andrea; Pircher, Haymo; Koziel, Rafal; von Grafenstein, Susanne; Baraldo, Giorgia; Palikaras, Konstantinos; Liedl, Klaus R.; Tavernarakis, Nektarios; Jansen-Dürr, Pidder

    2015-01-01

    The fumarylacetoacetate hydrolase (FAH) protein superfamily of metabolic enzymes comprises a diverse set of enzymatic functions, including ß-diketone hydrolases, decarboxylases, and isomerases. Of note, the FAH superfamily includes many prokaryotic members with very distinct functions that lack homologs in eukaryotes. A prokaryotic member of the FAH superfamily, referred to as Cg1458, was shown to encode a soluble oxaloacetate decarboxylase (ODx). Based on sequence homologies to Cg1458, we recently identified human FAH domain containing protein-1 (FAHD1) as the first eukaryotic oxaloacetate decarboxylase. The physiological functions of ODx in eukaryotes remain unclear. Here we have probed the function of fahd-1, the nematode homolog of FAHD1, in the context of an intact organism. We found that mutation of fahd-1 resulted in reduced brood size, a deregulation of the egg laying process and a severe locomotion deficit, characterized by a reduced frequency of body bends, reduced exploratory movements and reduced performance in an endurance exercise test. Notably, mitochondrial function was altered in the fahd-1(tm5005) mutant strain, as shown by a reduction of mitochondrial membrane potential and a reduced oxygen consumption of fahd-1(tm5005) animals. Mitochondrial dysfunction was accompanied by lifespan extension in worms grown at elevated temperature; however, unlike in mutant worms with a defect in the electron transport chain, the mitochondrial unfolded protein response was not upregulated in worms upon inactivation of fahd-1. Together these data establish a role of fahd-1 to maintain mitochondrial function and consequently physical activity in nematodes. PMID:26266933

  12. Distinct Functional Properties of Isoamylase-Type Starch Debranching Enzymes in Monocot and Dicot Leaves1[C][W][OPEN

    PubMed Central

    Facon, Maud; Lin, Qiaohui; Azzaz, Abdelhamid M.; Hennen-Bierwagen, Tracie A.; Myers, Alan M.; Putaux, Jean-Luc; Roussel, Xavier; D’Hulst, Christophe; Wattebled, Fabrice

    2013-01-01

    Isoamylase-type starch debranching enzymes (ISA) play important roles in starch biosynthesis in chloroplast-containing organisms, as shown by the strict conservation of both catalytically active ISA1 and the noncatalytic homolog ISA2. Functional distinctions exist between species, although they are not understood yet. Numerous plant tissues require both ISA1 and ISA2 for normal starch biosynthesis, whereas monocot endosperm and leaf exhibit nearly normal starch metabolism without ISA2. This study took in vivo and in vitro approaches to determine whether organism-specific physiology or evolutionary divergence between monocots and dicots is responsible for distinctions in ISA function. Maize (Zea mays) ISA1 was expressed in Arabidopsis (Arabidopsis thaliana) lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functioned in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2. Analysis of recombinant enzymes showed that Arabidopsis ISA1 requires ISA2 as a partner for enzymatic function, whereas maize ISA1 was active by itself. The electrophoretic mobility of recombinant and native maize ISA differed, suggestive of posttranslational modifications in vivo. Sedimentation equilibrium measurements showed recombinant maize ISA1 to be a dimer, in contrast to previous gel permeation data that estimated the molecular mass as a tetramer. These data demonstrate that evolutionary divergence between monocots and dicots is responsible for the distinctions in ISA1 function. PMID:24027240

  13. T3 glycoprotein is functional although structurally distinct on human T-cell receptor. gamma. T lymphocytes

    SciTech Connect

    Krangel, M.S.; Bierer, B.E.; Devlin, P.; Clabby, M.; Strominger, J.L.; McLean, J.; Brenner, M.B.

    1987-06-01

    The T-cell receptor (TCR) ..gamma.. gene product occurs in association with T3 (CD3) polypeptides on the surface of human T lymphocytes. TCR ..gamma.. lymphocytes express arrays of T3 polypeptides distinct from those typically observed on TCR ..cap alpha beta.. lymphocytes. This report demonstrates that identical T3 ..gamma.., delta, and element of polypeptides are synthesized by TCR ..gamma.. lymphocytes and TCR ..cap alpha beta.. lymphocytes. However, the processing of T3 delta oligosaccharides is distinct in the two cell types. This observation may suggest distinct quaternary structures of these receptor complexes. Despite these structural differences, the T3 molecule on TCR ..gamma.. lymphocytes is functional. It is associated with and comodulates with TCR ..gamma.. and it serves as a substrate from protein kinase C-mediated phosphorylation. Anti-T3 monoclonal antibodies induce a rapid increase in cytoplasmic free calcium, indicating that the receptor complex is involved in signal transduction and triggering of TCR ..gamma.. lymphocytes.

  14. Role of the PB2 627 Domain in Influenza A Virus Polymerase Function

    PubMed Central

    Nilsson, Benjamin E.; te Velthuis, Aartjan J. W.

    2017-01-01

    ABSTRACT The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap snatching, but the role of the auxiliary PB2 627 domain, implicated in host range restriction of influenza A viruses, is still poorly understood. In this study, we used structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627 domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we showed that a recombinant viral polymerase lacking the PB2 627 domain is able to carry out cap snatching, cap-dependent transcription initiation, and cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627 domain of the influenza virus RNA polymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627 domain is essential for both transcription and replication. In particular, we showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome. IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627 domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these

  15. 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.

