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Sample records for cysteine-rich domain protein

  1. Cysteine-rich domains related to Frizzled receptors and Hedgehog-interacting proteins

    PubMed Central

    Pei, Jimin; Grishin, Nick V

    2012-01-01

    Frizzled and Smoothened are homologous seven-transmembrane proteins functioning in the Wnt and Hedgehog signaling pathways, respectively. They harbor an extracellular cysteine-rich domain (FZ-CRD), a mobile evolutionary unit that has been found in a number of other metazoan proteins and Frizzled-like proteins in Dictyostelium. Domains distantly related to FZ-CRDs, in Hedgehog-interacting proteins (HHIPs), folate receptors and riboflavin-binding proteins (FRBPs), and Niemann-Pick Type C1 proteins (NPC1s), referred to as HFN-CRDs, exhibit similar structures and disulfide connectivity patterns compared with FZ-CRDs. We used computational analyses to expand the homologous set of FZ-CRDs and HFN-CRDs, providing a better understanding of their evolution and classification. First, FZ-CRD-containing proteins with various domain compositions were identified in several major eukaryotic lineages including plants and Chromalveolata, revealing a wider phylogenetic distribution of FZ-CRDs than previously recognized. Second, two new and distinct groups of highly divergent FZ-CRDs were found by sensitive similarity searches. One of them is present in the calcium channel component Mid1 in fungi and the uncharacterized FAM155 proteins in metazoans. Members of the other new FZ-CRD group occur in the metazoan-specific RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) proteins that are putative tumor suppressors acting as inhibitors of matrix metalloproteases. Finally, sequence and three-dimensional structural comparisons helped us uncover a divergent HFN-CRD in glypicans, which are important morphogen-binding heparan sulfate proteoglycans. Such a finding reinforces the evolutionary ties between the Wnt and Hedgehog signaling pathways and underscores the importance of gene duplications in creating essential signaling components in metazoan evolution. PMID:22693159

  2. Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain

    SciTech Connect

    Suzuki, Nobuhiro; Yamazaki, Yasuo; Brown, R. Lane; Fujimoto, Zui; Morita, Takashi; Mizuno, Hiroshi

    2008-10-01

    The structures of pseudechetoxin and pseudecin suggest that both proteins bind to cyclic nucleotide-gated ion channels in a manner in which the concave surface occludes the pore entrance. Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinities on CNG channels are different: PsTx blocks both the olfactory and retinal channels with ∼15–30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn{sup 2+}-bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn{sup 2+} ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn{sup 2+} binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels.

  3. Biochemical and molecular characterization of the chicken cysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with the actin cytoskeleton.

    PubMed

    Crawford, A W; Pino, J D; Beckerle, M C

    1994-01-01

    LIM domains are present in a number of proteins including transcription factors, a proto-oncogene product, and the adhesion plaque protein zyxin. The LIM domain exhibits a characteristic arrangement of cysteine and histidine residues and represents a novel zinc binding sequence (Michelsen et al., 1993). Previously, we reported the identification of a 23-kD protein that interacts with zyxin in vitro (Sadler et al., 1992). In this report, we describe the purification and characterization of this 23-kD zyxin-binding protein from avian smooth muscle. Isolation of a cDNA encoding the 23-kD protein has revealed that it consists of 192 amino acids and exhibits two copies of the LIM motif. The 23-kD protein is 91% identical to the human cysteine-rich protein (hCRP); therefore we refer to it as the chicken cysteine-rich protein (cCRP). Examination of a number of chick embryonic tissues by Western immunoblot analysis reveals that cCRP exhibits tissue-specific expression. cCRP is most prominent in tissues that are enriched in smooth muscle cells, such as gizzard, stomach, and intestine. In primary cell cultures derived from embryonic gizzard, differentiated smooth muscle cells exhibit the most striking staining with anti-cCRP antibodies. We have performed quantitative Western immunoblot analysis of cCRP, zyxin, and alpha-actinin levels during embryogenesis. By this approach, we have demonstrated that the expression of cCRP is developmentally regulated.

  4. Biochemical and molecular characterization of the chicken cysteine-rich protein, a developmentally regulated LIM-domain protein that is associated with the actin cytoskeleton

    PubMed Central

    1994-01-01

    LIM domains are present in a number of proteins including transcription factors, a proto-oncogene product, and the adhesion plaque protein zyxin. The LIM domain exhibits a characteristic arrangement of cysteine and histidine residues and represents a novel zinc binding sequence (Michelsen et al., 1993). Previously, we reported the identification of a 23-kD protein that interacts with zyxin in vitro (Sadler et al., 1992). In this report, we describe the purification and characterization of this 23-kD zyxin-binding protein from avian smooth muscle. Isolation of a cDNA encoding the 23-kD protein has revealed that it consists of 192 amino acids and exhibits two copies of the LIM motif. The 23-kD protein is 91% identical to the human cysteine-rich protein (hCRP); therefore we refer to it as the chicken cysteine-rich protein (cCRP). Examination of a number of chick embryonic tissues by Western immunoblot analysis reveals that cCRP exhibits tissue-specific expression. cCRP is most prominent in tissues that are enriched in smooth muscle cells, such as gizzard, stomach, and intestine. In primary cell cultures derived from embryonic gizzard, differentiated smooth muscle cells exhibit the most striking staining with anti-cCRP antibodies. We have performed quantitative Western immunoblot analysis of cCRP, zyxin, and alpha-actinin levels during embryogenesis. By this approach, we have demonstrated that the expression of cCRP is developmentally regulated. PMID:8294495

  5. The cysteine-rich secretory protein domain of Tpx-1 is related to ion channel toxins and regulates ryanodine receptor Ca2+ signaling.

    PubMed

    Gibbs, Gerard M; Scanlon, Martin J; Swarbrick, James; Curtis, Suzanne; Gallant, Esther; Dulhunty, Angela F; O'Bryan, Moira K

    2006-02-17

    The cysteine-rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract as well as in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionary diverse N-terminal domain and a characteristic cysteine-rich C-terminal domain, which we refer to as the Crisp domain. We presented the NMR solution structure of the Crisp domain of mouse Tpx-1, and we showed that it contains two subdomains, one of which has a similar fold to the ion channel regulators BgK and ShK. Furthermore, we have demonstrated for the first time that the ion channel regulatory activity of Crisp proteins is attributed to the Crisp domain. Specifically, the Tpx-1 Crisp domain inhibited cardiac ryanodine receptor (RyR) 2 with an IC(50) between 0.5 and 1.0 microM and activated the skeletal RyR1 with an AC(50) between 1 and 10 microM when added to the cytoplasmic domain of the receptor. This activity was nonvoltage-dependent and weakly voltage-dependent, respectively. Furthermore, the Tpx-1 Crisp domain activated both RyR forms at negative bilayer potentials and showed no effect at positive bilayer potentials when added to the luminal domain of the receptor. These data show that the Tpx-1 Crisp domain on its own can regulate ion channel activity and provide compelling evidence for a role for Tpx-1 in the regulation of Ca(2+) fluxes observed during sperm capacitation.

  6. Cysteine rich secretory proteins in reproduction and venom.

    PubMed

    Gibbs, Gerard M; O'Bryan, Moira K

    2007-01-01

    The cysteine rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract and in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionarily diverse N-terminal domain and a characteristic cysteine rich C-terminal domain which we refer to as the Crisp domain. Since their identification 30 years ago Crisp research in mammals has focused on the characterisation of their expression localization to infer function. While no doubt important observations, these have not substantially led to an understanding of the biochemical activity of the Crisps and their role in sperm function or fertilisation. Recently, we demonstrated that the Crisp-2 Crisp domain has a structure similar to ion channel toxins ShK and BgK and was itself able to regulate Ca2+ flux through ryanodine receptors. These data build upon the previous characterizations of reptile venom Crisps as regulators of several types of ion channels and permits for the first time a dissection of the biochemical activity of mammalian Crisps.

  7. Molecular cloning, genomic structure, and tissue distribution of EW135, a novel chicken egg white protein with group B scavenger receptor cysteine-rich domains.

    PubMed

    Yoo, Whayoung; Nakamura, Tomohiro; Asanuma, Hideki; Matsushita, Misao

    2013-11-01

    Approximately 80 proteins are reported to be present in chicken egg white. The major function of egg white proteins isolated so far is to defend the egg yolk against infections. We recently isolated a novel protein termed EW135 from chicken egg white. In this paper, we have determined the complete amino acid sequence of EW135 based on cDNA cloning. EW135 consists of 970 amino acids with a putative signal peptide of 17 amino acids. It is composed exclusively of tandem repeats of nine group B scavenger receptor cysteine-rich (SRCR) domains separated by eight seven-amino acid peptides. The features of consensus sequences found in the group B SRCR domain were well conserved in EW135. The EW135 gene consists of putative 11 exons, with each SRCR domain being encoded by a single exon. Reverse transcription PCR showed that EW135 is expressed in only the oviduct among the 11 types of tissues tested. EW135 is a second soluble protein belonging to the group B SRCR domain superfamily identified in chickens. One of the important functions of proteins belonging to the group B SRCR domain superfamily is to recognize pathogens in innate immunity. It is, therefore, conceivable that EW135 could be involved in host defense in egg white.

  8. Five amino acid residues in cysteine-rich domain of human T1R3 were involved in the response for sweet-tasting protein, thaumatin.

    PubMed

    Masuda, Tetsuya; Taguchi, Wakana; Sano, Ayane; Ohta, Keisuke; Kitabatake, Naofumi; Tani, Fumito

    2013-07-01

    Thaumatin, a sweet-tasting plant protein, elicits a sweet taste sensation at 50 nM in humans but not rodents. Although it was shown that the cysteine-rich domain (CRD) of human T1R3 (hT1R3) is important for the response to thaumatin, the amino acid residues within CRD critical for response are still unknown. A comparison of the amino acid sequence (69 amino acid residues) of CRD between hT1R3 and mouse T1R3 (mT1R3) revealed sixteen amino acids that differ. In the present study, we converted each of these sixteen amino acids in hT1R3 to their mouse counterpart and examined the response to thaumatin and sucralose using a cell-based assay. No significant decrease in the response to sucralose was seen among any of the sixteen mutants. However, five mutants (Q504K, A537T, R556P, S559P, and R560K) exhibited a significantly diminished response to thaumatin. The five critical residues involved in the response to thaumatin were dispersed in the CRD of hT1R3 and widely distributed when compared to brazzein. The unique intense sweet-taste of thaumatin might be attributed to the different receptor activation mechanism compared to the small molecule sweetener sucralose. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  9. Cysteine-rich protein 2 accelerates actin filament cluster formation

    PubMed Central

    Shinohara, Satoko; Takaoka, Shunpei; Miyake, Jun

    2017-01-01

    Filamentous actin (F-actin) forms many types of structures and dynamically regulates cell morphology and movement, and plays a mechanosensory role for extracellular stimuli. In this study, we determined that the smooth muscle-related transcription factor, cysteine-rich protein 2 (CRP2), regulates the supramolecular networks of F-actin. The structures of CRP2 and F-actin in solution were analyzed by small-angle X-ray solution scattering (SAXS). The general shape of CRP2 was partially unfolded and relatively ellipsoidal in structure, and the apparent cross sectional radius of gyration (Rc) was about 15.8 Å. The predicted shape, derived by ab initio modeling, consisted of roughly four tandem clusters: LIM domains were likely at both ends with the middle clusters being an unfolded linker region. From the SAXS analysis, the Rc of F-actin was about 26.7 Å, and it was independent of CRP2 addition. On the other hand, in the low angle region of the CRP2-bound F-actin scattering, the intensities showed upward curvature with the addition of CRP2, which indicates increasing branching of F-actin following CRP2 binding. From biochemical analysis, the actin filaments were augmented and clustered by the addition of CRP2. This F-actin clustering activity of CRP2 was cooperative with α-actinin. Thus, binding of CRP2 to F-actin accelerates actin polymerization and F-actin cluster formation. PMID:28813482

  10. Recovery of paramyxovirus simian virus 5 with a V protein lacking the conserved cysteine-rich domain: the multifunctional V protein blocks both interferon-beta induction and interferon signaling.

    PubMed

    He, Biao; Paterson, Reay G; Stock, Nicola; Durbin, Joan E; Durbin, Russell K; Goodbourn, Stephen; Randall, Richard E; Lamb, Robert A

    2002-11-10

    The V protein of the Paramyxovirus simian virus 5 (SV5) is a multifunctional protein containing an N-terminal 164 residue domain that is shared with the P protein and a distinct C-terminal domain that is cysteine-rich and which is highly conserved among Paramyxoviruses. We report the recovery from Vero cells [interferon (IFN) nonproducing cells] of a recombinant SV5 (rSV5) that lacks the V protein C-terminal specific domain (rSV5VDeltaC). In Vero cells rSV5VDeltaC forms large plaques and grows at a rate and titer similar to those of rSV5. In BHK or CV-1 cells rSV5VDeltaC forms small plaques and grows poorly. However, even when grown in Vero cells rSV5VDeltaC reverts to pseudo-wild-type virus in four to five passages, indicating the importance of the V protein for successful replication of SV5. Whereas rSV5 grows in many cell types with minimal cytopathic effect (CPE), rSV5VDeltaC causes extensive CPE in the same cell types. To overcome the antiviral state induced by IFN, many viruses have evolved mechanisms to counteract the effects of IFN by blocking the production of IFN and abrogating IFN signaling. Whereas rSV5 blocks IFN signaling by mediating the degradation of STAT1, rSV5VDeltaC does not cause the degradation of STAT1 and IFN signaling occurs through formation of the ISGF3 transcription complex. Furthermore, we find that rSV5 infection of cells prevents production of IFN-beta. The transcription factor IRF-3 which is required for transcription of the IFN-beta gene is not translocated from the cytoplasm to the nucleus in rSV5-infected cells. In contrast, in rSV5VDeltaC-infected cells IRF-3 is localized predominantly in the nucleus and IFN-beta is produced. By using ectopic expression of IRF-3, it was shown that after dsRNA treatment and expression of the V protein IRF-3 remained in the cytoplasm, whereas after dsRNA treatment and expression of the P protein (which lacks the C-terminal cysteine-rich domain) IRF-3 was localized predominantly in the nucleus. Thus

  11. Structure-based Discovery of Novel Small Molecule Wnt Signaling Inhibitors by Targeting the Cysteine-rich Domain of Frizzled*

    PubMed Central

    Lee, Ho-Jin; Bao, Ju; Miller, Ami; Zhang, Chi; Wu, Jibo; Baday, Yiressy C.; Guibao, Cristina; Li, Lin; Wu, Dianqing; Zheng, Jie J.

    2015-01-01

    Frizzled is the earliest discovered glycosylated Wnt protein receptor and is critical for the initiation of Wnt signaling. Antagonizing Frizzled is effective in inhibiting the growth of multiple tumor types. The extracellular N terminus of Frizzled contains a conserved cysteine-rich domain that directly interacts with Wnt ligands. Structure-based virtual screening and cell-based assays were used to identify five small molecules that can inhibit canonical Wnt signaling and have low IC50 values in the micromolar range. NMR experiments confirmed that these compounds specifically bind to the Wnt binding site on the Frizzled8 cysteine-rich domain with submicromolar dissociation constants. Our study confirms the feasibility of targeting the Frizzled cysteine-rich domain as an effective way of regulating canonical Wnt signaling. These small molecules can be further optimized into more potent therapeutic agents for regulating abnormal Wnt signaling by targeting Frizzled. PMID:26504084

  12. Cysteine-rich protein 61 (CCN1) domain-specific stimulation of matrix metalloproteinase-1 expression through αVβ3 integrin in human skin fibroblasts.

    PubMed

    Qin, Zhaoping; Fisher, Gary J; Quan, Taihao

    2013-04-26

    Human skin largely comprises collagenous extracellular matrix. The hallmark of skin aging is fragmentation of collagen fibrils. Matrix metalloproteinases (MMPs) are largely responsible for collagen degradation. MMP-1, principally derived from dermal fibroblasts, is the major protease capable of initiating degradation of native fibrillar collagens. Presently, we report that CCN1, a secreted and extracellular matrix-associated protein, is elevated in aged human skin dermal fibroblasts in vivo and stimulates MMP-1 expression through functional interaction with αVβ3 integrin in human dermal fibroblasts. CCN1 contains four conserved structural domains. Our results indicate that the three N-terminal domains (IGFBP, VWC, and TSP1), but not the C-terminal CT domain, are required for CCN1 to stimulate MMP-1 expression. This stimulation is dependent on interaction between the active structural domains and αVβ3 integrin. The interaction of VWC domain with integrin αVβ3 is necessary and requires functional cooperation with adjacent IGFBP and TSP1 domains to stimulate MMP-1 expression. Finally, induction of MMP-1 expression in dermal fibroblasts by CCN1 N-terminal domains resulted in fragmentation of type I collagen fibrils in a three-dimensional collagen lattice model. These data suggest that domain-specific interactions of CCN1 with αVβ3 integrin contribute to human skin aging by stimulating MMP-1-mediated collagen fibril fragmentation.

  13. Structural divergence of cysteine-rich secretory proteins in snake venoms.

    PubMed

    Matsunaga, Yukiko; Yamazaki, Yasuo; Hyodo, Fumiko; Sugiyama, Yusuke; Nozaki, Masatoshi; Morita, Takashi

    2009-03-01

    Cysteine-rich secretory proteins (CRISPs) are expressed in spermatocytes and granules of neutrophils in mammals, and are associated with sperm maturation and host defense. Related proteins have recently been recovered in snake venoms, and some of the snake venom-derived CRISPs exhibit ion channel blocking activity. Here we isolated and identified two novel CRISPs (kaouthin-1 and kaouthin-2) from the venom of Naja kaouthia (Elapidae), and cloned the encoding cDNAs. Kaouthin-1 and kaouthin-2 were classified into two broad sister groups of Elapidae, the Asian species and the marine/Australian species, respectively. Sequence comparisons reveal that the high-frequency variable regions among snake venom CRISPs define a continuous line on the molecular surface of the N-terminal pathogenesis-related protein-1 (PR-1) domain and the C-terminal cysteine-rich domain (CRD). Snake venom proteins generally display efficient molecular diversity around functionally key regions, suggesting that the PR-1 domain of CRISPs is important for the recognition of target molecules.

  14. The cysteine-rich domain regulates ADAM protease function in vivo.

    PubMed

    Smith, Katherine M; Gaultier, Alban; Cousin, Helene; Alfandari, Dominique; White, Judith M; DeSimone, Douglas W

    2002-12-09

    ADAMs are membrane-anchored proteases that regulate cell behavior by proteolytically modifying the cell surface and ECM. Like other membrane-anchored proteases, ADAMs contain candidate "adhesive" domains downstream of their metalloprotease domains. The mechanism by which membrane-anchored cell surface proteases utilize these putative adhesive domains to regulate protease function in vivo is not well understood. We address this important question by analyzing the relative contributions of downstream extracellular domains (disintegrin, cysteine rich, and EGF-like repeat) of the ADAM13 metalloprotease during Xenopus laevis development. When expressed in embryos, ADAM13 induces hyperplasia of the cement gland, whereas ADAM10 does not. Using chimeric constructs, we find that the metalloprotease domain of ADAM10 can substitute for that of ADAM13, but that specificity for cement gland expansion requires a downstream extracellular domain of ADAM13. Analysis of finer resolution chimeras indicates an essential role for the cysteine-rich domain and a supporting role for the disintegrin domain. These and other results reveal that the cysteine-rich domain of ADAM13 cooperates intramolecularly with the ADAM13 metalloprotease domain to regulate its function in vivo. Our findings thus provide the first evidence that a downstream extracellular adhesive domain plays an active role in regulating ADAM protease function in vivo. These findings are likely relevant to other membrane-anchored cell surface proteases.

  15. The role of cysteine-rich secretory proteins in male fertility.

    PubMed

    Koppers, Adam J; Reddy, Thulasimala; O'Bryan, Moira K

    2011-01-01

    The cysteine-rich secretory proteins (CRISPs) are a subgroup of the CRISP, antigen 5 and Pr-1 (CAP) protein superfamily, and are found only in vertebrates. They show a strong expression bias to the mammalian male reproductive tract and the venom of poisonous reptiles. Within the male reproductive tract CRISPs have been implicated in many aspects of male germ cell biology spanning haploid germ cell development, epididymal maturation, capacitation, motility and the actual processes of fertilization. At a structural level, CRISPs are composed of two domains, a CAP domain, which has been implicated in cell-cell adhesion, and a CRISP domain, which has been shown to regulate several classes of ion channels across multiple species. Herein, we will review the current literature on the role of CRISPs in male fertility, and by inference to related non-mammalian protein, infer potential biochemical functions.

  16. The role of cysteine-rich secretory proteins in male fertility

    PubMed Central

    Koppers, Adam J; Reddy, Thulasimala; O'Bryan, Moira K

    2011-01-01

    The cysteine-rich secretory proteins (CRISPs) are a subgroup of the CRISP, antigen 5 and Pr-1 (CAP) protein superfamily, and are found only in vertebrates. They show a strong expression bias to the mammalian male reproductive tract and the venom of poisonous reptiles. Within the male reproductive tract CRISPs have been implicated in many aspects of male germ cell biology spanning haploid germ cell development, epididymal maturation, capacitation, motility and the actual processes of fertilization. At a structural level, CRISPs are composed of two domains, a CAP domain, which has been implicated in cell–cell adhesion, and a CRISP domain, which has been shown to regulate several classes of ion channels across multiple species. Herein, we will review the current literature on the role of CRISPs in male fertility, and by inference to related non-mammalian protein, infer potential biochemical functions. PMID:20972450

  17. Characterization of a scavenger receptor cysteine-rich-domain-containing protein of the starfish, Asterina pectinifera: ApSRCR1 acts as an opsonin in the larval and adult innate immune systems.

    PubMed

    Furukawa, Ryohei; Matsumoto, Midori; Kaneko, Hiroyuki

    2012-01-01

    Proteins containing a scavenger receptor cysteine-rich (SRCR) domain (SRCR proteins) play an important role in the innate immune system of various metazoan animals. In the starfish Asterina pectinifera, mesenchyme cells and coelomocytes govern the two distinct innate immune systems of the larvae and adults, respectively. Here we identify a cDNA encoding a protein containing nine SRCR domains termed ApSRCR1, and present characterization of the molecular structure, expression, subcellular localization and function of ApSRCR1 protein during ontogenesis of this animal. ApSRCR1 protein is a membrane-type protein with a predicted molecular mass of approximately 120 kDa. During ontogenesis, ApSRCR1 protein is de novo synthesized and localizes to cytoplasmic vesicles in both mesenchyme cells and coelomocytes without translation of maternal mRNA; however, the net production and modification by N-glycosylation of ApSRCR1 protein differs in each cell type. In both types of cell, functional inhibition of ApSRCR1 protein leads to incompetent bacterial clearance and failure of aggregate formation. However, this inhibitory effect is weaker in the mesenchyme cells than in the coelomocytes. In the bacteria-sensitized adult, ApSRCR1 protein is up-regulated and digested to enable its secretion into the coelomic fluid. This secreted form of ApSRCR1 protein can apparently bind to bacteria. Overall, we show that ApSRCR1 protein is finely regulated for expression not only during development but also in a sensitive innate immunological situation, and thereupon acts as an opsonin for bacteria to different extents in the larvae and adults of A. pectinifera. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Crystallization and preliminary X-ray analysis of ginkbilobin-2 from Ginkgo biloba seeds: a novel antifungal protein with homology to the extracellular domain of plant cysteine-rich receptor-like kinases

    SciTech Connect

    Miyakawa, Takuya; Sawano, Yoriko; Miyazono, Ken-ichi; Hatano, Ken-ichi; Tanokura, Masaru

    2007-09-01

    Purification and crystallization of ginkbilobin-2 and its selenomethionine derivative allowed the collection of complete data to 2.38 Å resolution and multiwavelength anomalous diffraction data sets, respectively. The antifungal protein ginkbilobin-2 (Gnk2) from Ginkgo biloba seeds does not show homology to other pathogenesis-related proteins, but does show homology to the extracellular domain of plant cysteine-rich receptor-like kinases. Native Gnk2 purified from ginkgo nuts and the selenomethionine derivative of recombinant Gnk2 (SeMet-rGnk2) were crystallized by the sitting-drop vapour-diffusion method using different precipitants. X-ray diffraction data were collected from Gnk2 at 2.38 Å resolution and from SeMet-rGnk2 at 2.79 Å resolution using a synchrotron-radiation source. The crystals of both proteins belonged to the primitive cubic space group P2{sub 1}3, with unit-cell parameters a = b = c = 143.2 Å.

  19. The cysteine-rich domain of human T1R3 is necessary for the interaction between human T1R2-T1R3 sweet receptors and a sweet-tasting protein, thaumatin.

    PubMed

    Ohta, Keisuke; Masuda, Tetsuya; Tani, Fumito; Kitabatake, Naofumi

    2011-03-18

    Thaumatin is an intensely sweet-tasting protein perceived by humans but not rodents. Its threshold value of sweetness in humans is 50nM, the lowest of any sweet-tasting protein. In the present study, the sites where sweet receptors interact with thaumatin were investigated using human embryonic kidney 293 (HEK293) cells expressing the sweet receptors T1R2-T1R3. Chimeric human- mouse sweet receptors were constructed and their responses to sweeteners were investigated. The human (h) T1R2- mouse (m) T1R3 combination responded to sucralose but not to thaumatin, clearly indicating that a T1R3 subunit from humans is necessary for the interaction with thaumatin. Furthermore, results obtained from using chimeric T1R3s showed that the cysteine-rich domain (CRD) of human T1R3 is important for the interaction with thaumatin. The CRD of T1R3 would be a prominent target for designing new sweeteners. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of beta-carotene accumulation.

    PubMed

    Lu, Shan; Van Eck, Joyce; Zhou, Xiangjun; Lopez, Alex B; O'Halloran, Diana M; Cosman, Kelly M; Conlin, Brian J; Paolillo, Dominick J; Garvin, David F; Vrebalov, Julia; Kochian, Leon V; Küpper, Hendrik; Earle, Elizabeth D; Cao, Jun; Li, Li

    2006-12-01

    Despite recent progress in our understanding of carotenogenesis in plants, the mechanisms that govern overall carotenoid accumulation remain largely unknown. The Orange (Or) gene mutation in cauliflower (Brassica oleracea var botrytis) confers the accumulation of high levels of beta-carotene in various tissues normally devoid of carotenoids. Using positional cloning, we isolated the gene representing Or and verified it by functional complementation in wild-type cauliflower. Or encodes a plastid-associated protein containing a DnaJ Cys-rich domain. The Or gene mutation is due to the insertion of a long terminal repeat retrotransposon in the Or allele. Or appears to be plant specific and is highly conserved among divergent plant species. Analyses of the gene, the gene product, and the cytological effects of the Or transgene suggest that the functional role of Or is associated with a cellular process that triggers the differentiation of proplastids or other noncolored plastids into chromoplasts for carotenoid accumulation. Moreover, we demonstrate that Or can be used as a novel genetic tool to induce carotenoid accumulation in a major staple food crop. We show here that controlling the formation of chromoplasts is an important mechanism by which carotenoid accumulation is regulated in plants.

  1. Identification of the amino acid residues and domains in the cysteine-rich protein of Chinese wheat mosaic virus that are important for RNA silencing suppression and subcellular localization.

    PubMed

    Sun, Liying; Andika, Ida Bagus; Kondo, Hideki; Chen, Jianping

    2013-04-01

    Cysteine-rich proteins (CRPs) encoded by some plant viruses in diverse genera function as RNA silencing suppressors. Within the N-terminal portion of CRPs encoded by furoviruses, there are six conserved cysteine residues and a Cys-Gly-X-X-His motif (Cys, cysteine; Gly, glycine; His, histidine; X, any amino acid residue) with unknown function. The central domains contain coiled-coil heptad amino acid repeats that usually mediate protein dimerization. Here, we present evidence that the conserved cysteine residues and Cys-Gly-X-X-His motif in the CRP of Chinese wheat mosaic virus (CWMV) are critical for protein stability and silencing suppression activity. Mutation of a leucine residue in the third coiled-coil heptad impaired CWMV CRP activity for suppression of local silencing, but not for the promotion of cell-to-cell movement of Potato virus X (PVX). In planta and in vitro analysis of wild-type and mutant proteins indicated that the ability of the CRP to self-interact was correlated with its suppression activity. Deletion of up to 40 amino acids at the C-terminus did not abolish suppression activity, but disrupted the association of CRP with endoplasmic reticulum (ER), and reduced its activity in the enhancement of PVX symptom severity. Interestingly, a short region in the C-terminal domain, predicted to form an amphipathic α-helical structure, was responsible for the association of CWMV CRP with ER. Overall, our results demonstrate that the N-terminal and central regions are the functional domains for suppression activity, whereas the C-terminal region primarily functions to target CWMV CRP to the ER. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

  2. The disulfide bond pattern of catrocollastatin C, a disintegrin-like/cysteine-rich protein isolated from Crotalus atrox venom.

    PubMed Central

    Calvete, J. J.; Moreno-Murciano, M. P.; Sanz, L.; Jürgens, M.; Schrader, M.; Raida, M.; Benjamin, D. C.; Fox, J. W.

    2000-01-01

    The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass spectrometry. The N-terminal disintegrin-like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin-like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn-metalloproteinases (SVMPs). The C-terminal cysteine-rich domain of catrocollastatin-C contains five disulfide bonds between nearest-neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin-like and cysteine-rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed. PMID:10933502

  3. Identification and characterization of a cell surface scavenger receptor cysteine-rich protein of Sciaenops ocellatus: bacterial interaction and its dependence on the conserved structural features of the SRCR domain.

    PubMed

    Qiu, Reng; Sun, Bo-Guang; Li, Jun; Liu, Xiao; Sun, Li

    2013-03-01

    The scavenger receptor cysteine-rich (SRCR) proteins are secreted or membrane-bound receptors with one or multiple SRCR domains. Members of the SRCR superfamily are known to have diverse functions that include pathogen recognition and immunoregulation. In teleost, although protein sequences with SRCR structure have been identified in some species, very little functional investigation has been carried out. In this study, we identified and characterized a teleost SRCR protein from red drum Sciaenops ocellatus. The protein was named S. ocellatus SRCR1 (SoSRCRP1). SoSRCRP1 is 410-residue in length and was predicted to be a transmembrane protein, with the extracellular region containing a collagen triple helix repeat and a SRCR domain. The SRCR domain has six conserved cysteines, of which, C338 and C399, C351 and C409, and C379 and C389 were predicted to form three disulfide bonds. SoSRCRP1 expression was detected mainly in immune-relevant tissues and upregulated by bacterial and viral infection. In head kidney leukocytes, bacterial infection stimulated the expression of SoSRCRP1, and the expressed SoSRCRP1 was localized on cell surface. Recombinant SoSRCRP1 (rSoSRCRP1) corresponding to the SRCR domain was purified from Escherichia coli and found to be able to bind Gram-negative and Gram-positive bacteria. To examine the structure-function relationship of SoSRCRP1, the mutant proteins SoSRCRP1M1, SoSRCRP1M2, SoSRCRP1M3, and SoSRCRP1M4 were created, which bear C351S and C409S, C338S, C379S, and R325A mutations respectively. Compared to rSoSRCRP1, all mutants were significantly reduced in the ability of bacterial interaction, with the highest reduction observed with SoSRCRP1M4. Taken together, these results indicate that SoSRCRP1 is a cell surface-localized SRCR protein that binds bacterial ligands in a manner that depends on the conserved structural features of the SRCR domain.

  4. NOA36/ZNF330 is a conserved cystein-rich protein with proapoptotic activity in human cells.

    PubMed

    de Melo, Ivan S; Iglesias, Concepción; Benítez-Rondán, Alicia; Medina, Francisco; Martínez-Barberá, Juan Pedro; Bolívar, Jorge

    2009-12-01

    Translocations of regulator proteins from or to the mitochondria are key events in apoptosis regulation. NOA36/ZNF330 is a highly evolutionary conserved protein with a characteristic cystein-rich domain. In this work we address its mitochondrial localization and we demonstrate that a blockage of endogenous NOA36/ZNF330 expression by small-interfering RNA (siRNA) reduced apoptotic response to etoposide (ETO), camptothecin (CPT) and staurosporine (STS) but not to CH11 anti-Fas antibody or tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) in HeLa cells. In contrast, when ectopically expressed in the cytoplasm, NOA36/ZNF330 induces apoptotic cell death. We also found that the domain responsible for this proapoptotic activity is located its cystein-rich region. We propose that NOA36/ZNF330 is translocated from the mitochondria to the cytoplasm when apoptosis is induced and that it contributes to cytochrome c release.

  5. 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

  6. Metagenomics analysis reveals a new metallothionein family: Sequence and metal-binding features of new environmental cysteine-rich proteins.