  16. The DEP domain-containing protein TOE-2 promotes apoptosis in the Q lineage of C. elegans through two distinct mechanisms

    PubMed Central

    Gurling, Mark; Talavera, Karla; Garriga, Gian

    2014-01-01

    Neuroblast divisions in the nematode Caenorhabditis elegans often give rise to a larger neuron and a smaller cell that dies. We have previously identified genes that, when mutated, result in neuroblast divisions that generate daughter cells that are more equivalent in size. This effect correlates with the survival of daughter cells that would normally die. We now describe a role for the DEP domain-containing protein TOE-2 in promoting the apoptotic fate in the Q lineage. TOE-2 localized at the plasma membrane and accumulated in the cleavage furrow of the Q.a and Q.p neuroblasts, suggesting that TOE-2 might position the cleavage furrow asymmetrically to generate daughter cells of different sizes. This appears to be the case for Q.a divisions where loss of TOE-2 led to a more symmetric division and to survival of the smaller Q.a daughter. Localization of TOE-2 to the membrane is required for this asymmetry, but, surprisingly, the DEP domain is dispensable. By contrast, loss of TOE-2 led to loss of the apoptotic fate in the smaller Q.p daughter but did not affect the size asymmetry of the Q.p daughters. This function of TOE-2 required the DEP domain but not localization to the membrane. We propose that TOE-2 ensures an apoptotic fate for the small Q.a daughter by promoting asymmetry in the daughter cell sizes of the Q.a neuroblast division but by a mechanism that is independent of cell size in the Q.p division. PMID:24961802

  17. Comparative Analysis of the Biochemical and Functional Properties of C-Terminal Domains of Autotransporters ▿

    PubMed Central

    Marín, Elvira; Bodelón, Gustavo; Fernández, Luis Ángel

    2010-01-01

    Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-terminal domain that is inserted in the outer membrane (OM) and is essential for the translocation of an N-terminal passenger domain to the extracellular milieu. Several mechanisms have been proposed for AT secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the β-barrel or by the oligomerization of C-terminal domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam complex. In this work we have investigated AT secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-terminal domains selected from ATs of gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria. Our results indicate that C-terminal domains having an N-terminal α-helix and a β-barrel constitute functional transport units for the translocation of peptides and immunoglobulin domains with disulfide bonds. In vivo and in vitro analyses show that multimerization is not a conserved feature in AT C-terminal domains. Furthermore, we demonstrate that the deletion of the conserved α-helix severely impairs β-barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that the AT β-barrel without its α-helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for an understanding of AT secretion are discussed. PMID:20802036

  18. Janus-faced Sestrin2 controls ROS and mTOR signalling through two separate functional domains

    NASA Astrophysics Data System (ADS)

    Kim, Hanseong; An, Sojin; Ro, Seung-Hyun; Teixeira, Filipa; Jin Park, Gyeong; Kim, Cheal; Cho, Chun-Seok; Kim, Jeong-Sig; Jakob, Ursula; Hee Lee, Jun; Cho, Uhn-Soo

    2015-11-01

    Sestrins are stress-inducible metabolic regulators with two seemingly unrelated but physiologically important functions: reduction of reactive oxygen species (ROS) and inhibition of the mechanistic target of rapamycin complex 1 (mTORC1). How Sestrins fulfil this dual role has remained elusive so far. Here we report the crystal structure of human Sestrin2 (hSesn2), and show that hSesn2 is twofold pseudo-symmetric with two globular subdomains, which are structurally similar but functionally distinct from each other. While the N-terminal domain (Sesn-A) reduces alkylhydroperoxide radicals through its helix-turn-helix oxidoreductase motif, the C-terminal domain (Sesn-C) modified this motif to accommodate physical interaction with GATOR2 and subsequent inhibition of mTORC1. These findings clarify the molecular mechanism of how Sestrins can attenuate degenerative processes such as aging and diabetes by acting as a simultaneous inhibitor of ROS accumulation and mTORC1 activation.

  19. 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

  20. Transactivation domains are not functionally conserved between vertebrate and invertebrate serum response factors.

    PubMed

    Avila, Sonia; Casero, Marie-Carmen; Fernandez-Cantón, Rocío; Sastre, Leandro

    2002-08-01

    The transcription factor serum response factor (SRF) regulates expression of growth factor-dependent genes and muscle-specific genes in vertebrates. Homologous factors regulate differentiation of some ectodermic tissues in invertebrates. To explore the molecular basis of these different physiological functions, the functionality of human, Drosophila melanogaster and Artemia franciscana SRFs in mammalian cells has been compared in this article. D. melanogaster and, to a lesser extend, A. franciscana SRF co-expression represses the activity of strong SRF-dependent promoters, such as those of the mouse c-fos and A. franciscana actin 403 genes. Domain-exchange experiments showed that these results can be explained by the absence of a transactivation domain, functional in mammalian cells, in D. melanogaster and A. franciscana SRFs. Both invertebrate SRFs can dimerize with endogenous mouse SRF through the conserved DNA-binding and dimerization domain. Co-expression of human and A. franciscana SRFs activate expression of weaker SRF-dependent promoters, such as those of the human cardiac alpha-actin gene or an A. franciscana actin 403 promoter where the SRF-binding site has been mutated. Mapping of A. franciscana SRF domains involved in transcriptional activation has shown that the conserved DNA-binding and dimerization domain is neccessary, but not sufficient, for promoter activation in mammalian cells.

  1. 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

  2. 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.

  3. Structure-function studies of the lustrin A polyelectrolyte domains, RKSY and D4.

    PubMed

    Wustman, Brandon A; Weaver, James C; Morse, Daniel E; Evans, John Spencer

    2003-01-01

    The lustrin superfamily represents a unique group of biomineralization proteins localized between layered aragonite mineral plates (i.e., nacre layers) in mollusk shell. These proteins not only exhibit elastomeric behavior within the mineralized matrix, but also adhesion to the aragonite-containing composite layer. One member of the lustrin superfamily, Lustrin A, has been sequenced; the protein is organized into defined, modular sequence domains that are hypothesized to perform separate functions (i.e., force unfolding, mineral adhesion, intermolecular binding) within the Lustrin A protein. Using nuclear magnetic resonance (NMR) and in vitro mineralization assays, we investigated structure-function relationships for two Lustrin A putative mineral binding domains, the 30 AA Arg, Lys, Tyr, Ser-rich (RKSY) and the 24 AA Asp-rich (D4) sequence regions domain of the Lustrin A protein. The results indicate that both sequences adopt open, unfolded structures that represent either extended or random coil states. Using geologic calcite overgrowth assays and scanning electron microscopic analyses, we observe that the RKSY polypeptide does not significantly perturb calcium carbonate growth. However, the D4 domain does influence crystal growth in a concentration-dependent manner. Collectively, our data indicate that D4, and not the RKSY domain, exhibits structure-function activity consistent with a mineral binding region.