    PubMed

    Ziller, Antoine; Yadav, Rajiv Kumar; Capdevila, Mercè; Reddy, Mondem Sudhakara; Vallon, Laurent; Marmeisse, Roland; Atrian, Silvia; Palacios, Òscar; Fraissinet-Tachet, Laurence

    2017-02-01

    Metallothioneins are cysteine-rich proteins, which function as (i) metal carriers in basal cell metabolism and (ii) protective metal chelators in conditions of metal excess. Metallothioneins have been characterized from different eukaryotic model and cultivable species. Presently, they are categorized in 15 families but evolutionary relationships between these metallothionein families remain unresolved. Several cysteine-rich protein encoding genes that conferred Cd-tolerance in Cd-sensitive yeast mutants have previously been isolated from soil eukaryotic metatranscriptomes. They were called CRPs for "cysteine-rich proteins". These proteins, of unknown taxonomic origins, share conserved cysteine motifs and could be considered as metallothioneins. In the present work, we analyzed these CRPs with respect to their amino acid sequence features and their metal-binding abilities towards Cd, Zn and Cu metal ions. Sequence analysis revealed that they share common features with different known metallothionein families, but also exhibit unique specific features. Noticeably, CRPs display two separate cysteine-rich domains which, when expressed separately in yeast, confer Cd-tolerance. The N-terminal domain contains some conserved atypical Cys motifs, such as one CCC and two CXCC ones. Five CRPs were expressed and purified as recombinant proteins and their metal-binding characteristics were studied. All these CRPs chelated Cd(II), Zn(II) and Cu(I), although displaying a better capacity for Zn(II) coordination. All CRPs are able to confer Cd-tolerance, and four of them confer Zn-tolerance in the Zn-sensitive zrc1Δ yeast mutant. We designated these CRPs as environmental metallothioneins belonging to a new formerly undescribed metallothionein family. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Amplified pathogenic actions of angiotensin II in cysteine-rich LIM-only protein 4-negative mouse hearts.

    PubMed

    Straubinger, Julia; Boldt, Karsten; Kuret, Anna; Deng, Lisa; Krattenmacher, Diana; Bork, Nadja; Desch, Matthias; Feil, Robert; Feil, Susanne; Nemer, Mona; Ueffing, Marius; Ruth, Peter; Just, Steffen; Lukowski, Robert

    2017-04-01

    LIM domain proteins have been identified as essential modulators of cardiac biology and pathology; however, it is unclear which role the cysteine-rich LIM-only protein (CRP)4 plays in these processes. In studying CRP4 mutant mice, we found that their hearts developed normally, but lack of CRP4 exaggerated multiple parameters of the cardiac stress response to the neurohormone angiotensin II (Ang II). Aiming to dissect the molecular details, we found a link between CRP4 and the cardioprotective cGMP pathway, as well as a multiprotein complex comprising well-known hypertrophy-associated factors. Significant enrichment of the cysteine-rich intestinal protein (CRIP)1 in murine hearts lacking CRP4, as well as severe cardiac defects and premature death of CRIP1 and CRP4 morphant zebrafish embryos, further support the notion that depleting CRP4 is incompatible with a proper cardiac development and function. Together, amplified Ang II signaling identified CRP4 as a novel antiremodeling factor regulated, at least to some extent, by cardiac cGMP.-Straubinger, J., Boldt, K., Kuret, A., Deng, L., Krattenmacher, D., Bork, N., Desch, M., Feil, R., Feil, S., Nemer, M., Ueffing, M., Ruth, P., Just, S., Lukowski, R. Amplified pathogenic actions of angiotensin II in cysteine-rich LIM-only protein 4 negative mouse hearts.

  8. Structure and function of snake venom cysteine-rich secretory proteins.

    PubMed

    Yamazaki, Yasuo; Morita, Takashi

    2004-09-01

    Cysteine-rich secretory proteins (CRISPs) are primarily found in the epididymis of mammals and are expressed in diverse organisms. However, the functions of most CRISPs remain unknown. Recent studies reveal that CRISPs are widely distributed in snake venoms and that they inhibit smooth muscle contraction and cyclic nucleotide-gated ion channels. In this review, we discuss recent findings on several snake venom-derived CRISPs.

  9. The Arabidopsis cysteine-rich protein GASA4 promotes GA responses and exhibits redox activity in bacteria and in planta.

    PubMed

    Rubinovich, Lior; Weiss, David

    2010-12-01

    Although the gibberellin (GA) signaling pathway has been elucidated, very little is known about the steps linking first transcriptional activation to physiological responses. Among the few identified GA-induced genes are the plant-specific GAST1-like genes, which encode small proteins with a conserved cysteine-rich domain. The role of these proteins in plant development and GA responses is not yet clear. The Arabidopsis GAST1-like gene family consists of 14 members, GASA1-14. Here we show that over-expression of the GA-induced GASA4 gene in Arabidopsis promoted GA responses such as flowering and seed germination. Suppression of several GASA genes using synthetic microRNA (miR(GASA) ) also promoted seed germination. This was probably caused by suppression of GASA5, which acts as a repressor of GA responses. Previously, we proposed that GAST1-like proteins are involved in redox reactions via their cysteine-rich domain. The results of this study support this hypothesis, as over-expression of GASA4 suppressed ROS accumulation and the transgenic seeds were partially resistant to the NO donor sodium nitroprusside (SNP). Moreover, Escherichia coli expressing intact GASA4 or a truncated version containing only the cysteine-rich domain were resistant to SNP. Mutated GASA4, in which conserved cysteines were replaced by alanines, lost its redox activity and the ability to promote GA responses, suggesting that the two functions are linked. We propose that GA induces some GAST1-like genes and suppresses others to regulate its own responses. We also suggest that the encoded proteins regulate the redox status of specific components to promote or suppress these responses.

  10. The role of the ADAMTS13 cysteine-rich domain in VWF binding and proteolysis.

    PubMed

    de Groot, Rens; Lane, David A; Crawley, James T B

    2015-03-19

    ADAMTS13 proteolytically regulates the platelet-tethering function of von Willebrand factor (VWF). ADAMTS13 function is dependent upon multiple exosites that specifically bind the unraveled VWF A2 domain and enable proteolysis. We carried out a comprehensive functional analysis of the ADAMTS13 cysteine-rich (Cys-rich) domain using engineered glycans, sequence swaps, and single point mutations in this domain. Mutagenesis of Cys-rich domain-charged residues had no major effect on ADAMTS13 function, and 5 out of 6 engineered glycans on the Cys-rich domain also had no effect on ADAMTS13 function. However, a glycan attached at position 476 appreciably reduced both VWF binding and proteolysis. Substitution of Cys-rich sequences for the corresponding regions in ADAMTS1 identified a hydrophobic pocket involving residues Gly471-Val474 as being of critical importance for both VWF binding and proteolysis. Substitution of hydrophobic VWF A2 domain residues to serine in a region (residues 1642-1659) previously postulated to interact with the Cys-rich domain revealed the functional importance of VWF residues Ile1642, Trp1644, Ile1649, Leu1650, and Ile1651. Furthermore, the functional deficit of the ADAMTS13 Cys-rich Gly471-Val474 variant was dependent on these same hydrophobic VWF residues, suggesting that these regions form complementary binding sites that directly interact to enhance the efficiency of the proteolytic reaction.

  11. The role of the ADAMTS13 cysteine-rich domain in VWF binding and proteolysis

    PubMed Central

    Lane, David A.; Crawley, James T. B.

    2015-01-01

    ADAMTS13 proteolytically regulates the platelet-tethering function of von Willebrand factor (VWF). ADAMTS13 function is dependent upon multiple exosites that specifically bind the unraveled VWF A2 domain and enable proteolysis. We carried out a comprehensive functional analysis of the ADAMTS13 cysteine-rich (Cys-rich) domain using engineered glycans, sequence swaps, and single point mutations in this domain. Mutagenesis of Cys-rich domain–charged residues had no major effect on ADAMTS13 function, and 5 out of 6 engineered glycans on the Cys-rich domain also had no effect on ADAMTS13 function. However, a glycan attached at position 476 appreciably reduced both VWF binding and proteolysis. Substitution of Cys-rich sequences for the corresponding regions in ADAMTS1 identified a hydrophobic pocket involving residues Gly471-Val474 as being of critical importance for both VWF binding and proteolysis. Substitution of hydrophobic VWF A2 domain residues to serine in a region (residues 1642-1659) previously postulated to interact with the Cys-rich domain revealed the functional importance of VWF residues Ile1642, Trp1644, Ile1649, Leu1650, and Ile1651. Furthermore, the functional deficit of the ADAMTS13 Cys-rich Gly471-Val474 variant was dependent on these same hydrophobic VWF residues, suggesting that these regions form complementary binding sites that directly interact to enhance the efficiency of the proteolytic reaction. PMID:25564400

  12. Isolation and developmental expression of a rat cDNA encoding a cysteine-rich zinc finger protein.

    PubMed Central

    McLaughlin, C R; Tao, Q; Abood, M E

    1994-01-01

    A number of cysteine-rich proteins have recently been isolated by homology screening, differential library screens, and association with other proteins. In this report, we describe the isolation of the rat cysteine-rich protein from a rat brain library during a search for clones with homology to the delta-opioid receptor. One of the cDNAs isolated hybridized to a 1.8 kb mRNA abundantly expressed throughout the rat brain as well as in rat liver. In situ hybridization reveals a wide distribution in rat brain; in particular, abundant hybridization was detected in the hippocampus, cerebellum, habenula, reticular thalamic nucleus and interposed nucleus. Nucleotide sequence analysis of a 1403 bp cDNA clone indicated 77% identity with the cDNA for human cysteine-rich protein (hCRP), that translates into a 99% identity at the amino acid level. The predicted amino acid sequence suggests four zinc fingers, two of the C4 class and two of the C2HC class. This structural motif is characteristic of members of the LIM domain protein family. The mRNA is serum-inducible in Balb/c 3T3 cells. Additional study suggests that its expression is not induced by either NGF treatment of PC12h pheochromocytoma cells, or inflammation-induced injury in the spinal cord at up to 60 min after injury. It does appear to be developmentally expressed in rat brain, consistent with a potential role in neuronal development. The rat CRP clone will be useful for studying the function of CRPs in rodent models. Images PMID:7816640

  13. Sclerostin binds and regulates the activity of cysteine-rich protein 61

    SciTech Connect

    Craig, Theodore A.; Bhattacharya, Resham; Mukhopadhyay, Debabrata; Kumar, Rajiv

    2010-01-29

    Sclerostin, a secreted glycoprotein, regulates osteoblast function. Using yeast two-hybrid and direct protein interaction analyses, we demonstrate that sclerostin binds the Wnt-modulating and Wnt-modulated, extracellular matrix protein, cysteine-rich protein 61 (Cyr61, CCN1), which regulates mesenchymal stem cell proliferation and differentiation, osteoblast and osteoclast function, and angiogenesis. Sclerostin was shown to inhibit Cyr61-mediated fibroblast attachment, and Cyr61 together with sclerostin increases vascular endothelial cell migration and increases osteoblast cell division. The data show that sclerostin binds to and influences the activity of Cyr61.

  14. Scavenger receptor cysteine-rich domains 9 and 11 of WC1 are receptors for the WC1 counter receptor.

    PubMed

    Ahn, J S; Konno, A; Gebe, J A; Aruffo, A; Hamilton, M J; Park, Y H; Davis, W C

    2002-08-01

    Workshop cluster 1 (WC1) is a member of the scavenger receptor cysteine-rich (SRCR) superfamily that includes CD5, CD6, CD163, and M160. Bovine WC1 consists of 11 SRCR domains, a unique domain 1, and two homologous 5 SRCR domain cassettes, WC1 domains 2-6 and 7-11. The porcine orthologue of WC1 contains five SRCR domains with a different domain arrangement. Although the function of WC1 is unknown, WC1 is proposed to be an accessory or homing molecule. Thus, identification of cells that express the counter receptor for WC1 (WC1-CR) is critical to understanding the function of WC1. For this reason, we constructed WC1-human immunoglobulin G1 fusion proteins to identify the binding domain of WC1 and cells that express the WC1-CR. Immunohistochemical analysis revealed WC1 domains 9 and 11 bind cells with macrophage and dendritic cell morphology and cells in ellipsoids in the spleen. These results and the finding of conserved signaling motifs in the cytoplasmic tail suggest WC1 may be an accessory molecule.

  15. Cysteine-rich intestinal protein binds zinc during transmucosal zinc transport.

    PubMed Central

    Hempe, J M; Cousins, R J

    1991-01-01

    The mechanism of zinc absorption has not been delineated, but kinetic studies show that both passive and carrier-mediated processes are involved. We have identified a low molecular mass zinc-binding protein in the soluble fraction of rat intestinal mucosa that could function as an intracellular zinc carrier. The protein was not detected in liver or pancreas, suggesting a role specific to the intestine. The protein binds zinc during transmucosal zinc transport and shows signs of saturation at higher luminal zinc concentrations, characteristics consistent with a role in carrier-mediated zinc absorption. Microsequence analysis of the protein purified by gel-filtration HPLC and SDS/PAGE showed complete identity within the first 41 N-terminal amino acids with the deduced protein sequence of cysteine-rich intestinal protein [Birkenmeier, E. H. & Gordon, J. I. (1986) Proc. Natl. Acad. Sci. USA 83, 2516-2520]. These investigators showed that the gene for this protein is developmentally regulated in neonates during the suckling period, conserved in many vertebrate species, and predominantly expressed in the small intestine. Cysteine-rich intestinal protein contains a recently identified conserved sequence of histidine and cysteine residues, the LIM motif, which our results suggest confers metal-binding properties that are important for zinc transport and/or functions of this micronutrient. Images PMID:1946385

  16. A novel cysteine-rich sequence-specific DNA-binding protein interacts with the conserved X-box motif of the human major histocompatibility complex class II genes via a repeated Cys-His domain and functions as a transcriptional repressor

    PubMed Central

    1994-01-01

    The class II major histocompatibility complex (MHC) molecules function in the presentation of processed peptides to helper T cells. As most mammalian cells can endocytose and process foreign antigen, the critical determinant of an antigen-presenting cell is its ability to express class II MHC molecules. Expression of these molecules is usually restricted to cells of the immune system and dysregulated expression is hypothesized to contribute to the pathogenesis of a severe combined immunodeficiency syndrome and certain autoimmune diseases. Human complementary DNA clones encoding a newly identified, cysteine-rich transcription factor, NF-X1, which binds to the conserved X-box motif of class II MHC genes, were obtained, and the primary amino acid sequence deduced. The major open reading frame encodes a polypeptide of 1,104 amino acids with a symmetrical organization. A central cysteine-rich portion encodes the DNA-binding domain, and is subdivided into seven repeated motifs. This motif is similar to but distinct from the LIM domain and the RING finger family, and is reminiscent of known metal-binding regions. The unique arrangement of cysteines indicates that the consensus sequence CX3CXL-XCGX1- 5HXCX3CHXGXC represents a novel cysteine-rich motif. Two lines of evidence indicate that the polypeptide encodes a potent and biologically relevant repressor of HLA-DRA transcription: (a) overexpression of NF-X1 from a retroviral construct strongly decreases transcription from the HLA-DRA promoter; and (b) the NF-X1 transcript is markedly induced late after induction with interferon gamma (IFN- gamma), coinciding with postinduction attenuation of HLA-DRA transcription. The NF-X1 protein may therefore play an important role in regulating the duration of an inflammatory response by limiting the period in which class II MHC molecules are induced by IFN-gamma. PMID:7964459

  17. Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms.

    PubMed

    Jin, Yang; Lu, Qiumin; Zhou, Xingding; Zhu, Shaowen; Li, Rui; Wang, Wanyu; Xiong, Yuliang

    2003-10-01

    Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes.

  18. The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc*

    PubMed Central

    Gottlieb, Colin D.; Zhang, Sheng; Linder, Maurine E.

    2015-01-01

    DHHC palmitoyltransferases catalyze the addition of the fatty acid palmitate to proteins on the cytoplasmic leaflet of cell membranes. There are 23 members of the highly diverse mammalian DHHC protein family, all of which contain a conserved catalytic domain called the cysteine-rich domain (CRD). DHHC proteins transfer palmitate via a two-step catalytic mechanism in which the enzyme first modifies itself with palmitate in a process termed autoacylation. The enzyme then transfers palmitate from itself onto substrate proteins. The number and location of palmitoylated cysteines in the autoacylated intermediate is unknown. In this study, we present evidence using mass spectrometry that DHHC3 is palmitoylated at the cysteine in the DHHC motif. Mutation of highly conserved CRD cysteines outside the DHHC motif resulted in activity deficits and a structural perturbation revealed by limited proteolysis. Treatment of DHHC3 with chelating agents in vitro replicated both the specific structural perturbations and activity deficits observed in conserved cysteine mutants, suggesting metal ion-binding in the CRD. Using the fluorescent indicator mag-fura-2, the metal released from DHHC3 was identified as zinc. The stoichiometry of zinc binding was measured as 2 mol of zinc/mol of DHHC3 protein. Taken together, our data demonstrate that coordination of zinc ions by cysteine residues within the CRD is required for the structural integrity of DHHC proteins. PMID:26487721

  19. Crystal Structure of the Frizzled-Like Cysteine-Rich Domain of the Receptor Tyrosine Kinase MuSK

    SciTech Connect

    Stiegler, A.; Burden, S; Hubbard, S

    2009-01-01

    Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 {angstrom} resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteins but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ.

  20. Cysteine-rich secretory proteins are not exclusively expressed in the male reproductive tract.

    PubMed

    Reddy, Thulasimala; Gibbs, Gerard M; Merriner, D Jo; Kerr, Jeffrey B; O'Bryan, Moira K

    2008-11-01

    The Cysteine-RIch Secretory Proteins (CRISPs) are abundantly produced in the male reproductive tract of mammals and within the venom of reptiles and have been shown to regulate ion channel activity. CRISPs, along with the Antigen-5 proteins and the Pathogenesis related-1 (Pr-1) proteins, form the CAP superfamily of proteins. Analyses of EST expression databases are increasingly suggesting that mammalian CRISPs are expressed more widely than in the reproductive tract. We, therefore, conducted a reverse transcription PCR expression profile and immunohistochemical analyses of 16 mouse tissues to define the sites of production of each of the four murine CRISPs. These data showed that each of the CRISPs have distinct and sometimes overlapping expression profiles, typically associated with the male and female reproductive tract, the secretory epithelia of exocrine glands, and immune tissues including the spleen and thymus. These investigations raise the potential for a role for CRISPs in general mammalian physiology.

  1. Cobra venom contains a pool of cysteine-rich secretory proteins.

    PubMed

    Osipov, Alexey V; Levashov, Mikhail Yu; Tsetlin, Victor I; Utkin, Yuri N

    2005-03-04

    A large family of cysteine-rich secretory proteins (CRISPs) includes proteins of different origin, the function of the majority of CRISPs being unknown. For CRISPs isolated from snake venom, two types of activities were found: two proteins blocked cyclic nucleotide-gated ion channels, several others blocked potassium-stimulated smooth muscle contraction. Thus, snake CRISPs represent potentially valuable tools for studies of ion channels, which makes promising a search for new CRISPs. Here we report on the isolation of several novel CRISPs from the venoms of Asian cobra Naja kaouthia and African cobra Naja haje using a combination of different types of liquid chromatography. Four CRISP variants were identified in N. kaouthia venom and three proteins, one of them acidic, were found in N. haje venom. Acidic CRISP was found in a reptilian venom for the first time. Our data suggest that each cobra venom contains a pool of different CRISPs.

  2. Crystal Structure of the Cysteine-Rich Domain of Mannose Receptor Complexed with a Sulfated Carbohydrate Ligand

    PubMed Central

    Liu, Yang; Chirino, Arthur J.; Misulovin, Ziva; Leteux, Christine; Feizi, Ten; Nussenzweig, Michel C.; Bjorkman, Pamela J.

    2000-01-01

    The macrophage and epithelial cell mannose receptor (MR) binds carbohydrates on foreign and host molecules. Two portions of MR recognize carbohydrates: tandemly arranged C-type lectin domains facilitate carbohydrate-dependent macrophage uptake of infectious organisms, and the NH2-terminal cysteine-rich domain (Cys-MR) binds to sulfated glycoproteins including pituitary hormones. To elucidate the mechanism of sulfated carbohydrate recognition, we determined crystal structures of Cys-MR alone and complexed with 4-sulfated-N-acetylgalactosamine at 1.7 and 2.2 Å resolution, respectively. Cys-MR folds into an approximately three-fold symmetric β-trefoil shape resembling fibroblast growth factor. The sulfate portions of 4-sulfated-N-acetylgalactosamine and an unidentified ligand found in the native crystals bind in a neutral pocket in the third lobe. We use the structures to rationalize the carbohydrate binding specificities of Cys-MR and compare the recognition properties of Cys-MR with other β-trefoil proteins. PMID:10748229

  3. C-Cell Neoplasia in Asymptomatic Carriers of RET Mutation in Extracellular Cysteine-Rich and Intracellular Tyrosine Kinase Domain.

    PubMed

    Abi-Raad, Rita; Virk, Renu K; Dinauer, Catherine A; Prasad, A; Morotti, Raffaella A; Breuer, Christopher K; Sosa, Julie A; Udelsman, Robert; Rivkees, Scott A; Prasad, Manju L

    2015-08-01

    Germline mutations in RET proto-oncogene associated with multiple endocrine neoplasia type 2 (MEN2) may affect codons for the extracellular cysteine-rich (ECR) or the intracellular tyrosine kinase (ITK) domain of the transmembrane receptor tyrosine kinase protein. We compared C-cell pathology in asymptomatic carriers of RET mutation affecting the 2 domains. Twenty-two asymptomatic carriers (median age, 9.5 years), 10 with mutations in the ECR (codons 634, 611, 618, and 620) and 12 with mutations in the ITK domain (codons 804, 790, 891, and 918), underwent total thyroidectomy. C-cell hyperplasia was identified in 16 (73%), was multifocal and/or bilateral in 11, and was associated with medullary thyroid carcinoma (MTC) in 10 thyroids. When comparing the ECR and ITK groups in 21 carriers from MEN2A/familial MTC families, C-cell hyperplasia was more frequent in the former (90% versus 55%), as was multifocality (70% versus 27%) and MTC (60% versus 27%), despite the significantly younger median age in the former group (5 versus 23 years, P = .04). One asymptomatic carrier had de novo codon 918 mutation (MEN2B) and showed bilateral microcarcinoma with lymph node metastasis at presentation and progressive disease on follow-up. In conclusion, asymptomatic carriers of high-risk RET mutations affecting the ECR were significantly younger and frequently showed C-cell neoplasia, multifocality, and MTC when compared with mutations affecting the ITK domain in the MEN2A/familial MTC families. The presence of C-cell disease, its severity, and aggressiveness correlated with the mutated codon and with increasing age. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Small cysteine-rich antifungal proteins from radish: their role in host defense.

    PubMed Central

    Terras, F R; Eggermont, K; Kovaleva, V; Raikhel, N V; Osborn, R W; Kester, A; Rees, S B; Torrekens, S; Van Leuven, F; Vanderleyden, J

    1995-01-01

    Radish seeds have previously been shown to contain two homologous, 5-kD cysteine-rich proteins designated Raphanus sativus-antifungal protein 1 (Rs-AFP1) and Rs-AFP2, both of which exhibit potent antifungal activity in vitro. We now demonstrate that these proteins are located in the cell wall and occur predominantly in the outer cell layers lining different seed organs. Moreover, Rs-AFPs are preferentially released during seed germination after disruption of the seed coat. The amount of released proteins is sufficient to create a microenvironment around the seed in which fungal growth is suppressed. Both the cDNAs and the intron-containing genomic regions encoding the Rs-AFP preproteins were cloned. Transcripts (0.55 kb) hybridizing with an Rs-AFP1 cDNA-derived probe were present in near-mature and mature seeds. Such transcripts as well as the corresponding proteins were barely detectable in healthy uninfected leaves but accumulated systemically at high levels after localized fungal infection. The induced leaf proteins (designated Rs-AFP3 and Rs-AFP4) were purified and shown to be homologous to seed Rs-AFPs and to exert similar antifungal activity in vitro. A chimeric Rs-AFP2 gene under the control of the constitutive cauliflower mosaic virus 35S promoter conferred enhanced resistance to the foliar pathogen Alternaria longipes in transgenic tobacco. The term "plant defensins" is proposed to denote these defense-related proteins. PMID:7780308

  5. Cysteine-Rich Atrial Secretory Protein from the Snail Achatina achatina: Purification and Structural Characterization

    PubMed Central

    Shabelnikov, Sergey; Kiselev, Artem

    2015-01-01

    Despite extensive studies of cardiac bioactive peptides and their functions in molluscs, soluble proteins expressed in the heart and secreted into the circulation have not yet been reported. In this study, we describe an 18.1-kDa, cysteine-rich atrial secretory protein (CRASP) isolated from the terrestrial snail Achatina achatina that has no detectable sequence similarity to any known protein or nucleotide sequence. CRASP is an acidic, 158-residue, N-glycosylated protein composed of eight alpha-helical segments stabilized with five disulphide bonds. A combination of fold recognition algorithms and ab initio folding predicted that CRASP adopts an all-alpha, right-handed superhelical fold. CRASP is most strongly expressed in the atrium in secretory atrial granular cells, and substantial amounts of CRASP are released from the heart upon nerve stimulation. CRASP is detected in the haemolymph of intact animals at nanomolar concentrations. CRASP is the first secretory protein expressed in molluscan atrium to be reported. We propose that CRASP is an example of a taxonomically restricted gene that might be responsible for adaptations specific for terrestrial pulmonates. PMID:26444993

  6. Dragline silk: a fiber assembled with low-molecular-weight cysteine-rich proteins.

    PubMed

    Pham, Thanh; Chuang, Tyler; Lin, Albert; Joo, Hyun; Tsai, Jerry; Crawford, Taylor; Zhao, Liang; Williams, Caroline; Hsia, Yang; Vierra, Craig

    2014-11-10

    Dragline silk has been proposed to contain two main protein constituents, MaSp1 and MaSp2. However, the mechanical properties of synthetic spider silks spun from recombinant MaSp1 and MaSp2 proteins have yet to approach natural fibers, implying the natural spinning dope is missing critical factors. Here we report the discovery of novel molecular constituents within the spinning dope that are extruded into dragline silk. Protein studies of the liquid spinning dope from the major ampullate gland, coupled with the analysis of dragline silk fibers using mass spectrometry, demonstrate the presence of a new family of low-molecular-weight cysteine-rich proteins (CRPs) that colocalize with the MA fibroins. Expression of the CRP family members is linked to dragline silk production, specifically MaSp1 and MaSp2 mRNA synthesis. Biochemical data support that CRP molecules are secreted into the spinning dope and assembled into macromolecular complexes via disulfide bond linkages. Sequence analysis supports that CRP molecules share similarities to members that belong to the cystine slipknot superfamily, suggesting that these factors may have evolved to increase fiber toughness by serving as molecular hubs that dissipate large amounts of energy under stress. Collectively, our findings provide molecular details about the components of dragline silk, providing new insight that will advance materials development of synthetic spider silk for industrial applications.

  7. Purification and characterization of a cysteine-rich secretory protein from Philodryas patagoniensis snake venom.

    PubMed

    Peichoto, María E; Mackessy, Stephen P; Teibler, Pamela; Tavares, Flávio L; Burckhardt, Paula L; Breno, María C; Acosta, Ofelia; Santoro, Marcelo L

    2009-07-01

    Cysteine-rich secretory proteins (CRiSPs) are widespread in reptile venoms, but most have functions that remain unknown. In the present study we describe the purification and characterization of a CRiSP (patagonin) from the venom of the rear-fanged snake Philodryas patagoniensis, and demonstrate its biological activity. Patagonin is a single-chain protein, exhibiting a molecular mass of 24,858.6 Da, whose NH(2)-terminal and MS/MS-derived sequences are nearly identical to other snake venom CRiSPs. The purified protein hydrolyzed neither azocasein nor fibrinogen, and it could induce no edema, hemorrhage or inhibition of platelet adhesion and aggregation. In addition, patagonin did not inhibit contractions of rat aortic smooth muscle induced by high K(+). However, it caused muscular damage to murine gastrocnemius muscle, an action that has not been previously described for any snake venom CRiSPs. Thus, patagonin will be important for studies of the structure-function and evolutionary relationships of this family of proteins that are widely distributed among snake venoms.

  8. Expression of transcripts for cysteine-rich secretory proteins (CRISPs) in the murine lacrimal gland.

    PubMed

    Haendler, B; Toda, I; Sullivan, D A; Schleuning, W D

    1999-03-01

    Cysteine-rich secretory proteins (CRISPs) represent a family of evolutionarily conserved proteins which may play a role in the innate immune system and are transcriptionally regulated by androgens in several tissues. Transcripts for all three members of the CRISP family have now been identified in the murine lacrimal gland. RT-PCR using primers able to discriminate between the related CRISP forms allowed the amplification of fragments with the expected length. DNA sequencing revealed a complete identity with the hitherto characterized epididymal CRISP-1, testicular CRISP-2, and salivary gland CRISP-3. An analysis of several mouse strains indicated that all expressed the three CRISP forms, but in differing amounts. RT-PCR analysis of RNA isolated from acinar cells of lacrimal glands revealed that they expressed CRISP-1 and CRISP-2. Semiquantitative and quantitative analyses furthermore showed higher CRISP-1 and CRISP-3 mRNA levels in the lacrimal glands of male BALB/c and NOD mice when compared to females. Testosterone treatment of C3H/HeJ female mice was followed by an upregulation of the steady-state CRISP-1 but not CRISP-2 transcript levels. A comparable stimulation was observed for the mRNAs coding for parotid secretory protein (PSP), a factor previously shown to exhibit sexual dimorphism in the murine lacrimal gland. The expression of CRISP transcripts in the lacrimal gland is consistent with a function in the innate immune system.

  9. Redox modulation of the expression of bacterial genes encoding cysteine-rich proteins in plant protoplasts.

    PubMed Central

    Piñeiro, M; García-Olmedo, F; Diaz, I

    1994-01-01

    Activity of neomycin phosphotransferase II (NPTII; gene, neo; five cysteines) in tobacco protoplasts transfected with fusions of the octopine TR2' or cauliflower mosaic virus 35S promoter and the neo gene, with or without a signal peptide, increased up to 8-fold in response to externally added dithiothreitol at concentrations that did not affect protoplast viability (up to 2.5 mM). Activity of phosphinothricin acetyltransferase (PAT; gene, bar; one cysteine) expressed under control of the TR1' or 35S promoter was not similarly affected, thus excluding a redox modulation of transcription as the mechanism of NPTII activation by dithiothreitol. Western-blot analyses showed an increase in the amount of protein in response to dithiothreitol, whereas neither the steady-state level of NPTII mRNA nor the specific activity of the purified enzyme was affected. The same type of modulation was observed for transiently expressed beta-glucuronidase (nine cysteines) produced from a fusion with the 35S promoter, with or without a signal peptide. Limitation of cotranslational and/or early posttranslational steps by excessively oxidizing sulfhydryl/disulfide redox potentials is postulated to explain the low net accumulation of cysteine-rich proteins of bacterial origin (i.e., NPTII and beta-glucuronidase) when expressed in plant protoplasts, and the marked increase in such proteins in response to externally added dithiothreitol. Images PMID:8171004

  10. The cysteine-rich region of T1R3 determines responses to intensely sweet proteins.

    PubMed

    Jiang, Peihua; Ji, Qingzhou; Liu, Zhan; Snyder, Lenore A; Benard, Lumie M J; Margolskee, Robert F; Max, Marianna

    2004-10-22

    A wide variety of chemically diverse compounds taste sweet, including natural sugars such as glucose, fructose, sucrose, and sugar alcohols, small molecule artificial sweeteners such as saccharin and acesulfame K, and proteins such as monellin and thaumatin. Brazzein, like monellin and thaumatin, is a naturally occurring plant protein that humans, apes, and Old World monkeys perceive as tasting sweet but that is not perceived as sweet by other species including New World monkeys, mouse, and rat. It has been shown that heterologous expression of T1R2 plus T1R3 together yields a receptor responsive to many of the above-mentioned sweet tasting ligands. We have determined that the molecular basis for species-specific sensitivity to brazzein sweetness depends on a site within the cysteine-rich region of human T1R3. Other mutations in this region of T1R3 affected receptor activity toward monellin, and in some cases, overall efficacy to multiple sweet compounds, implicating this region as a previously unrecognized important determinant of sweet receptor function.

  11. Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein

    PubMed Central

    Londono-Renteria, Berlin; Troupin, Andrea; Conway, Michael J; Vesely, Diana; Ledizet, Michael; Roundy, Christopher M.; Cloherty, Erin; Jameson, Samuel; Vanlandingham, Dana; Higgs, Stephen; Fikrig, Erol; Colpitts, Tonya M.

    2015-01-01

    Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses. PMID:26491875

  12. Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein.

    PubMed

    Londono-Renteria, Berlin; Troupin, Andrea; Conway, Michael J; Vesely, Diana; Ledizet, Michael; Roundy, Christopher M; Cloherty, Erin; Jameson, Samuel; Vanlandingham, Dana; Higgs, Stephen; Fikrig, Erol; Colpitts, Tonya M

    2015-10-01

    Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

  13. Increased Susceptibility of  Thymocytes to Apoptosis in Mice Lacking AIM, a Novel Murine Macrophage-derived Soluble Factor Belonging to the Scavenger Receptor Cysteine-rich Domain Superfamily

    PubMed Central

    Miyazaki, Toru; Hirokami, Yumiko; Matsuhashi, Nobuyuki; Takatsuka, Hisakazu; Naito, Makoto

    1999-01-01

    Apoptosis of cells must be regulated both positively and negatively in response to a variety of stimuli in the body. Various environmental stresses are known to initiate apoptosis via differential signal transduction cascades. However, induction of signals that may inhibit apoptosis is poorly understood, although a number of intracellular molecules that mediate inhibition of apoptosis have been identified. Here we present a novel murine macrophage-specific 54-kD secreted protein which inhibits apoptosis (termed AIM, for apoptosis inhibitor expressed by macrophages). AIM belongs to the macrophage scavenger receptor cysteine-rich domain superfamily (SRCR-SF), members of which share a highly homologous conserved cysteine-rich domain. In AIM-deficient mice, the thymocyte numbers were diminished to half those in wild-type mice, and CD4/CD8 double-positive (DP) thymocytes were strikingly more susceptible to apoptosis induced by both dexamethasone and irradiation in vivo. Recombinant AIM protein significantly inhibited cell death of DP thymocytes in response to a variety of stimuli in vitro. These results indicate that in the thymus, AIM functions in trans to induce resistance to apoptosis within DP cells, and thus supports the viability of DP thymocytes before thymic selection. PMID:9892623

  14. Characterization of a family of cysteine rich proteins and development of a MaSp1 derived miniature fibroin

    NASA Astrophysics Data System (ADS)

    Chuang, Tyler Casey

    Spider silk displays a unique balance of high tensile strength and extensibility, making it one of the toughest materials on the planet. Dragline silk, also known as the lifeline of the spider, represents one of the best studied fiber types and many labs are attempting to produce synthetic dragline silk fibers for commercial applications. In these studies, we develop a minifibroin for expression studies in bacteria. Using recombinant DNA methodology and protein expression studies, we develop a natural minifibroin that contains the highly conserved N- and C-terminal domains, along with several internal block repeats of MaSp1. We also characterize a family of small cysteine-rich proteins (CRPs) and demonstrate that these factors are present within the spinning dope of the major ampullate gland using MS analysis. Biochemical studies and characterization of one of the family members, CRP1, demonstrate that this factor can self-polymerize into higher molecular weight complexes under oxidizing conditions, but can be converted into a monomeric species under reducing conditions. Self-polymerization of CRP1 is also shown to be independent of pH and salt concentration, two important chemical cues that help fibroin aggregation. Overall, our data demonstrate that the polymerization state of CRP1 is dependent upon redox state, suggesting that the redox environment during fiber extrusion may help regulate the oligomerization of CRP molecules during dragline silk production.