  4. 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.

  5. Structural and functional comparisons of retroviral envelope protein C-terminal domains: still much to learn.

    PubMed

    Steckbeck, Jonathan D; Kuhlmann, Anne-Sophie; Montelaro, Ronald C

    2014-01-16

    Retroviruses are a family of viruses that cause a broad range of pathologies in animals and humans, from the apparently harmless, long-term genomic insertion of endogenous retroviruses, to tumors induced by the oncogenic retroviruses and acquired immunodeficiency syndrome (AIDS) resulting from human immunodeficiency virus infection. Disease can be the result of diverse mechanisms, including tumorigenesis induced by viral oncogenes or immune destruction, leading to the gradual loss of CD4 T-cells. Of the virally encoded proteins common to all retroviruses, the envelope (Env) displays perhaps the most diverse functionality. Env is primarily responsible for binding the cellular receptor and for effecting the fusion process, with these functions mediated by protein domains localized to the exterior of the virus. The remaining C-terminal domain may have the most variable functionality of all retroviral proteins. The C-terminal domains from three prototypical retroviruses are discussed, focusing on the different structures and functions, which include fusion activation, tumorigenesis and viral assembly and lifecycle influences. Despite these genetic and functional differences, however, the C-terminal domains of these viruses share a common feature in the modulation of Env ectodomain conformation. Despite their differences, perhaps each system still has information to share with the others.

  6. Age-related disorders of sleep and motor control in the rat models of functionally distinct cholinergic neuropathology.

    PubMed

    Ciric, Jelena; Lazic, Katarina; Petrovic, Jelena; Kalauzi, Aleksandar; Saponjic, Jasna

    2016-03-15

    We studied the impact of aging during sleep in the rat models of Alzheimer's (AD) and Parkinson's (PD) disease cholinergic neuropathology to determine the possible different and earlier onset of age-related sleep disorder during the neurodegenerative diseases vs. healthy aging. We used the bilateral nucleus basalis (NB) and pedunculopontine tegmental nucleus (PPT) lesioned rats as the in vivo models of functionally distinct cholinergic neuropathology, and we followed the impact of aging on sleep architecture, the electroencephalographic (EEG) microstructure and motor control across sleep/wake states. Our results have shown for the first time that the earliest signs of aging during distinct cholinergic neuropathology were expressed through a different and topographically specific EEG microstructure during rapid eye movement sleep (REM). EEG delta amplitude attenuation within the sensorimotor cortex (SMCx) during REM was the earliest sign of aging in the NB lesion. EEG sigma amplitude augmentation within the motor cortex (MCx) during REM was the earliest sign of aging in the PPT lesion. In addition, aging was differently expressed through the SMCx drive alterations, but it was commonly expressed through the MCx drive alterations during all sleep/wake states. Our study provided evidence of distinct REM sleep disorders and sleep state related cortical drives as the signs of aging onset during functionally distinct cholinergic neuropathologies (NB lesion vs. PPT lesion).

  7. The domain organization of streptokinase: nuclear magnetic resonance, circular dichroism, and functional characterization of proteolytic fragments.

    PubMed Central

    Parrado, J.; Conejero-Lara, F.; Smith, R. A.; Marshall, J. M.; Ponting, C. P.; Dobson, C. M.

    1996-01-01

    Streptococcus equisimilis streptokinase (SK) is a bacterial protein of unknown tertiary structure and domain organization that is used extensively to treat acute myocardial infarction following coronary thrombosis. Six fragments of SK were generated by limited proteolysis with chymotrypsin and purified. NMR and CD experiments have shown that the secondary and tertiary structure present in the native molecule is preserved within all fragments, except the N-terminal fragment SK7. NMR spectra demonstrate the presence in SK of three structurally autonomous domains and a less structured C-terminal "tail." Cleavage within the N-terminal domain generates an N-terminal fragment, SK7, which remains noncovalently associated with the remainder of the molecule; in isolation, SK7 adopts an unfolded conformation. The abilities of these fragments to induce active site formation within human plasminogen upon formation of their heterodimeric complex were assayed. The lowest mass SK fragment exhibiting Plg-dependent activator activity was shown to be SK27 (mass 27,000, residues 147-380), which contains both central and C-terminal domains, although this activity was reduced approximately 6,000-fold relative to that of full-length SK. The activity of a 36,000 mass fragment, SK36 (residues 64-380), which differs from SK27 in possessing a portion of the N-terminal domain, was reduced to 0.1-1.0% of that of SK. Other fragments (masses 7,000, 11,000, 16,000, 17,000, 25,000, and 26,000), representing either single domains or single domains extended by portions of other domains, were inactive. However, SK7 (residues 1-63), at a 100-fold molar excess concentration, greatly potentiated the activities of SK27 and SK36, by up to 50- and > 130-fold, respectively. These findings demonstrate that all of SK's three domains are essential for native-like SK activity. The central and C-terminal domains mediate plasminogen-binding and active site-generating functions, whereas the N-terminal domain

  8. Prediction of the repeat domain structures and impact of parkinsonism-associated variations on structure and function of all functional domains of leucine-rich repeat kinase 2 (LRRK2).

    PubMed

    Mills, Ryan D; Mulhern, Terrence D; Liu, Fei; Culvenor, Janetta G; Cheng, Heung-Chin

    2014-04-01

    Genetic variations of leucine-rich repeat kinase 2 (LRRK2) are the major cause of dominantly inherited Parkinson disease (PD). LRRK2 protein contains seven predicted domains: a tandem Ras-like GTPase (ROC) domain and C-terminal of Roc (COR) domain, a protein kinase domain, and four repeat domains. PD-causative variations arise in all domains, suggesting that aberrant functioning of any domain can contribute to neurotoxic mechanisms of LRRK2. Determination of the three-dimensional structure of LRRK2 is one of the best avenues to decipher its neurotoxic mechanism. However, with the exception of the Roc domain, the three-dimensional structures of the functional domains of LRRK2 have yet to be determined. Based on the known three-dimensional structures of repeat domains of other proteins, the tandem Roc-COR domains of the Chlorobium tepidum Rab family protein, and the kinase domain of the Dictyostelium discoideum Roco4 protein, we predicted (1) the motifs essential for protein-protein interactions in all domains, (2) the motifs critical for catalysis and substrate recognition in the tandem Roc-COR and kinase domains, and (3) the effects of some PD-associated missense variations on the neurotoxic action of LRRK2. Results of our analysis provide a conceptual framework for future investigation into the regulation and the neurotoxic mechanism of LRRK2.

  9. Analysis of Arabidopsis thioredoxin-h isotypes identifies discrete domains that confer specific structural and functional properties.

    PubMed

    Jung, Young Jun; Chi, Yong Hun; Chae, Ho Byoung; Shin, Mi Rim; Lee, Eun Seon; Cha, Joon-Yung; Paeng, Seol Ki; Lee, Yuno; Park, Jin Ho; Kim, Woe Yeon; Kang, Chang Ho; Lee, Kyun Oh; Lee, Keun Woo; Yun, Dae-Jin; Lee, Sang Yeol

    2013-11-15

    Multiple isoforms of Arabidopsis thaliana h-type thioredoxins (AtTrx-hs) have distinct structural and functional specificities. AtTrx-h3 acts as both a disulfide reductase and as a molecular chaperone. We prepared five representative AtTrx-hs and compared their protein structures and disulfide reductase and molecular chaperone activities. AtTrx-h2 with an N-terminal extension exhibited distinct functional properties with respect to other AtTrx-hs. AtTrx-h2 formed low-molecular-mass structures and exhibited only disulfide reductase activity, whereas the other AtTrx-h isoforms formed high-molecular-mass complexes and displayed both disulfide reductase and molecular chaperone activities. The domains that determine the unique structural and functional properties of each AtTrx-hs protein were determined by constructing a domain-swap between the N- and C-terminal regions of AtTrx-h2 and AtTrx-h3 (designated AtTrx-h-2N3C and AtTrx-h-3N2C respectively), an N-terminal deletion mutant of AtTrx-h2 [AtTrx-h2-N(∆19)] and site-directed mutagenesis of AtTrx-h3. AtTrx-h2-N(∆19) and AtTrx-h-3N2C exhibited similar properties to those of AtTrx-h2, but AtTrx-h-2N3C behaved more like AtTrx-h3, suggesting that the structural and functional specificities of AtTrx-hs are determined by their C-terminal regions. Hydrophobicity profiling and molecular modelling revealed that Ala100 and Ala106 in AtTrx-h3 play critical roles in its structural and functional regulation. When these two residues in AtTrx-h3 were replaced with lysine, AtTrx-h3 functioned like AtTrx-h2. The chaperone function of AtTrx-hs conferred enhanced heat-shock-resistance on a thermosensitive trx1/2-null yeast mutant.

  10. Functional domains and motifs of bacterial type III effector proteins and their roles in infection.

    PubMed

    Dean, Paul

    2011-11-01

    A key feature of the virulence of many bacterial pathogens is the ability to deliver effector proteins into eukaryotic cells via a dedicated type three secretion system (T3SS). Many bacterial pathogens, including species of Chlamydia, Xanthomonas, Pseudomonas, Ralstonia, Shigella, Salmonella, Escherichia and Yersinia, depend on the T3SS to cause disease. T3SS effectors constitute a large and diverse group of virulence proteins that mimic eukaryotic proteins in structure and function. A salient feature of bacterial effectors is their modular architecture, comprising domains or motifs that confer an array of subversive functions within the eukaryotic cell. These domains/motifs therefore represent a fascinating repertoire of molecular determinants with important roles during infection. This review provides a snapshot of our current understanding of bacterial effector domains and motifs where a defined role in infection has been demonstrated.

  11. Cell surface expression and function of an HLA class II molecule with class I domain configuration

    PubMed Central

    1993-01-01

    Recombinant major histocompatibility complex (MHC) class II molecules were expressed with extracellular polypeptide domains reorganized to form heavy (H) and light (L) chains (alpha 1-beta 1-beta 2 and alpha 2) analogous to class I. Accurate protein folding and dimerization is demonstrated by the ability of this 3+1-DR1 construct to bind class II- restricted peptides and stimulate CD4+ T cells. Cell surface expression of a functional class II molecule consisting of H and L chains supports the validity of current class II models and affirms the evolutionary relatedness of class I/II. MHC functions that differ between class I/II may be influenced by domain configuration, and the use of domain- shifted constructs will allow examination of this possibility. PMID:8340763

  12. Distinct Z-DNA binding mode of a PKR-like protein kinase containing a Z-DNA binding domain (PKZ)

    PubMed Central

    Kim, Doyoun; Hur, Jeonghwan; Park, Kwangsoo; Bae, Sangsu; Shin, Donghyuk; Ha, Sung Chul; Hwang, Hye-Yeon; Hohng, Sungchul; Lee, Joon-Hwa; Lee, Sangho; Kim, Yang-Gyun; Kim, Kyeong Kyu

    2014-01-01

    Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and functional studies of the Z-DNA binding domain (Zα) of PKZ from Carassius auratus (caZαPKZ). The 1.7-Å resolution crystal structure of caZαPKZ:Z-DNA revealed that caZαPKZ shares the overall fold with other Zα, but has discrete structural features that differentiate its DNA binding mode from others. Functional analyses of caZαPKZ and its mutants revealed that caZαPKZ mediates the fastest B-to-Z transition of DNA among Zα, and the minimal interaction for Z-DNA recognition is mediated by three backbone phosphates and six residues of caZαPKZ. Structure-based mutagenesis and B-to-Z transition assays confirmed that Lys56 located in the β-wing contributes to its fast B-to-Z transition kinetics. Investigation of the DNA binding kinetics of caZαPKZ further revealed that the B-to-Z transition rate is positively correlated with the association rate constant. Taking these results together, we conclude that the positive charge in the β-wing largely affects fast B-to-Z transition activity by enhancing the DNA binding rate. PMID:24682817

  13. Functional Genetic Screen to Identify Interneurons Governing Behaviorally Distinct Aspects of Drosophila Larval Motor Programs

    PubMed Central

    Clark, Matt Q.; McCumsey, Stephanie J.; Lopez-Darwin, Sereno; Heckscher, Ellie S.; Doe, Chris Q.