  15. Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins*

    PubMed Central

    Mehus, Aaron A.; Muhonen, Wallace W.; Garrett, Scott H.; Somji, Seema; Sens, Donald A.; Shabb, John B.

    2014-01-01

    Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with 14N- or 15N-iodoacetamide. Absolute quantitation was achieved using 15N-iodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue- and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptor-negative cell lines than in estrogen-receptor-positive cells. These

  16. Blocking effect and crystal structure of natrin toxin, a cysteine-rich secretory protein from Naja atra venom that targets the BKCa channel.

    PubMed

    Wang, Jing; Shen, Bing; Guo, Min; Lou, Xiaohua; Duan, Yuanyuan; Cheng, Xin Ping; Teng, Maikun; Niu, Liwen; Liu, Qun; Huang, Qingqiu; Hao, Quan

    2005-08-02

    Cysteine-rich secretory proteins (CRISPs) are widespread in snake venoms. Some members of these CRISPs recently have been found to block L-type Ca(2+) channels or cyclic nucleotide-gated ion (CNG) channels. Here, natrin purified from Naja atra venom, a member of the CRISP family, can induce a further contractile response in the endothelium-denuded thoracic aorta of mouse which has been contracted by a high-K(+) solution. Further experiments show it can block the high-conductance calcium-activated potassium (BK(Ca)) channel in a concentration-dependent manner with an IC(50) of 34.4 nM and a Hill coefficient of 1.02, which suggests that only a single natrin molecule is required to bind an ion channel to block BK(Ca) current. The crystal structure of natrin displaying two domains in tandem shows its cysteine-rich domain (CRD) has relatively independent flexibility, especially for the C-terminal long loop (loop I) of CRD to participate in the interface of two domains. On the basis of previous studies of CNG channel and L-Ca(2+) channel blockers, and the sequence and structural comparison of natrin and stecrisp, the deviation of the vital loop I of CRD is suggested to contribute to different effects of some CRISPs in protein-protein interaction.

  17. Putative DHHC-Cysteine-Rich Domain S-Acyltransferase in Plants

    PubMed Central

    Sun, Meihong; Liu, Shiyang; Qi, Baoxiu; Li, Xinzheng

    2013-01-01

    Protein S-acyltransferases (PATs) containing Asp-His-His-Cys within a Cys-rich domain (DHHC-CRD) are polytopic transmembrane proteins that are found in eukaryotic cells and mediate the S-acylation of target proteins. S-acylation is an important secondary and reversible modification that regulates the membrane association, trafficking and function of target proteins. However, little is known about the characteristics of PATs in plants. Here, we identified 804 PATs from 31 species with complete genomes. The analysis of the phylogenetic relationships suggested that all of the PATs fell into 8 groups. In addition, we analysed the phylogeny, genomic organization, chromosome localisation and expression pattern of PATs in Arabidopsis, Oryza sative, Zea mays and Glycine max. The microarray data revealed that PATs genes were expressed in different tissues and during different life stages. The preferential expression of the ZmPATs in specific tissues and the response of Zea mays to treatments with phytohormones and abiotic stress demonstrated that the PATs play roles in plant growth and development as well as in stress responses. Our data provide a useful reference for the identification and functional analysis of the members of this protein family. PMID:24155879

  18. HmCRIP, a cysteine-rich intestinal protein, is expressed by an identified regenerating nerve cell.

    PubMed

    Emes, R D; Wang, W-Z; Lanary, K; Blackshaw, S E

    2003-01-02

    A Hirudo medicinalis cDNA isolated from regenerating CNS tissue at 24 h post-axotomy was identified as a leech homologue of the mammalian cysteine-rich intestinal proteins (CRIPs) and named HmCRIP. HmCRIP is up-regulated within 6 h of axotomy, peaking at 24 h. This is the first demonstration of a CRIP homologue in regenerating CNS and in a serotonergic neurone. In rodents CRIP is an important factor in the regulation of the inflammatory immune response through control of Th1/Th2 differentiation. The role of HmCRIP in the regeneration competent environment of the annelid central nervous system is discussed.

  19. The Disintegrin-like and Cysteine-rich domains of ADAM-9 Mediate Interactions between Melanoma Cells and Fibroblasts*

    PubMed Central

    Zigrino, Paola; Nischt, Roswitha; Mauch, Cornelia

    2011-01-01

    A characteristic of malignant cells is their capacity to invade their surrounding and to metastasize to distant organs. During these processes, proteolytic activities of tumor and stromal cells modify the extracellular matrix to produce a microenvironment suitable for their growth and migration. In recent years the family of ADAM proteases has been ascribed important roles in these processes. ADAM-9 is expressed in human melanoma at the tumor-stroma border where direct or indirect interactions between tumor cells and fibroblasts occur. To analyze the role of ADAM-9 for the interaction between melanoma cells and stromal fibroblasts, we produced the recombinant disintegrin-like and cysteine-rich domain of ADAM-9 (DC-9). Melanoma cells and human fibroblasts adhered to immobilized DC-9 in a Mn2+-dependent fashion suggesting an integrin-mediated process. Inhibition studies showed that adhesion of fibroblasts was mediated by several β1 integrin receptors independent of the RGD and ECD recognition motif. Furthermore, interaction of fibroblasts and high invasive melanoma cells with soluble recombinant DC-9 resulted in enhanced expression of MMP-1 and MMP-2. Silencing of ADAM-9 in melanoma cells significantly reduced cell adhesion to fibroblasts. Ablation of ADAM-9 in fibroblasts almost completely abolished these cellular interactions and melanoma cell invasion in vitro. In summary, these results suggest that ADAM-9 expression plays an important role in mediating cell-cell contacts between fibroblasts and melanoma cells and that these interactions contribute to proteolytic activities required during invasion of melanoma cells. PMID:21135106

  20. Genome-wide analysis of small secreted cysteine-rich proteins identifies candidate effector proteins potentially involved in Fusarium graminearum-wheat interactions

    USDA-ARS?s Scientific Manuscript database

    Pathogen-derived, small secreted cysteine-rich proteins (SSCPs) are known to be a common source of fungal effectors that trigger resistance or susceptibility in specific host plants. This group of proteins has not been well studied in Fusarium graminearum, the primary cause of Fusarium head blight ...

  1. Comparative expression analysis of cysteine-rich intestinal protein family members crip1, 2 and 3 during Xenopus laevis embryogenesis.

    PubMed

    Hempel, Annemarie; Kühl, Susanne J

    2014-01-01

    Members of the cysteine-rich intestinal protein (Crip) family belong to the group 2 LIM proteins. Crip proteins are widely expressed in adult mammals but their expression profile and function during embryonic development are still mostly unknown. In this study, we have described for the first time the spatio-temporal expression pattern of the three family members crip1, crip2 and crip3 during Xenopus laevis embryogenesis by RT-PCR and whole mount in situ hybridization approaches. We observed that all three genes are expressed in the pronephros, branchial arches and the eye. Furthermore, crip1 transcripts could be visualized in the developing cranial ganglia and neural tube. In contrast, crip2 could be detected in the cardiovascular system, the brain and the neural tube while crip3 was expressed in the cranial ganglions and the heart. Based on these findings, we suggest that each crip family member may play an important role during embryonic development.

  2. Developmental expression, intracellular localization, and selenium content of the cysteine-rich protein associated with the mitochondrial capsules of mouse sperm.

    PubMed

    Cataldo, L; Baig, K; Oko, R; Mastrangelo, M A; Kleene, K C

    1996-11-01

    The outer membranes of mitochondria of mammalian sperm are encased in a keratinous structure known as the mitochondrial capsule. The experiments in the present study were designed to resolve a controversy surrounding the intracellular localization, developmental expression, and selenium-content of a cysteine-rich 17-20 kD protein that has been reported to constitute the major structural protein in the mitochondrial capsule of mammals. An antibody to a synthetic oligopeptide based on the predicted sequence of mouse cysteinerich protein recognizes a 24 kD protein in epididymal sperm tails of mice. The 24 kD protein does not appear to be a selenoprotein because: (1) it is not labeled with 75Se-selenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and translation of T7 RNA polymerase transcripts in vitro indicate that the translation start site is located downstream of potential UGA selenocysteine codons in the mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich protein in rat lacks inphase UGA selenocysteine codons. Light and electron microscopy immunocytochemistry detects the cysteine-rich protein first during step 11 of spermiogenesis in the mouse demonstrating that the cysteine-rich protein mRNA is under temporal translational control. Electron microscope immunocytochemistry reveals that the cysteine-rich protein is evenly distributed in the cytoplasm in spermatids in steps 11 through early step 16 in mouse, and that it is associated with the outer mitochondrial membranes of spermatids in late step 16 and epididymal spermatozoa.

  3. Thrombin receptor and RhoA mediate cell proliferation through integrins and cysteine-rich protein 61

    PubMed Central

    Walsh, Colin T.; Radeff-Huang, Julie; Matteo, Rosalia; Hsiao, Albert; Subramaniam, Shankar; Stupack, Dwayne; Brown, Joan Heller

    2008-01-01

    A subset of G-protein coupled receptors (GPCRs), including the thrombin receptor (PAR1), elicits mitogenic responses. Thrombin also activates Ras homolog gene family member A (RhoA) and activating protein (AP-1) -mediated gene expression in 1321N1 astrocytoma cells, whereas the nonmitogenic agonist carbachol does not. Transcriptomic analysis was used to explore differential gene induction by these agonists and revealed that the matricellular protein cysteine-rich 61 (Cyr61/CCN1) is selectively induced by thrombin. The ability of GPCR agonists to induce Cyr61 parallels their ability to activate RhoA; agonist-stimulated Cyr61 expression is inhibited by C3 toxin. When Cyr61 is down-regulated using short interfering RNA (siRNA) or short-hairpin RNA (shRNA), thrombin-induced DNA synthesis is significantly attenuated. When Cyr61 expression is induced, it appears in the extracellular compartment and on the cell surface. Extracellular Cyr61 interacts with α5, α6, and β1 integrins on these cells, and monoclonal antibodies directed against α5 and β1 integrins inhibit thrombin-induced DNA synthesis. Functional blockade of Cyr61 with soluble heparin or anti-Cyr61 antibodies also inhibits thrombin-induced DNA synthesis. Thus Cyr61 is a highly inducible, secreted extracellular factor through which GPCR and RhoA signaling pathways engage integrins that contribute to GPCR-mediated proliferation.—Walsh, C. T., Radeff-Huang, J., Matteo, R., Hsiao, A., Subramaniam, S., Stupack, D., and Brown, J. H. Thrombin receptor and RhoA mediate cell proliferation through integrins and cysteine-rich protein 61. PMID:18687805

  4. A Secreted Protein with Plant-Specific Cysteine-Rich Motif Functions as a Mannose-Binding Lectin That Exhibits Antifungal Activity1[W

    PubMed Central

    Miyakawa, Takuya; Hatano, Ken-ichi; Miyauchi, Yumiko; Suwa, You-ichi; Sawano, Yoriko; Tanokura, Masaru

    2014-01-01

    Plants have a variety of mechanisms for defending against plant pathogens and tolerating environmental stresses such as drought and high salinity. Ginkbilobin2 (Gnk2) is a seed storage protein in gymnosperm that possesses antifungal activity and a plant-specific cysteine-rich motif (domain of unknown function26 [DUF26]). The Gnk2-homologous sequence is also observed in an extracellular region of cysteine-rich repeat receptor-like kinases that function in response to biotic and abiotic stresses. Here, we report the lectin-like molecular function of Gnk2 and the structural basis of its monosaccharide recognition. Nuclear magnetic resonance experiments showed that mannan was the only yeast (Saccharomyces cerevisiae) cell wall polysaccharide that interacted with Gnk2. Gnk2 also interacted with mannose, a building block of mannan, with a specificity that was similar to those of mannose-binding legume lectins, by strictly recognizing the configuration of the hydroxy group at the C4 position of the monosaccharide. The crystal structure of Gnk2 in complex with mannose revealed that three residues (asparagine-11, arginine-93, and glutamate-104) recognized mannose by hydrogen bonds, which defined the carbohydrate-binding specificity. These interactions were directly related to the ability of Gnk2 to inhibit the growth of fungi, including the plant pathogenic Fusarium spp., which were disrupted by mutation of arginine-93 or the presence of yeast mannan in the assay system. In addition, Gnk2 did not inhibit the growth of a yeast mutant strain lacking the α1,2-linked mannose moiety. These results provide insights into the molecular basis of the DUF26 protein family. PMID:25139159

  5. The RafC1 Cysteine-Rich Domain Contains Multiple Distinct Regulatory Epitopes Which Control Ras-Dependent Raf Activation

    PubMed Central

    Daub, Martina; Jöckel, Johannes; Quack, Thomas; Weber, Christoph K.; Schmitz, Frank; Rapp, Ulf R.; Wittinghofer, Alfred; Block, Christoph

    1998-01-01

    Activation of c-Raf-1 (referred to as Raf) by Ras is a pivotal step in mitogenic signaling. Raf activation is initiated by binding of Ras to the regulatory N terminus of Raf. While Ras binding to residues 51 to 131 is well understood, the role of the RafC1 cysteine-rich domain comprising residues 139 to 184 has remained elusive. To resolve the function of the RafC1 domain, we have performed an exhaustive surface scanning mutagenesis. In our study, we defined a high-resolution map of multiple distinct functional epitopes within RafC1 that are required for both negative control of the kinase and the positive function of the protein. Activating mutations in three different epitopes enhanced Ras-dependent Raf activation, while only some of these mutations markedly increased Raf basal activity. One contiguous inhibitory epitope consisting of S177, T182, and M183 clearly contributed to Ras-Raf binding energy and represents the putative Ras binding site of the RafC1 domain. The effects of all RafC1 mutations on Ras binding and Raf activation were independent of Ras lipid modification. The inhibitory mutation L160A is localized to a position analogous to the phorbol ester binding site in the protein kinase C C1 domain, suggesting a function in cofactor binding. Complete inhibition of Ras-dependent Raf activation was achieved by combining mutations K144A and L160A, which clearly demonstrates an absolute requirement for correct RafC1 function in Ras-dependent Raf activation. PMID:9774683

  6. Characterization of a cysteine-rich protein specifically expressed in the silk gland of caddisfly Stenopsyche marmorata (Trichoptera; Stenopsychidae).

    PubMed

    Wang, Yujun; Wang, Hong; Zhao, Tianfu; Nakagaki, Masao

    2010-01-01

    A novel protein, Smsp-72k, was found to be selectively expressed in the silk gland of aquatic larvae of the Stenopsychid caddisfly (Stenopsyche marmorata). The protein was characterized by an abundance of cysteine (13.97%) and charged residues (47.21%). Amino acids with hydroxyl side-chains accounted for an additional 10% of the Smsp-72k protein, with serine at 4.4% and threonine at 5.6%. A cysteine-rich repetitive sequence is common to many potential and known underwater adhesive/cement proteins and cell-cell adhesion molecules. We hypothesized that Smsp-72k is an adhesive/cement protein that increases the adhesiveness of the silk fiber of S. marmorata. The hydroxyl groups of Smsp-72k might form a link with the heavy chain fibroin of S. marmorata, removing the weak boundary-water layer and allowing the spreading of the silk protein onto the surface of the substratum during the process of adhesion.