    2016-01-01

    Drosophila larval crawling is an attractive system to study rhythmic motor output at the level of animal behavior. Larval crawling consists of waves of muscle contractions generating forward or reverse locomotion. In addition, larvae undergo additional behaviors, including head casts, turning, and feeding. It is likely that some neurons (e.g., motor neurons) are used in all these behaviors, but the identity (or even existence) of neurons dedicated to specific aspects of behavior is unclear. To identify neurons that regulate specific aspects of larval locomotion, we performed a genetic screen to identify neurons that, when activated, could elicit distinct motor programs. We used 165 Janelia CRM-Gal4 lines—chosen for sparse neuronal expression—to ectopically express the warmth-inducible neuronal activator TrpA1, and screened for locomotor defects. The primary screen measured forward locomotion velocity, and we identified 63 lines that had locomotion velocities significantly slower than controls following TrpA1 activation (28°). A secondary screen was performed on these lines, revealing multiple discrete behavioral phenotypes, including slow forward locomotion, excessive reverse locomotion, excessive turning, excessive feeding, immobile, rigid paralysis, and delayed paralysis. While many of the Gal4 lines had motor, sensory, or muscle expression that may account for some or all of the phenotype, some lines showed specific expression in a sparse pattern of interneurons. Our results show that distinct motor programs utilize distinct subsets of interneurons, and provide an entry point for characterizing interneurons governing different elements of the larval motor program. PMID:27172197

  14. 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.

  15. Statistics and frequency-domain moveout for multiple-taper receiver functions

    NASA Astrophysics Data System (ADS)

    Park, J.; Levin, V.

    2016-10-01

    The multiple-taper correlation (MTC) algorithm for the estimation of teleseismic receiver functions (RFs) has desirable statistical properties. This paper presents several adaptations to the MTC algorithm that exploit its frequency-domain uncertainty estimates to generate stable RFs that include moveout corrections for deeper interfaces. Narrow-band frequency averaging implicit in spectral cross-correlation restricts the MTC-based RF estimates to resolve Ps converted phases only at short delay times, appropriate to the upper 100 km of Earth's lithosphere. The Ps conversions from deeper interfaces can be reconstructed by the MTC algorithm in two ways. Event cross-correlation computes a cross-correlation of single-taper spectrum estimates for a cluster of events rather than for a set of eigenspectrum estimates of a single P coda. To extend the reach of the algorithm, pre-stack moveout corrections in the frequency domain preserves the formal uncertainties of the RF estimates, which are used to weight RF stacks. Moving-window migration retains the multiple-taper approach, but cross-correlates the P-polarized motion with time-delayed SH and SV motion to focus on a Ps phase of interest. The frequency-domain uncertainties of bin-averaged RFs do not translate directly into the time domain. A jackknife over data records in each bin stack offers uncertainty estimates in the time domain while preserving uncertainty weighting in the frequency-domain RF stack.

  16. 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

  17. Polymorphism, shared functions and convergent evolution of genes with sequences coding for polyalanine domains.

    PubMed

    Lavoie, Hugo; Debeane, Francois; Trinh, Quoc-Dien; Turcotte, Jean-Francois; Corbeil-Girard, Louis-Philippe; Dicaire, Marie-Josée; Saint-Denis, Anik; Pagé, Martin; Rouleau, Guy A; Brais, Bernard

    2003-11-15

    Mutations causing expansions of polyalanine domains are responsible for nine hereditary diseases. Other GC-rich sequences coding for some polyalanine domains were found to be polymorphic in human. These observations prompted us to identify all sequences in the human genome coding for polyalanine stretches longer than four alanines and establish their degree of polymorphism. We identified 494 annotated human proteins containing 604 polyalanine domains. Thirty-two percent (31/98) of tested sequences coding for more than seven alanines were polymorphic. The length of the polyalanine-coding sequence and its GCG or GCC repeat content are the major predictors of polymorphism. GCG codons are over-represented in human polyalanine coding sequences. Our data suggest that GCG and GCC codons play a key role in polyalanine-coding sequence appearance and polymorphism. The grouping by shared function of polyalanine-containing proteins in Homo sapiens, Drosophila melanogaster and Caenorhabditis elegans shows that the majority are involved in transcriptional regulation. Phylogenetic analyses of HOX, GATA and EVX protein families demonstrate that polyalanine domains arose independently in different members of these families, suggesting that convergent molecular evolution may have played a role. Finally polyalanine domains in vertebrates are conserved between mammals and are rarer and shorter in Gallus gallus and Danio rerio. Together our results show that the polymorphic nature of sequences coding for polyalanine domains makes them prime candidates for mutations in hereditary diseases and suggests that they have appeared in many different protein families through convergent evolution.

  18. Oxidation of the N-terminal domain of the wheat metallothionein Ec -1 leads to the formation of three distinct disulfide bridges.

    PubMed

    Tarasava, Katsiaryna; Chesnov, Serge; Freisinger, Eva

    2016-05-01

    Metallothioneins (MTs) are low molecular weight proteins, characterized by a high cysteine content and the ability to coordinate large amounts of d(10) metal ions, for example, Zn(II), Cd(II), and Cu(I), in form of metal-thiolate clusters. Depending on intracellular conditions such as redox potential or metal ion concentrations, MTs can occur in various states ranging from the fully metal-loaded holo- to the metal-free apo-form. The Cys thiolate groups in the apo-form can be either reduced or be involved in disulfide bridges. Although oxidation-mediated Zn(II) release might be a possible mechanism for the regulation of Zn(II) availability by MTs, no concise information regarding the associated pathways and the structure of oxidized apo-MT forms is available. Using the well-studied Zn2 γ-Ec -1 domain of the wheat Zn6 Ec -1 MT we attempt here to answer several question regarding the structure and biophysical properties of oxidized MT forms, such as: (1) does disulfide bond formation increase the stability against proteolysis, (2) is the overall peptide backbone fold similar for the holo- and the oxidized apo-MT form, and (3) are disulfide bridges specifically or randomly formed? Our investigations show that oxidation leads to three distinct disulfide bridges independently of the applied oxidation conditions and of the initial species used for oxidation, that is, the apo- or the holo-form. In addition, the oxidized apo-form is as stable against proteolysis as Zn2 γ-Ec -1, rendering the currently assumed degradation of oxidized MTs unlikely and suggesting a role of the oxidation process for the extension of protein lifetime in absence of sufficient amounts of metal ions. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 295-308, 2016.