  7. A Small Cysteine-Rich Protein from the Asian Soybean Rust Fungus, Phakopsora pachyrhizi, Suppresses Plant Immunity.

    PubMed

    Qi, Mingsheng; Link, Tobias I; Müller, Manuel; Hirschburger, Daniela; Pudake, Ramesh N; Pedley, Kerry F; Braun, Edward; Voegele, Ralf T; Baum, Thomas J; Whitham, Steven A

    2016-09-01

    The Asian soybean rust fungus, Phakopsora pachyrhizi, is an obligate biotrophic pathogen causing severe soybean disease epidemics. Molecular mechanisms by which P. pachyrhizi and other rust fungi interact with their host plants are poorly understood. The genomes of all rust fungi encode many small, secreted cysteine-rich proteins (SSCRP). While these proteins are thought to function within the host, their roles are completely unknown. Here, we present the characterization of P. pachyrhizi effector candidate 23 (PpEC23), a SSCRP that we show to suppress plant immunity. Furthermore, we show that PpEC23 interacts with soybean transcription factor GmSPL12l and that soybean plants in which GmSPL12l is silenced have constitutively active immunity, thereby identifying GmSPL12l as a negative regulator of soybean defenses. Collectively, our data present evidence for a virulence function of a rust SSCRP and suggest that PpEC23 is able to suppress soybean immune responses and physically interact with soybean transcription factor GmSPL12l, a negative immune regulator.

  8. The involvement of cysteine-rich intestinal protein in early development and innate immunity of Asiatic hard clam, Meretrix meretrix.

    PubMed

    Chen, Hongjian; Yang, Xue; Tang, Ting; Li, Juan; Liu, Baozhong; Liu, Fengsong; Xie, Song

    2014-10-01

    Cysteine-rich intestinal protein (CRIP), a Zn(2+)-binding protein, contains a single copy of the highly conserved double-zinc-finger structure known as the LIM (lin-11-isl-1-mec-3) motif. In this paper, a cDNA encoding MmCRIP was isolated from the Asiatic hard clam Meretrix meretrix. The full-length cDNA of MmCRIP consists of a 237-bp open reading frame that encodes a polypeptide of 78 amino acids with a predicted molecular weight (MW) of 8635.8 Da and theoretical isoelectric point (pI) of 9.01. Bioinformatics analysis showed that it belonged to a new member of the CRIP subfamily. Relationship analysis revealed that MmCRIP has high-levels of sequence similarity to many CRIPs reported in other animals, particularly in invertebrates. Real-time PCR analysis showed that the highest level of MmCRIP expression was in hemocyte tissue and at pediveligers stage. To investigate immune function, mature clams were challenged with Aeromonas hydrophila. During A. hydrophila infection, up-regulation of MmCRIP transcript in clam's hemocyte, gill and hepatopancreas was detected. DsRNAi (double-strand RNA interference) approach was employed to study the function of MmCRIP and the data showed that inactivation of the MmCRIP gene blocked larvae development and caused mass mortalities. The probable roles of MmCRIP in clam early development and innate immunity are presented for the first time.

  9. Cysteine-rich secretory proteins in snake venoms form high affinity complexes with human and porcine beta-microseminoproteins.

    PubMed

    Hansson, Karin; Kjellberg, Margareta; Fernlund, Per

    2009-08-01

    BETA-microseminoprotein (MSP), a 10 kDa protein in human seminal plasma, binds human cysteine-rich secretory protein-3 (CRISP-3) with high affinity. CRISP-3 is a member of the family of CRISPs, which are widespread among animals. In this work we show that human as well as porcine MSP binds catrin, latisemin, pseudecin, and triflin, which are CRISPs present in the venoms of the snakes Crotalus atrox, Laticauda semifasciata, Pseudechis porphyriacus, and Trimeresurus flavoviridis, respectively. The CRISPs were purified from the venoms by affinity chromatography on a human MSP column and their identities were settled by gel electrophoresis and mass spectrometry. Their interactions with human and porcine MSPs were studied with size exclusion chromatography and surface plasmon resonance measurements. The binding affinities at 25 degrees C were between 10(-10)M and 10(-7)M for most of the interactions, with higher affinities for the interactions with porcine MSP compared to human MSP and with Elapidae CRISPs compared to Viperidae CRISPs. The high affinities of the bindings in spite of the differences in amino acid sequence between the MSPs as well as between the CRISPs indicate that the binding is tolerant to amino acid sequence variation and raise the question how universal this cross-species reaction between MSPs and CRISPs is.

  10. VCRPs, small cysteine-rich proteins, might be involved in extracellular signaling in the green alga Volvox.

    PubMed

    Hallmann, Armin

    2008-02-01

    The sex-inducer of the spherical green alga Volvox carteri is one of the most potent biological effector molecules known: it is released into the medium by sexual males and triggers the switch to the sexual cleavage program in the reproductive cells of vegetatively grown males and females even at concentrations as low as 10(-16) M. In an adult Volvox alga, all cells are embedded in an extensive extracellular matrix (ECM), which constitutes >99% of the volume of the spheroid. There exist no cytoplasmic connections between the cells in an adult alga, so any signal transduction between different cells or from the organism's environment to a reproductive cell must involve the ECM. Recently, a small cysteine-rich extracellular protein, VCRP, was identified in Volvox and shown to be quickly synthesized by somatic cells in response to the sex-inducer. Due to its characteristics, VCRP was speculated to be an extracellular second messenger from somatic cells to reproductive cells. Here a related protein, VCRP2, is presented, exhibiting a 56% amino acid sequence identity with VCRP. Two possible scenarios for signal transduction from the sex-inducer to the reproductive cell are discussed.

  11. FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family

    PubMed Central

    Holcomb, Ilona N.; Kabakoff, Rhona C.; Chan, Betty; Baker, Thad W.; Gurney, Austin; Henzel, William; Nelson, Chris; Lowman, Henry B.; Wright, Barbara D.; Skelton, Nicholas J.; Frantz, Gretchen D.; Tumas, Daniel B.; Peale,Jr, Franklin V.; Shelton, David L.; Hébert, Caroline C.

    2000-01-01

    Bronchoalveolar lavage fluid from mice with experimentally induced allergic pulmonary inflammation contains a novel 9.4 kDa cysteine-rich secreted protein, FIZZ1 (found in inflammatory zone). Murine (m) FIZZ1 is the founding member of a new gene family including two other murine genes expressed, respectively, in intestinal crypt epithelium and white adipose tissue, and two related human genes. In control mice, FIZZ1 mRNA and protein expression occur at low levels in a subset of bronchial epithelial cells and in non-neuronal cells adjacent to neurovascular bundles in the peribronchial stroma, and in the wall of the large and small bowel. During allergic pulmonary inflammation, mFIZZ1 expression markedly increases in hypertrophic, hyperplastic bronchial epithelium and appears in type II alveolar pneumocytes. In vitro, recombinant mFIZZ1 inhibits the nerve growth factor (NGF)-mediated survival of rat embryonic day 14 dorsal root ganglion (DRG) neurons and NGF-induced CGRP gene expression in adult rat DRG neurons. In vivo, FIZZ1 may modulate the function of neurons innervating the bronchial tree, thereby altering the local tissue response to allergic pulmonary inflammation. PMID:10921885

  12. A Small Cysteine-Rich Protein from the Asian Soybean Rust Fungus, Phakopsora pachyrhizi, Suppresses Plant Immunity

    PubMed Central

    Qi, Mingsheng; Link, Tobias I.; Müller, Manuel; Hirschburger, Daniela; Pudake, Ramesh N.; Pedley, Kerry F.; Braun, Edward; Voegele, Ralf T.; Baum, Thomas J.

    2016-01-01

    The Asian soybean rust fungus, Phakopsora pachyrhizi, is an obligate biotrophic pathogen causing severe soybean disease epidemics. Molecular mechanisms by which P. pachyrhizi and other rust fungi interact with their host plants are poorly understood. The genomes of all rust fungi encode many small, secreted cysteine-rich proteins (SSCRP). While these proteins are thought to function within the host, their roles are completely unknown. Here, we present the characterization of P. pachyrhizi effector candidate 23 (PpEC23), a SSCRP that we show to suppress plant immunity. Furthermore, we show that PpEC23 interacts with soybean transcription factor GmSPL12l and that soybean plants in which GmSPL12l is silenced have constitutively active immunity, thereby identifying GmSPL12l as a negative regulator of soybean defenses. Collectively, our data present evidence for a virulence function of a rust SSCRP and suggest that PpEC23 is able to suppress soybean immune responses and physically interact with soybean transcription factor GmSPL12l, a negative immune regulator. PMID:27676173

  13. Role of cysteine-rich 61 protein (CCN1) in macrophage-mediated oncolytic herpes simplex virus clearance.

    PubMed

    Thorne, Amy Haseley; Meisen, Walter H; Russell, Luke; Yoo, Ji Young; Bolyard, Chelsea M; Lathia, Justin D; Rich, Jeremy; Puduvalli, Vinay K; Mao, Hsiaoyin; Yu, Jianhua; Caligiuri, Michael A; Tridandapani, Susheela; Kaur, Balveen

    2014-09-01

    Glioblastoma is a devastating disease, and there is an urgent need to develop novel therapies, such as oncolytic HSV1 (OV) to effectively target tumor cells. OV therapy depends on tumor-specific replication leading to destruction of neoplastic tissues. Host responses that curtail virus replication limit its efficacy in vivo. We have previously shown that cysteine-rich 61 protein (CCN1) activates a type 1 IFN antiviral defense response in glioblastoma cells. Incorporating TCGA data, we found CCN1 expression to be a negative prognostic factor for glioblastoma patients. Based on this, we used neutralizing antibodies against CCN1 to investigate its effect on OV therapy. Use of an anti-CCN1 antibody in mice bearing glioblastomas treated with OV led to enhanced virus expression along with reduced immune cell infiltration. OV-induced CCN1 increases macrophage migration toward infected glioblastoma cells by directly binding macrophages and also by enhancing the proinflammatory activation of macrophages inducing MCP-1 expression in glioblastoma cells. Activation of macrophages by CCN1 also increases viral clearance. Neutralization of integrin αMβ2 reversed CCN1-induced macrophage activation and migration, and reduced MCP-1 expression by glioblastoma cells. Our findings reveal that CCN1 plays a novel role in pathogen clearance; increasing macrophage infiltration and activation resulting in increased virus clearance in tumors.

  14. Genome-Wide Analysis of Small Secreted Cysteine-Rich Proteins Identifies Candidate Effector Proteins Potentially Involved in Fusarium graminearum-Wheat Interactions.

    PubMed

    Lu, Shunwen; Edwards, Michael C

    2016-02-01

    Pathogen-derived, small secreted cysteine-rich proteins (SSCPs) are known to be a common source of fungal effectors that trigger resistance or susceptibility in specific host plants. This group of proteins has not been well studied in Fusarium graminearum, the primary cause of Fusarium head blight (FHB), a devastating disease of wheat. We report here a comprehensive analysis of SSCPs encoded in the genome of this fungus and selection of candidate effector proteins through proteomics and sequence/transcriptional analyses. A total of 190 SSCPs were identified in the genome of F. graminearum (isolate PH-1) based on the presence of N-terminal signal peptide sequences, size (≤200 amino acids), and cysteine content (≥2%) of the mature proteins. Twenty-five (approximately 13%) SSCPs were confirmed to be true extracellular proteins by nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis of a minimal medium-based in vitro secretome. Sequence analysis suggested that 17 SSCPs harbor conserved functional domains, including two homologous to Ecp2, a known effector produced by the tomato pathogen Cladosporium fulvum. Transcriptional analysis revealed that at least 34 SSCPs (including 23 detected in the in vitro secretome) are expressed in infected wheat heads; about half are up-regulated with expression patterns correlating with the development of FHB. This work provides a solid candidate list for SSCP-derived effectors that may play roles in mediating F. graminearum-wheat interactions. The in vitro secretome-based method presented here also may be applicable for identifying candidate effectors in other ascomycete pathogens of crop plants.

  15. New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

    PubMed

    Jung, Woongsic; Campbell, Robert L; Gwak, Yunho; Kim, Jong Im; Davies, Peter L; Jin, EonSeon

    2016-01-01

    Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP's biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.

  16. Transcriptomic screening for cyclotides and other cysteine-rich proteins in the metallophyte Viola baoshanensis.

    PubMed

    Zhang, Jun; Li, Jintian; Huang, Zebo; Yang, Bing; Zhang, Xiaojie; Li, Dehua; Craik, David J; Baker, Alan J M; Shu, Wensheng; Liao, Bin

    2015-04-15

    Cysteine (Cys)-rich proteins (CRPs) are frequently associated with plant defense and stress resistance. Viola baoshanensis is a cadmium (Cd) hyper-accumulating plant whose CRPs-based defense systems are so far poorly understood. Next generation sequencing (NGS) techniques and a specialist searching tool, CrpExcel, were employed for identifying CRPs in V. baoshanensis. The transcriptome sequences of V. baoshanensis were assembled primarily from 454FLX/Hiseq2000 reads of plant cDNA sequencing libraries. CrpExcel was then used to search the ORFs and 9687 CRPs were identified, and included zinc finger (ZF) proteins, lipid transfer proteins, thaumatins and cyclotide precursors. Real-time PCR results showed that all CRP genes tested are constitutively expressed, but the genes of defensive peptides showed greater up-regulated expression than those of ZF-proteins in Cd- and/or wounding (Wd) treatments of V. baoshanensis seedlings. The NGS-derived sequences of cyclotide precursor genes were verified by RT-PCR and ABI3730 sequencing studies, and 32 novel cyclotides were identified in V. baoshanensis. In general, the metal-binding sites of ZF-containing CRPs also represented the potential vulnerable targets of toxic metals. This study provides broad insights into CRPs-based defense systems and stress-vulnerable targets in V. baoshanensis. It now brings the number of cyclotide sequences in V. baoshanensis to 53 and based on projections from this work, the number of cyclotides in the Violaceae is now conservatively estimated to be >30000. Copyright © 2015 Elsevier GmbH. All rights reserved.

  17. New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

    PubMed Central

    Jung, Woongsic; Gwak, Yunho; Kim, Jong Im; Davies, Peter L.; Jin, EonSeon

    2016-01-01

    Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP’s biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP. PMID:27097164

  18. Crovirin, a Snake Venom Cysteine-Rich Secretory Protein (CRISP) with Promising Activity against Trypanosomes and Leishmania

    PubMed Central

    Adade, Camila M.; Carvalho, Ana Lúcia O.; Tomaz, Marcelo A.; Costa, Tatiana F. R.; Godinho, Joseane L.; Melo, Paulo A.; Lima, Ana Paula C. A.; Rodrigues, Juliany C. F.; Zingali, Russolina B.; Souto-Padrón, Thaïs

    2014-01-01

    Background The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania. Methodology/Principal Findings Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10–2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells. Conclusions This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases. PMID:25330220

  19. Unfolding Thermodynamics of Cysteine-Rich Proteins and Molecular Thermal-Adaptation of Marine Ciliates

    PubMed Central

    Cazzolli, Giorgia; Škrbić, Tatjana; Guella, Graziano; Faccioli, Pietro

    2013-01-01

    Euplotes nobilii and Euplotes raikovi are phylogenetically closely allied species of marine ciliates, living in polar and temperate waters, respectively. Their evolutional relation and the sharply different temperatures of their natural environments make them ideal organisms to investigate thermal-adaptation. We perform a comparative study of the thermal unfolding of disulfide-rich protein pheromones produced by these ciliates. Recent circular dichroism (CD) measurements have shown that the two psychrophilic (E. nobilii) and mesophilic (E. raikovi) protein families are characterized by very different melting temperatures, despite their close structural homology. The enhanced thermal stability of the E. raikovi pheromones is realized notwithstanding the fact that these proteins form, as a rule, a smaller number of disulfide bonds. We perform Monte Carlo (MC) simulations in a structure-based coarse-grained (CG) model to show that the higher stability of the E. raikovi pheromones is due to the lower locality of the disulfide bonds, which yields a lower entropy increase in the unfolding process. Our study suggests that the higher stability of the mesophilic E. raikovi phermones is not mainly due to the presence of a strongly hydrophobic core, as it was proposed in the literature. In addition, we argue that the molecular adaptation of these ciliates may have occurred from cold to warm, and not from warm to cold. To provide a testable prediction, we identify a point-mutation of an E. nobilii pheromone that should lead to an unfolding temperature typical of that of E. raikovi pheromones. PMID:24970199

  20. Unfolding thermodynamics of cysteine-rich proteins and molecular thermal-adaptation of marine ciliates.

    PubMed

    Cazzolli, Giorgia; Skrbić, Tatjana; Guella, Graziano; Faccioli, Pietro

    2013-11-18

    Euplotes nobilii and Euplotes raikovi are phylogenetically closely allied species of marine ciliates, living in polar and temperate waters, respectively. Their evolutional relation and the sharply different temperatures of their natural environments make them ideal organisms to investigate thermal-adaptation. We perform a comparative study of the thermal unfolding of disulfide-rich protein pheromones produced by these ciliates. Recent circular dichroism (CD) measurements have shown that the two psychrophilic (E. nobilii) and mesophilic (E. raikovi) protein families are characterized by very different melting temperatures, despite their close structural homology. The enhanced thermal stability of the E. raikovi pheromones is realized notwithstanding the fact that these proteins form, as a rule, a smaller number of disulfide bonds. We perform Monte Carlo (MC) simulations in a structure-based coarse-grained (CG) model to show that the higher stability of the E. raikovi pheromones is due to the lower locality of the disulfide bonds, which yields a lower entropy increase in the unfolding process. Our study suggests that the higher stability of the mesophilic E. raikovi phermones is not mainly due to the presence of a strongly hydrophobic core, as it was proposed in the literature. In addition, we argue that the molecular adaptation of these ciliates may have occurred from cold to warm, and not from warm to cold. To provide a testable prediction, we identify a point-mutation of an E. nobilii pheromone that should lead to an unfolding temperature typical of that of E. raikovi pheromones.