  19. Flavin-binding and protein structural integrity studies on NADPH-cytochrome P450 reductase are consistent with the presence of distinct domains.

    PubMed

    Narayanasami, R; Horowitz, P M; Masters, B S

    1995-01-10

    NADPH-cytochrome P450 reductase (reductase) contains FMN and FAD in 1:1 stoichiometry as tightly bound cofactors. Electrons from NADPH are transferred to cytochrome P450 through the intermediacy of reductase. A knowledge of the interactions which must occur to allow the intermolecular and intramolecular transfer of electrons is not only of intrinsic interest but is necessary to understand the regulation of the overall oxidation-reduction processes in which cytochromes P450 participate in the endoplasmic reticulum of many organs. In the present study, urea has been employed as a chaotropic agent to study the dissociation of flavins from NADPH-cytochrome P450 reductase. The results show that dissociation of FMN occurs at concentrations of urea between 0 and 1 M and that, as the concentrations of urea approach 1 M, the intrinsic protein fluorescence increases, indicating a change in protein conformation. Above 2 M urea protein fluorescence increases, reaching a plateau at 3 M urea, and FAD begins to dissociate from the enzyme. In the range of 0-1 M urea, a completely reversible dissociation of FMN occurs and, at 3 M urea, the fluorescence values representing flavin dissociation and protein conformation changes have reached a maximum. Thus, the definition of various states of the flavoprotein with both, one, or no flavins bound and the ability to remove the flavins reversibly under specific conditions have permitted the construction of a simple model to explain the various unfolding intermediates of this enzyme. Our experiments suggest that reductase is composed of distinct domains which can be examined independently by the application of chaotropic agents.

  20. Differentiating plant cells switched to proliferation remodel the functional organization of nuclear domains.

    PubMed

    Testillano, P S; González-Melendi, P; Coronado, M J; Seguí-Simarro, J M; Moreno-Risueño, M A; Risueño, M C

    2005-01-01

    The immature pollen grain, the microspore, under stress conditions can switch its developmental program towards proliferation and embryogenesis. The comparison between the gametophytic and sporophytic pathways followed by the microspore permitted us to analyse the nuclear changes in plant differentiating cells when switched to proliferation. The nucleus is highly dynamic, the architecture of its well organised functional domains--condensed chromatin, interchromatin region, nuclear bodies and nucleolus--changing in response to DNA replication, RNA transcription, processing and transport. In the present work, the rearrangements of the nuclear domains during the switch to proliferation have been determined by in situ molecular identification methods for the subcellular localization of chromatin at different functional states, rDNA, elements of the nuclear machinery (PCNA, splicing factors), signalling and stress proteins. The study of the changes in the nuclear domains was determined by a correlative approach at confocal and electron microscopy levels. The results showed that the switch of the developmental program and the activation of the proliferative activity affected the functional organization of the nuclear domains, which accordingly changed their architecture and functional state. A redistribution of components, among them various signalling molecules which targeted structures within the interchromatin region upon translocation from the cytoplasm, was also observed.

  1. Wavelet Domain Image Reconstruction by Compactly-Supported Radial Basis Functions

    NASA Astrophysics Data System (ADS)

    Diago, Luis A.; Kitago, Masaki; Hagiwara, Ichiro

    In this paper we propose the use of wavelets to accelerate the solution of the System of Linear Algebraic Equations that arise from the formulation of the problem of image interpolation from scattered data by means of Compactly-Supported Radial Basis Functions. Examples demonstrate the superiority of the solution in the wavelet domain using preconditioned iterative Krylov methods.

  2. 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, ...

  3. An Examination of the Domain of Multivariable Functions Using the Pirie-Kieren Model

    ERIC Educational Resources Information Center

    Sengul, Sare; Yildiz, Sevda Goktepe

    2016-01-01

    The aim of this study is to employ the Pirie-Kieren model so as to examine the understandings relating to the domain of multivariable functions held by primary school mathematics preservice teachers. The data obtained was categorized according to Pirie-Kieren model and demonstrated visually in tables and bar charts. The study group consisted of…

  4. Implement of the Owner Distinction Function for Healing-Type Pet Robots

    NASA Astrophysics Data System (ADS)

    Nambo, Hidetaka; Kimura, Haruhiko; Hirose, Sadaki

    In recent years, a robotics technology is extremely progressive, and robots are widely applied in many fields. One of the most typical robots is a pet robot. The pet robot is based on an animal pet, such as a dog or a cat. Also, it is known that an animal pet has a healing effect. Therefore, the study to apply pet robots to Animal Assisted Therapy instead of an animal pet has begun to be investigated. We, also, have investigated a method of an owner distinction for pet robot, to emphasize a healing effect of pet robots. In this paper, taking account of implementation into pet robots, a real-time owner distinction method is proposed. In the concrete, the method provides a real-time matching algorithm and an oblivion mechanism. The real-time matching means that a matching and a data acquisition are processed simultaneously. The oblivion mechanism is deleting features of owners in the database of the pet robots. Additionally, the mechanism enables to reduce matching costs or size of database and it enables to follow a change of owners. Furthermore, effectivity and a practicality of the method are evaluated by experiments.

  5. Structure reveals function of the dual variable domain immunoglobulin (DVD-Ig™) molecule.

    PubMed

    Jakob, Clarissa G; Edalji, Rohinton; Judge, Russell A; DiGiammarino, Enrico; Li, Yingchun; Gu, Jijie; Ghayur, Tariq

    2013-01-01

    Several bispecific antibody-based formats have been developed over the past 25 years in an effort to produce a new generation of immunotherapeutics that target two or more disease mechanisms simultaneously. One such format, the dual-variable domain immunoglobulin (DVD-Ig™), combines the target binding domains of two monoclonal antibodies via flexible naturally occurring linkers, which yields a tetravalent IgG - like molecule. We report the structure of an interleukin (IL)12-IL18 DVD-Ig™ Fab (DFab) fragment with IL18 bound to the inner variable domain (VD) that reveals the remarkable flexibility of the DVD-Ig™ molecule and how the DVD-Ig™ format can function to bind four antigens simultaneously. An understanding of how the inner variable domain retains function is of critical importance for designing DVD-Ig™ molecules, and for better understanding of the flexibility of immunoglobulin variable domains and linkers, which may aid in the design of improved bi- and multi-specific biologics in general.