  1. Autosomal dominant hypocalcemia: a novel activating mutation (E604K) in the cysteine-rich domain of the calcium-sensing receptor.

    PubMed

    Tan, Y M; Cardinal, J; Franks, A H; Mun, H-C; Lewis, N; Harris, L B; Prins, J B; Conigrave, A D

    2003-02-01

    We report a novel activating mutation (E604K) of the calcium-sensing receptor in a family with autosomal dominant hypocalcemia. Whereas all affected individuals exhibited marked hypocalcemia, some cases with untreated hypocalcemia exhibited seizures in infancy, whereas others were largely asymptomatic from birth into adulthood. The missense mutation E604K (G2182A; GenBank accession no. U20759), which affects an amino acid residue in the C terminus of the cysteine-rich domain of the extracellular head, cosegregated with hypocalcemia in all seven individuals for whom DNA was available. Two unaffected, normocalcemic members of the family did not exhibit the mutation. The molecular impact of the mutation on two key components of the signaling response was assessed in HEK-293 cells transiently transfected with cDNA corresponding to either the wild-type calcium-sensing receptor or the E604K mutation derived by site-directed mutagenesis. There was a significant leftward shift in the concentration response curves for the effects of extracellular Ca(2+) on both intracellular Ca(2+) mobilization (determined by aequorin luminescence) and MAPK activity (determined by luciferase expression). The C terminus of the cysteine-rich domain of the extracellular head may normally act to suppress receptor activity in the presence of low extracellular Ca(2+) concentrations.

  2. Cysteine-rich protein 61 (CCN1) mediates replicative senescence-associated aberrant collagen homeostasis in human skin fibroblasts.

    PubMed

    Quan, Taihao; Qin, Zhaoping; Voorhees, John J; Fisher, Gary J

    2012-09-01

    Dermal fibroblasts produce a collagen-rich extracellular matrix, which confers mechanical strength and resiliency to human skin. During aging, collagen production is reduced and collagen fragmentation is increased, which is initiated by matrix metalloproteinase-1 (MMP-1). This aberrant collagen homeostasis results in net collagen deficiency, which impairs the structural integrity and function of skin. Cysteine-rich protein 61 (CCN1), a member of the CCN family, negatively regulates collagen homeostasis, in primary human skin dermal fibroblasts. As replicative senescence is a form of cellular aging, we have utilized replicative senescent dermal fibroblasts to further investigate the connection between elevated CCN1 and aberrant collagen homeostasis. CCN1 mRNA and protein levels were significantly elevated in replicative senescent dermal fibroblasts. Replicative senescent dermal fibroblasts also expressed significantly reduced levels of type I procollagen and increased levels of MMP-1. Knockdown of elevated CCN1 in senescent dermal fibroblasts partially normalized both type I procollagen and MMP-1 expression. These data further support a key role of CCN1 in regulation of collagen homeostasis. Elevated expression of CCN1 substantially increased collagen lattice contraction and fragmentation caused by replicative senescent dermal fibroblasts. Atomic force microscopy (AFM) further revealed collagen fibril fragmentation and disorganization were largely prevented by knockdown of CCN1 in replicative senescent dermal fibroblasts, suggesting CCN1 mediates MMP-1-induced alterations of collagen fibrils by replicative senescent dermal fibroblasts. Given the ability of CCN1 to regulate both production and degradation of type I collagen, it is likely that elevated-CCN1 functions as an important mediator of collagen loss, which is observed in aged human skin.

  3. Characterization of toxins from the broad-banded water snake Helicops angulatus (Linnaeus, 1758): isolation of a cysteine-rich secretory protein, Helicopsin.

    PubMed

    Estrella, Amalid; Sánchez, Elda E; Galán, Jacob A; Tao, W Andy; Guerrero, Belsy; Navarrete, Luis F; Rodríguez-Acosta, Alexis

    2011-04-01

    Helicops angulatus (broad-banded water snake) according to recent proposals is presently cited in the family Dipsadidae, subfamily Xenodontinae, forming the tribe Hydropsini along with the genera Hydrops and Pseudoeryx. The current work characterizes the proteolytic and neurotoxic activities of H. angulatus crude toxins from salivary excretion (SE) and describes the isolation and identification of a cysteine-rich secretory protein (CRISP) called helicopsin. The SE lethal dose (LD₅₀) was 5.3 mg/kg; however, the SE did not contain hemorrhagic activity. Helicopsin was purified using activity-guided, Superose 12 10/300 GL molecular exclusion, Mono Q10 ion exchange, and Protein Pak 60 molecular exclusion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a highly purified band of approximately 20 kDa. The minimal lethal dose for helicopsin was 0.4 mg/kg. Liquid chromatography mass spectrometry (LC-MS/MS) analysis identified 2 unique peptides MEWYPEAAANAER and YTQIVWYK, representing a protein sequence (deleted homology) belonging to cysteine-rich secretory proteins, which are conserved in snake venoms (CRISPs). CRISPs are a large family of cysteine-rich secretory proteins found in various organisms and participate in diverse biological processes. Helicopsin exhibited robust neurotoxic activity as evidenced by immediate death (~8 min) due to respiratory paralysis in NIH mice. These observations for helicopsin purified from H. angulatus provide further evidence of the extensive distribution of highly potent neurotoxins in the Colubroidea superfamily of snakes than previously described.

  4. Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function.

    PubMed

    Gibbs, Gerard M; Orta, Gerardo; Reddy, Thulasimala; Koppers, Adam J; Martínez-López, Pablo; de la Vega-Beltràn, José Luis; Lo, Jennifer C Y; Veldhuis, Nicholas; Jamsai, Duangporn; McIntyre, Peter; Darszon, Alberto; O'Bryan, Moira K

    2011-04-26

    The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function.

  5. Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function

    PubMed Central

    Gibbs, Gerard M.; Orta, Gerardo; Reddy, Thulasimala; Koppers, Adam J.; Martínez-López, Pablo; Luis de la Vega-Beltràn, José; Lo, Jennifer C. Y.; Veldhuis, Nicholas; Jamsai, Duangporn; McIntyre, Peter; Darszon, Alberto; O'Bryan, Moira K.

    2011-01-01

    The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function. PMID:21482758

  6. P-domains as shuffled cysteine-rich modules in integumentary mucin C.1 (FIM-C.1) from Xenopus laevis. Polydispersity and genetic polymorphism.

    PubMed

    Hauser, F; Hoffmann, W

    1992-12-05

    A new frog integumentary mucin (FIM-C.1) has been discovered by molecular cloning. This mucin contains at least six typical P-domains as cysteine-rich modules. Shuffled P-domains have previously been detected in FIM-A.1, and they also form the basis of various P-domain peptides, which presumably have growth-modulating activities. Furthermore, FIM-C.1 contains at least three threonine-rich clusters, which consist of semi-repetitive cassettes. Various polydisperse transcripts have been characterized. They originate from two genes only and differ by deletions/insertions that are congruent with the semi-repetitive cassettes. Thus, polydispersities are probably generated by alternative splicing. Southern analysis revealed genetic polymorphism between different individuals. Furthermore, a specific antiserum was generated against a synthetic peptide deduced from the COOH-terminal end of FIM-C.1 and used for Western analysis.

  7. Overexpression of an Arabidopsis cysteine-rich receptor-like protein kinase, CRK5, enhances abscisic acid sensitivity and confers drought tolerance

    PubMed Central

    Lu, Kai; Liang, Shan; Wu, Zhen; Bi, Chao; Yu, Yong-Tao; Wang, Xiao-Fang; Zhang, Da-Peng

    2016-01-01

    Receptor-like kinases (RLKs) have been reported to regulate many developmental and defense process, but only a few members have been functionally characterized. In the present study, our observations suggest that one of the RLKs, a membrane-localized cysteine-rich receptor-like protein kinase, CRK5, is involved in abscisic acid (ABA) signaling in Arabidopsis thaliana. Overexpression of CRK5 increases ABA sensitivity in ABA-induced early seedling growth arrest and promotion of stomatal closure and inhibition of stomatal opening. Interestingly, and importantly, overexpression of CRK5 enhances plant drought tolerance without affecting plant growth at the mature stages and plant productivity. Transgenic lines overexpressing a mutated form of CRK5, CRK5 K372E with the change of the 372nd conserved amino acid residue from lysine to glutamic acid in its kinase domain, result in wild-type ABA and drought responses, supporting the role of CRK5 in ABA signaling. The loss-of-function mutation of the CRK5 gene does not affect the ABA response, while overexpression of two homologs of CRK5, CRK4 and CRK19, confers ABA responses, suggesting that these CRK members function redundantly. We further showed that WRKY18, WRKY40 and WRKY60 transcription factors repress the expression of CRK5, and that CRK5 likely functions upstream of ABI2 in ABA signaling. These findings help in understanding the complex ABA signaling network. PMID:27406784

  8. Convergent solid-phase and solution approaches in the synthesis of the cysteine-rich Mdm2 RING finger domain.

    PubMed

    Vasileiou, Zoe; Barlos, Kostas; Gatos, Dimitrios

    2009-12-01

    The RING finger domain of the Mdm2, located at the C-terminus of the protein, is necessary for regulation of p53, a tumor suppressor protein. The 48-residues long Mdm2 peptide is an important target for studying its interaction with small anticancer drug candidates. For the chemical synthesis of the Mdm2 RING finger domain, the fragment condensation on solid-phase and the fragment condensation in solution were studied. The latter method was performed using either protected or free peptides at the C-terminus as the amino component. Best results were achieved using solution condensation where the N-component was applied with the C-terminal carboxyl group left unprotected. The developed method is well suited for large-scale synthesis of Mdm2 RING finger domain, combining the advantages of both solid-phase and solution synthesis.

  9. Immunogold labeling shows that glycine-cysteine-rich beta-proteins are deposited in the Oberhäutchen layer of snake epidermis in preparation to shedding.

    PubMed

    Alibardi, Lorenzo

    2015-02-01

    Shedding in snakes is cyclical and derives from the differentiation of an intraepidermal shedding complex made of two different layers, termed clear and Oberhäutchen that determine the separation between the outer from the inner epidermal generation that produces a molt. The present comparative immunocytochemical study on the epidermis and molts of different species of snakes shows that a glycine-cysteine-rich corneous beta-protein in a snake is prevalently accumulated in cells of the Oberhäutchen layer and decreases in those of the beta-layer. The protein is variably distributed in the mature beta-layer of species representing some snake families when the beta-layer merges with the Oberhäutchen but disappears in alpha-layers. Therefore, this protein represents an early marker of the transition between the outer and the inner epidermal generations in the epidermis of snakes in general. It is hypothesized that specific gene activation for glycine-cysteine-rich corneous beta-proteins occurs during the passage from the clear layer of the outer epidermal generation to the Oberhäutchen layer of the replacing inner epidermal generation. It is suggested that in the epidermis of most species glycine-cysteine-rich corneous beta-proteins form part of the dense corneous material that rapidly accumulates in the differentiating Oberhäutchen cells but decreases in the following beta-layer of the inner epidermal generation destined to be separated from the previous outer generation in the process of shedding. The regulation of the synthesis of these and other proteins is, therefore, crucial in timing the different stages of the shedding cycle in lepidosaurian reptiles.

  10. Characterization of the human mucin gene MUC5AC: a consensus cysteine-rich domain for 11p15 mucin genes?

    PubMed Central

    Guyonnet Duperat, V; Audie, J P; Debailleul, V; Laine, A; Buisine, M P; Galiegue-Zouitina, S; Pigny, P; Degand, P; Aubert, J P; Porchet, N

    1995-01-01

    To date five human mucin cDNAs (MUC2, 5A, 5B, 5C and 6) mapped to 11p15.3-15.5, so it appears that this chromosome region might contain several distinct gene loci for mucins. Three of these cDNAs, MUC5A, B and C, were cloned in our laboratory and previously published. A common number, 5, was recommended by the Human Gene Mapping Nomenclature Committee to designate them because of their common provenance from human tracheobronchial mucosa. In order to define whether they are products of the same gene locus or distinct loci, we describe in this paper physical mapping of these cDNAs using the strategy of analysis of CpG islands by pulse-field gel electrophoresis. The data suggest that MUC5A and MUC5C are part of the same gene (called MUC5AC) which is distinct from MUC5B. In the second part of this work, complete sequences of the inserts corresponding to previously described (JER47, JER58) and novel (JER62, JUL32, MAR2, MAR10 and MAR11) cDNAs of the so-called MUC5AC gene are presented and analysed. The data show that in this mucin gene, the tandem repeat domain is interrupted several times with a subdomain encoding a 130 amino acid cysteine-rich peptide in which the TR3A and TR3B peptides previously isolated by Rose et al. [Rose, Kaufman and Martin (1989) J. Biol. Chem., 264, 8193-8199] from airway mucins are found. A consensus peptide sequence for these subdomains involving invariant positions of most of the cysteines is proposed. The consensus nucleotide sequence of this subdomain is also found in the MUC2 gene and in the MUC5B gene, two other mucin genes mapped to 11p15. The functional significance for secreted mucins of these cysteine-rich subdomains and the modular organization of mucin peptides are discussed. Images Figure 3 Figure 4 Figure 5 Figure 8 PMID:7826332

  11. A two component chitin-binding protein from French bean -- association of a proline-rich protein with a cysteine-rich polypeptide.

    PubMed

    Bindschedler, Laurence V; Whitelegge, Julian P; Millar, David J; Bolwell, G Paul

    2006-03-06

    A 42kDa chitin-binding proline-rich protein (PRP) from French bean has been previously characterised through its involvement in plant-pathogen interactions. It is located at the plasmalemma-wall interface, intercellular spaces and binds to the pathogen Colletotrichum lindemuthianum in vitro and in planta. It is also present in cell wall appositions formed in response to an hrp mutant of Xanthomonas campestris. We now show that the 42kDa protein is composed of two components, a 25kDa polypeptide member of the PRP family of legumes and a 6.8kDa cysteine-rich peptide with high similarity to snakin-2 from potato. Snakins bind to pathogens and are antimicrobial. Molecular cloning of the longest PRP corresponding to the N-terminal sequence of the purified protein and the 6.8kDa component is reported. The cognate mRNAs show coordinate expression. The two-component protein complex has already been shown to be involved in binding and immobilising pathogens through oxidative cross-linking of the PRP components but could also function as a two-component chitin-receptor involved in plant-pathogen interactions through antimicrobial activity and/or signalling.

  12. Genetic diversity and identification of human infection by amplification of the chlamydial 60-kilodalton cysteine-rich outer membrane protein gene.

    PubMed Central

    Watson, M W; Lambden, P R; Clarke, I N

    1991-01-01

    The 60-kDa cysteine-rich outer membrane protein genes of Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis have very different 5' ends, but two areas flanking this variable region show absolute sequence conservation. This observation permitted differentiation of the three species of Chlamydia by the polymerase chain reaction (PCR), forming the basis of a diagnostic test for chlamydial infections. The PCR product containing the variable region of the respective 60-kDa CrP genes was also subjected to restriction endonuclease digestion, enabling differentiation of individual type strains of C. psittaci. Differentiation was possible between lymphogranuloma venereum and trachoma isolates of C. trachomatis. The PCR-based diagnostic test was successful with all strains of chlamydiae studied. The PCR primers showed high specificity and did not produce any product with common bacterial pathogens that may share the same sites of infection. Images PMID:1864938

  13. Introgression of leginsulin, a cysteine-rich protein, and high-protein trait from an Asian soybean plant introduction genotype into a North American experimental soybean line.