  6. 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

  7. 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

  8. Functional hierarchy of two L domains in Porcine Endogenous Retrovirus (PERV) that influence release and infectivity

    PubMed Central

    Marcucci, Katherine T.; Martina, Yuri; Harrison, Frank; Wilson, Carolyn A.

    2008-01-01

    The Porcine Endogenous Retrovirus (PERV) Gag protein contains two late (L) domain motifs, PPPY and P(F/S)AP. Using viral release assays we demonstrate that PPPY is the dominant L domain involved in PERV release. PFAP represents a novel retroviral L domain variant and is defined by abnormal viral assembly phenotypes visualized by electron microscopy and attenuation of early PERV release as measured by viral genomes. PSAP is functionally dominant over PFAP in early PERV release. PSAP virions are 3.5-fold more infectious in vitro by TCID50 and in vivo results in more RNA positive tissues and higher levels of proviral DNA using our human PERV-A receptor (HuPAR-2) transgenic mouse model (Martina et al., 2006. Journal of Virology. 80: 3135-46). The functional hierarchies displayed by PERV L domains, demonstrates that L domain selection in viral evolution exists to promote efficient viral assembly, release and infectivity in the virus-host context. PMID:18355887

  9. Nonclinical and Clinical Enterococcus faecium Strains, but Not Enterococcus faecalis Strains, Have Distinct Structural and Functional Genomic Features

    PubMed Central

    Kim, Eun Bae

    2014-01-01

    Certain strains of Enterococcus faecium and Enterococcus faecalis contribute beneficially to animal health and food production, while others are associated with nosocomial infections. To determine whether there are structural and functional genomic features that are distinct between nonclinical (NC) and clinical (CL) strains of those species, we analyzed the genomes of 31 E. faecium and 38 E. faecalis strains. Hierarchical clustering of 7,017 orthologs found in the E. faecium pangenome revealed that NC strains clustered into two clades and are distinct from CL strains. NC E. faecium genomes are significantly smaller than CL genomes, and this difference was partly explained by significantly fewer mobile genetic elements (ME), virulence factors (VF), and antibiotic resistance (AR) genes. E. faecium ortholog comparisons identified 68 and 153 genes that are enriched for NC and CL strains, respectively. Proximity analysis showed that CL-enriched loci, and not NC-enriched loci, are more frequently colocalized on the genome with ME. In CL genomes, AR genes are also colocalized with ME, and VF are more frequently associated with CL-enriched loci. Genes in 23 functional groups are also differentially enriched between NC and CL E. faecium genomes. In contrast, differences were not observed between NC and CL E. faecalis genomes despite their having larger genomes than E. faecium. Our findings show that unlike E. faecalis, NC and CL E. faecium strains are equipped with distinct structural and functional genomic features indicative of adaptation to different environments. PMID:24141120

  10. Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases

    PubMed Central

    Masuda, Yuji; Kanao, Rie; Kaji, Kentaro; Ohmori, Haruo; Hanaoka, Fumio; Masutani, Chikahide

    2015-01-01

    Translesion DNA synthesis (TLS) by the Y-family DNA polymerases Polη, Polι and Polκ, mediated via interaction with proliferating cell nuclear antigen (PCNA), is a crucial pathway that protects human cells against DNA damage. We report that Polη has three PCNA-interacting protein (PIP) boxes (PIP1, 2, 3) that contribute differentially to two distinct functions, stimulation of DNA synthesis and promotion of PCNA ubiquitination. The latter function is strongly associated with formation of nuclear Polη foci, which co-localize with PCNA. We also show that Polκ has two functionally distinct PIP boxes, like Polη, whereas Polι has a single PIP box involved in stimulation of DNA synthesis. All three polymerases were additionally stimulated by mono-ubiquitinated PCNA in vitro. The three PIP boxes and a ubiquitin-binding zinc-finger of Polη exert redundant and additive effects in vivo via distinct molecular mechanisms. These findings provide an integrated picture of the orchestration of TLS polymerases. PMID:26170230

  11. Distinct patterns of functional and effective connectivity between perirhinal cortex and other cortical regions in recognition memory and perceptual discrimination.

    PubMed

    O'Neil, Edward B; Protzner, Andrea B; McCormick, Cornelia; McLean, D Adam; Poppenk, Jordan; Cate, Anthony D; Köhler, Stefan

    2012-01-01

    Traditionally, the medial temporal lobe (MTL) is thought to be dedicated to declarative memory. Recent evidence challenges this view, suggesting that perirhinal cortex (PrC), which interfaces the MTL with the ventral visual pathway, supports highly integrated object representations in recognition memory and perceptual discrimination. Even with comparable representational demands, perceptual and memory tasks differ in numerous task demands and the subjective experience they evoke. Here, we tested whether such differences are reflected in distinct patterns of connectivity between PrC and other cortical regions, including differential involvement of prefrontal control processes. We examined functional magnetic resonance imaging data for closely matched perceptual and recognition memory tasks for faces that engaged right PrC equivalently. Multivariate seed analyses revealed distinct patterns of interactions: Right ventrolateral prefrontal and posterior cingulate cortices exhibited stronger functional connectivity with PrC in recognition memory; fusiform regions were part of the pattern that displayed stronger functional connectivity with PrC in perceptual discrimination. Structural equation modeling revealed distinct patterns of effective connectivity that allowed us to constrain interpretation of these findings. Overall, they demonstrate that, even when MTL structures show similar involvement in recognition memory and perceptual discrimination, differential neural mechanisms are reflected in the interplay between the MTL and other cortical regions.

  12. Higher order smooth fitting for the convex cavities in the multiply connected plane domain by multiple functions

    NASA Astrophysics Data System (ADS)

    Cao, Jialian; Wan, Chaoyan; Zhao, Wenzhong

    2013-07-01

    In order to satisfy different engineering applications, a new higher order smooth fitting method for the convex cavities in the multiply connected plane domain by multiple functions is put forward. For the boundaries of convex cavities in the multiply connected domain--every close domain, the unique fitting function KS, is represented with some fitting precision controlled by only one single parameter (Rho). The fitted smooth figure can be drawn according to the function.

  13. Distinct Aging Effects on Functional Networks in Good and Poor Cognitive Performers

    PubMed Central

    Lee, Annie; Tan, Mingzhen; Qiu, Anqi

    2016-01-01

    Brain network hubs are susceptible to normal aging processes and disruptions of their functional connectivity are detrimental to decline in cognitive functions in older adults. However, it remains unclear how the functional connectivity of network hubs cope with cognitive heterogeneity in an aging population. This study utilized cognitive and resting-state functional magnetic resonance imaging data, cluster analysis, and graph network analysis to examine age-related alterations in the network hubs’ functional connectivity of good and poor cognitive performers. Our results revealed that poor cognitive performers showed age-dependent disruptions in the functional connectivity of the right insula and posterior cingulate cortex (PCC), while good cognitive performers showed age-related disruptions in the functional connectivity of the left insula and PCC. Additionally, the left PCC had age-related declines in the functional connectivity with the left medial prefrontal cortex (mPFC) and anterior cingulate cortex (ACC). Most interestingly, good cognitive performers showed age-related declines in the functional connectivity of the left insula and PCC with their right homotopic structures. These results may provide insights of neuronal correlates for understanding individual differences in aging. In particular, our study suggests prominent protection roles of the left insula and PCC and bilateral ACC in good performers. PMID:27667972

  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. In situ dissection of RNA functional subunits by domain-specific chromatin isolation by RNA purification (dChIRP).

    PubMed

    Quinn, Jeffrey J; Chang, Howard Y

    2015-01-01

    Here we describe domain-specific chromatin isolation by RNA purification (dChIRP), a technique for dissecting the functional domains of a target RNA in situ. For an RNA of interest, dChIRP can identify domain-level intramolecular and intermolecular RNA-RNA, RNA-protein, and RNA-DNA interactions and maps the RNA's genomic binding sites with higher precision than domain-agnostic methods. We illustrate how this technique has been applied to the roX1 lncRNA to resolve its domain-level architecture, discover its protein- and chromatin-interacting domains, and map its occupancy on the X chromosome.

  16. Myocardial Perfusion and Function Are Distinctly Altered by Sevoflurane Anesthesia in Diet-Induced Prediabetic Rats.

    PubMed

    van den Brom, Charissa E; Boly, Chantal A; Bulte, Carolien S E; van den Akker, Rob F P; Kwekkeboom, Rick F J; Loer, Stephan A; Boer, Christa; Bouwman, R Arthur

    2016-01-01

    Preservation of myocardial perfusion during surgery is particularly important in patients with increased risk for perioperative complications, such as diabetes. Volatile anesthetics, like sevoflurane, have cardiodepressive effects and may aggravate cardiovascular complications. We investigated the effect of sevoflurane on myocardial perfusion and function in prediabetic rats. Rats were fed a western diet (WD; n = 18) or control diet (CD; n = 18) for 8 weeks and underwent (contrast) echocardiography to determine perfusion and function during baseline and sevoflurane exposure. Myocardial perfusion was estimated based on the product of microvascular filling velocity and blood volume. WD-feeding resulted in a prediabetic phenotype characterized by obesity, hyperinsulinemia, hyperlipidemia, glucose intolerance, and hyperglycemia. At baseline, WD-feeding impaired myocardial perfusion and systolic function compared to CD-feeding. Exposure of healthy rats to sevoflurane increased the microvascular filling velocity without altering myocardial perfusion but impaired systolic function. In prediabetic rats, sevoflurane did also not affect myocardial perfusion; however, it further impaired systolic function. Diet-induced prediabetes is associated with impaired myocardial perfusion and function in rats. While sevoflurane further impaired systolic function, it did not affect myocardial perfusion in prediabetic rats. Our findings suggest that sevoflurane anesthesia leads to uncoupling of myocardial perfusion and function, irrespective of the metabolic state.

  17. Myocardial Perfusion and Function Are Distinctly Altered by Sevoflurane Anesthesia in Diet-Induced Prediabetic Rats

    PubMed Central

    van den Brom, Charissa E.; Boly, Chantal A.; Bulte, Carolien S. E.; van den Akker, Rob F. P.; Kwekkeboom, Rick F. J.; Loer, Stephan A.; Boer, Christa; Bouwman, R. Arthur

    2016-01-01

    Preservation of myocardial perfusion during surgery is particularly important in patients with increased risk for perioperative complications, such as diabetes. Volatile anesthetics, like sevoflurane, have cardiodepressive effects and may aggravate cardiovascular complications. We investigated the effect of sevoflurane on myocardial perfusion and function in prediabetic rats. Rats were fed a western diet (WD; n = 18) or control diet (CD; n = 18) for 8 weeks and underwent (contrast) echocardiography to determine perfusion and function during baseline and sevoflurane exposure. Myocardial perfusion was estimated based on the product of microvascular filling velocity and blood volume. WD-feeding resulted in a prediabetic phenotype characterized by obesity, hyperinsulinemia, hyperlipidemia, glucose intolerance, and hyperglycemia. At baseline, WD-feeding impaired myocardial perfusion and systolic function compared to CD-feeding. Exposure of healthy rats to sevoflurane increased the microvascular filling velocity without altering myocardial perfusion but impaired systolic function. In prediabetic rats, sevoflurane did also not affect myocardial perfusion; however, it further impaired systolic function. Diet-induced prediabetes is associated with impaired myocardial perfusion and function in rats. While sevoflurane further impaired systolic function, it did not affect myocardial perfusion in prediabetic rats. Our findings suggest that sevoflurane anesthesia leads to uncoupling of myocardial perfusion and function, irrespective of the metabolic state. PMID:26824042

  18. Harmonic Analysis and H2-Functions on Siegel Domains of Type II