    PubMed

    Krishnan, Hari B; Kim, Won-Seok; Oehrle, Nathan W; Alaswad, Alaa A; Baxter, Ivan; Wiebold, William J; Nelson, Randall L

    2015-03-25

    Soybean is an important protein source for both humans and animals. However, soybean proteins are relatively poor in the sulfur-containing amino acids, cysteine and methionine. Improving the content of endogenous proteins rich in sulfur-containing amino acids could enhance the nutritive value of soybean meal. Leginsulin, a cysteine-rich peptide, predominantly accumulates in Asian soybean accessions but not in most North American cultivars. By screening diverse soybean accessions from the USDA Soybean Germplasm Collection, we were able to identify one plant introduction, PI 427138, as a high-protein line with relatively high amounts of both elemental sulfur and leginsulin. We introgressed these desirable traits from PI 427138 into an experimental line with the aim of improving the overall protein content and quality of seed proteins. Biochemical characterization of inbred progenies from the cross of LD00-3309 with PI 427138 grown at six locations revealed stable ingression of high protein, high elemental sulfur, and high leginsulin accumulation. Comparison of soybean seed proteins resolved by high-resolution 2-D gel electrophoresis in combination with Delta2D image analysis software revealed preferential accumulation of a few glycinin subunits contributed to the increased protein content in the introgressed lines. Amino acid analysis revealed that even though the leginsulin introgressed lines had higher protein, leginsulin, and elemental sulfur, the overall concentration of sulfur-containing amino acids was not significantly altered when compared with the parental lines. The experimental soybean lines developed during this study (Leg-3, Leg-7, and Leg-8) lack A5, A4, and B3 glycinin subunits and could be utilized in breeding programs to develop high-quality tofu cultivars.

  14. The corneous layer of the claw in the lizard Anolis carolinensis mainly contains the glycine-cysteine-rich beta-protein HgGC3 in addition to hard keratins.

    PubMed

    Alibardi, L

    2014-10-01

    The localization of specific claw beta-proteins among the 40 total corneous beta-proteins present in the lizard Anolis carolinensis is not known. The hardness of claws likely depends on glycine-cysteine-rich beta-proteins content, as suggested by previous immunoblot studies. Previous studies have indicated that glycine-cysteine-rich corneous beta-proteins in addition to cysteine-rich alpha-keratins are present in the claw. In order to detect at the ultrastructural level the presence of claw-specific corneous proteins immunofluorescence and electron microscopy immunogold have been utilized. More intense immunoreactivity is obtained for the HgGC3 beta-protein while less intense immunolabeling is seen for HgGC10 and HgG5 beta-proteins and no labeling for the cysteine-rich beta-protein HgC1. The HgGC3 beta-protein appears the prevalent type present in the claw and its numerous cysteines likely form intermolecular disulphide bonds while glycine contributes hydrophobic properties to the corneous material. Other antibodies tagging the core-box and pre-core box regions of beta-proteins label with less intensity the corneous layer. The presence of cysteine-rich alpha-keratins with high homology to some human hair keratins in the dorsal part of the claw suggests that HgGC3-like beta-proteins form numerous disulphide bonds with the larger alpha-keratins giving rise to the hard corneous material of the claw.

  15. Involvement of cysteine-rich protein 61 in the epidermal growth factor-induced migration of human anaplastic thyroid cancer cells.

    PubMed

    Chin, Li-Han; Hsu, Sung-Po; Zhong, Wen-Bin; Liang, Yu-Chih

    2016-05-01

    Anaplastic thyroid cancer (ATC) is among the most aggressive types of malignant cancer. Epidermal growth factor (EGF) plays a crucial role in the pathogenesis of ATC, and patients with thyroid carcinoma typically exhibit increased cysteine-rich protein 61 (Cyr61). In this study, we found that EGF treatment induced cell migration, stress fiber formation, Cyr61 mRNA and protein expressions, and Cyr61 protein secretion in ATC cells. The recombinant Cyr61 protein significantly induced cell migration; however, inhibition of Cyr61 activity by a Cyr61-specific antibody abrogated EGF-induced cell migration. EGF treatment also affected epithelial-to-mesenchymal transition (EMT)-related marker protein expression, as evidenced by an increase in vimentin and a decrease in E-cadherin expression. Inhibition of Cyr61 expression by Cyr61 siRNA decreased cell migration and reversed the EMT-related marker protein expression. EGF treatment increased the phosphorylation of the extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB), and finally activated Cyr61 promoter plasmid activity. Our results suggest that Cyr61 is induced by EGF through the ERK/CREB signal pathway and that it plays a crucial role in the migration and invasion of ATC cells; moreover, Cyr61 might be a therapeutic target for metastatic ATC.

  16. The matricellular protein cysteine-rich protein 61 (CCN1/Cyr61) enhances physiological adaptation of retinal vessels and reduces pathological neovascularization associated with ischemic retinopathy.

    PubMed

    Hasan, Adeel; Pokeza, Nataliya; Shaw, Lynn; Lee, Hyun-Seung; Lazzaro, Douglas; Chintala, Hemabindu; Rosenbaum, Daniel; Grant, Maria B; Chaqour, Brahim

    2011-03-18

    Retinal vascular damages are the cardinal hallmarks of retinopathy of prematurity (ROP), a leading cause of vision impairment and blindness in childhood. Both angiogenesis and vasculogenesis are disrupted in the hyperoxia-induced vaso-obliteration phase, and recapitulated, although aberrantly, in the subsequent ischemia-induced neovessel formation phase of ROP. Yet, whereas the histopathological features of ROP are well characterized, many key modulators with a therapeutic potential remain unknown. The CCN1 protein also known as cysteine-rich protein 61 (Cyr61) is a dynamically expressed, matricellular protein required for proper angiogenesis and vasculogenesis during development. The expression of CCN1 becomes abnormally reduced during the hyperoxic and ischemic phases of ROP modeled in the mouse eye with oxygen-induced retinopathy (OIR). Lentivirus-mediated re-expression of CCN1 enhanced physiological adaptation of the retinal vasculature to hyperoxia and reduced pathological angiogenesis following ischemia. Remarkably, injection into the vitreous of OIR mice of hematopoietic stem cells (HSCs) engineered to express CCN1 harnessed ischemia-induced neovessel outgrowth without adversely affecting the physiological adaptation of retinal vessels to hyperoxia. In vitro exposure of HSCs to recombinant CCN1 induced integrin-dependent cell adhesion, migration, and expression of specific endothelial cell markers as well as many components of the Wnt signaling pathway including Wnt ligands, their receptors, inhibitors, and downstream targets. CCN1-induced Wnt signaling mediated, at least in part, adhesion and endothelial differentiation of cultured HSCs, and inhibition of Wnt signaling interfered with normalization of the retinal vasculature induced by CCN1-primed HSCs in OIR mice. These newly identified functions of CCN1 suggest its possible therapeutic utility in ischemic retinopathy.

  17. Molecular mechanisms involved in gamete interaction: evidence for the participation of cysteine-rich secretory proteins (CRISP) in sperm-egg fusion.

    PubMed

    Da Ros, V; Busso, D; Cohen, D J; Maldera, J; Goldweic, N; Cuasnicu, P S

    2007-01-01

    Epididymal protein DE and testicular protein Tpx-1 are two cysteine-rich secretory proteins also known as CRISP-1 and CRISP-2, respectively. DE/ CRISP-1 is localised on the equatorial segment of acrosome-reacted sperm and participates in rat gamete fusion through its binding to egg-complementary sites. Recent results using bacterially-expressed recombinant fragments of DE as well as synthetic peptides revealed that the ability of DE to bind to the egg surface and inhibit gamete fusion resides in a region of 12 amino acids corresponding to an evolutionary conserved motif of the CRISP family (Signature 2). Given the high degree of homology between DE/CRISP-1 and Tpx-1/CRISP-2, we also explored the potential participation of the testicular intra-acrosomal protein in gamete fusion. Results showing the ability of recombinant Tpx-1 to bind to the surface of rat eggs (evaluated by indirect immunofluorescence) and to significantly inhibit zona-free egg penetration, support the participation of this protein in gamete fusion through its interaction with egg-binding sites. Interestingly, rat Tpx-1 exhibits only two substitutions in Signature 2 when compared to this region in DE. Together, these results provide evidence for the involvement of both epididymal DE/CRISP-1 and testicular Tpx-1/CRISP-2 in gamete fusion suggesting the existence of a functional cooperation between homologue molecules as a mechanism to ensure the success of fertilisation.

  18. Cysteine-rich protein 2, a novel substrate for cGMP kinase I in enteric neurons and intestinal smooth muscle.

    PubMed

    Huber, A; Neuhuber, W L; Klugbauer, N; Ruth, P; Allescher, H D

    2000-02-25

    Nitric oxide/cGMP/cGMP kinase I (cGKI) signaling causes relaxation of intestinal smooth muscle. In the gastrointestinal tract substrates of cGKI have not been identified yet. In the present study a protein interacting with cGKIbeta has been isolated from a rat intestinal cDNA library using the yeast two-hybrid system. The protein was identified as cysteine-rich protein 2 (CRP2), recently cloned from rat brain (Okano, I., Yamamoto, T., Kaji, A., Kimura, T., Mizuno, K., and Nakamura, T. (1993) FEBS Lett. 333, 51-55). Recombinant CRP2 is specifically phosphorylated by cGKs but not by cAMP kinase in vitro. Co-transfection of CRP2 and cGKIbeta into COS cells confirmed the phosphorylation of CRP2 in vivo. Cyclic GMP kinase I phosphorylated CRP2 at Ser-104, because the mutation to Ala completely prevented the in vivo phosphorylation. Immunohistochemical analysis using confocal laser scan microscopy showed a co-localization of CRP2 and cGKI in the inner part of the circular muscle layer, in the muscularis mucosae, and in specific neurons of the myenteric and submucosal plexus. The co-localization together with the specific phosphorylation of CRP2 by cGKI in vitro and in vivo suggests that CRP2 is a novel substrate of cGKI in neurons and smooth muscle of the small intestine.

  19. Retinoids suppress cysteine-rich protein 61 (CCN1), a negative regulator of collagen homeostasis, in skin equivalent cultures and aged human skin in vivo.

    PubMed

    Quan, Taihao; Qin, Zhaoping; Shao, Yuan; Xu, Yiru; Voorhees, John J; Fisher, Gary J

    2011-07-01

    Alterations in connective tissue collagen are prominent features of both chronologically aged and photoaged (ageing because of sun exposure) human skin. These age-related abnormalities are mediated in part by cysteine-rich protein 61 (CCN1). CCN1 is elevated in the dermis of both chronologically aged and photoaged human skin in vivo and promotes aberrant collagen homeostasis by down-regulating type I collagen, the major structural protein in skin, and promoting collagen degradation. Vitamin A and its metabolites have been shown to improve chronologically aged and photoaged skin by promoting deposition of new collagen and preventing its degradation. Here, we investigated regulation of CCN1 expression by retinoids in skin equivalent cultures and chronologically aged and photoaged human skin in vivo. In skin equivalent cultures, all-trans retinoic acid (RA), the major bioactive form of vitamin A in skin, significantly increased type I procollagen and reduced collagenase (matrix metalloproteinases-1, MMP-1). Addition of recombinant human CCN1 to skin equivalent cultures significantly reduced type I procollagen and increased MMP-1. Importantly, RA significantly reduced CCN1 expression in skin equivalent cultures. Topical treatment with retinol (vitamin A, 0.4%) for 7days significantly reduced CCN1 mRNA and protein expression in both chronologically aged (80+years) and photoaged human skin in vivo, compared to vehicle-treated skin. These data indicate that the mechanism by which retinoids improve aged skin, through increased collagen production, involves down-regulation of CCN1.

  20. Characterization of Helicobacter pylori HP0231 (DsbK): role in disulfide bond formation, redox homeostasis and production of Helicobacter cystein-rich protein HcpE.

    PubMed

    Lester, Jeffrey; Kichler, Sari; Oickle, Brandon; Fairweather, Spencer; Oberc, Alexander; Chahal, Jaspreet; Ratnayake, Dinath; Creuzenet, Carole

    2015-04-01

    Helicobacter pylori is a human gastric pathogen that colonizes ∼ 50% of the world's population. It can cause gastritis, gastric or duodenal ulcers and also gastric cancer. The numerous side effects of available treatments and the emergence of antibiotic resistant strains are severe concerns that justify further research into H. pylori's pathogenic mechanisms. H. pylori produces secreted proteins that may play a role in virulence, including the Helicobacter cysteine-rich protein HcpE (aka HP0235). We demonstrate herein that HcpE is secreted in the culture supernatant both as a soluble protein and in association with outer membrane vesicles. We show that the structure of HcpE comprises an organized array of disulfide bonds. We identify DsbK (aka HP0231) as a folding factor necessary for HcpE production and secretion in H. pylori and show that recombinant DsbK can interact with and refold unprocessed, reduced HcpE in vitro. These experiments highlight the first biologically relevant substrate for DsbK. Furthermore, we show that DsbK has disulfide bond (Dsb) forming activity on reduced lysozyme and demonstrate a DsbA-type of activity for DsbK upon expression in E. coli, despite its similarity with DsbG. Finally, we show a role of DsbK in maintaining redox homeostasis in H. pylori.

  1. Investigating possible biological targets of Bj-CRP, the first cysteine-rich secretory protein (CRISP) isolated from Bothrops jararaca snake venom.

    PubMed

    Lodovicho, Marina E; Costa, Tássia R; Bernardes, Carolina P; Menaldo, Danilo L; Zoccal, Karina F; Carone, Sante E; Rosa, José C; Pucca, Manuela B; Cerni, Felipe A; Arantes, Eliane C; Tytgat, Jan; Faccioli, Lúcia H; Pereira-Crott, Luciana S; Sampaio, Suely V

    2017-01-04

    Cysteine-rich secretory proteins (CRISPs) are commonly described as part of the protein content of snake venoms, nevertheless, so far, little is known about their biological targets and functions. Our study describes the isolation and characterization of Bj-CRP, the first CRISP isolated from Bothrops jararaca snake venom, also aiming at the identification of possible targets for its actions. Bj-CRP was purified using three chromatographic steps (Sephacryl S-200, Source 15Q and C18) and showed to be an acidic protein of 24.6kDa with high sequence identity to other snake venom CRISPs. This CRISP was devoid of proteolytic, hemorrhagic or coagulant activities, and it did not affect the currents from 13 voltage-gated potassium channel isoforms. Conversely, Bj-CRP induced inflammatory responses characterized by increase of leukocytes, mainly neutrophils, after 1 and 4h of its injection in the peritoneal cavity of mice, also stimulating the production of IL-6. Bj-CRP also acted on the human complement system, modulating some of the activation pathways and acting directly on important components (C3 and C4), thus inducing the generation of anaphylatoxins (C3a, C4a and C5a). Therefore, our results for Bj-CRP open up prospects for better understanding this class of toxins and its biological actions.

  2. A small cysteine-rich extracellular protein, VCRP, is inducible by the sex-inducer of Volvox carteri and by wounding.

    PubMed

    Hallmann, Armin

    2007-08-01

    The green alga Volvox carteri represents one of the simplest multicellular organisms: it is composed of only two cell types, somatic and reproductive. Volvox is capable of both vegetative and sexual reproduction. Sexual development of males and females is triggered by a sex-inducer at concentrations as low as 10(-16) M. By differential screenings of cDNA libraries, a novel gene was identified that is under the control of this sex-inducer and that encodes a small cysteine-rich extracellular protein, named VCRP. Analysis of the VCRP polypeptide sequence suggests ten disulfide bonds and a dimetal-binding capacity. VCRP mRNA is detectable in males and females approximately 1 h after the spheroids' first contact with the sex-inducer, but transcription is restricted to the somatic cell-type. mRNA and protein synthesis is triggered not only by the sex-inducer, but also by wounding. VCRP does not share significant sequence similarity with any known protein sequence, but a potential EGF-like calcium-binding motif and a potential plant metallothionein family-15 motif have been identified. The characteristics of VCRP suggest a function as a signal transducer molecule, an extracellular second messenger from somatic cells to reproductive cells, or a role within the stress response.

  3. Oxidant Exposure Induces Cysteine-Rich Protein 61 (CCN1) via c-Jun/AP-1 to Reduce Collagen Expression in Human Dermal Fibroblasts

    PubMed Central

    Qin, Zhaoping; Robichaud, Patrick; He, Tianyuan; Fisher, Gary J.; Voorhees, John J.; Quan, Taihao

    2014-01-01

    Human skin is a primary target of oxidative stress from reactive oxygen species (ROS) generated from both extrinsic and intrinsic sources. Oxidative stress inhibits the production of collagen, the most abundant protein in skin, and thus contributes to connective tissue aging. Here we report that cysteine-rich protein 61 (CCN1), a negative regulator of collagen production, is markedly induced by ROS and mediates loss of type I collagen in human dermal fibroblasts. Conversely, antioxidant N-acetyl-L-cysteine significantly reduced CCN1 expression and prevented ROS-induced loss of type I collagen in both human dermal fibroblasts and human skin in vivo. ROS increased c-Jun, a critical member of transcription factor AP-1 complex, and increased c-Jun binding to the AP-1 site of the CCN1 promoter. Functional blocking of c-Jun significantly reduced CCN1 promoter and gene expression and thus prevented ROS-induced loss of type I collagen. Targeting the c-Jun/CCN1 axis may provide clinical benefit for connective tissue aging in human skin. PMID:25536346

  4. Oxidant exposure induces cysteine-rich protein 61 (CCN1) via c-Jun/AP-1 to reduce collagen expression in human dermal fibroblasts.

    PubMed

    Qin, Zhaoping; Robichaud, Patrick; He, Tianyuan; Fisher, Gary J; Voorhees, John J; Quan, Taihao

    2014-01-01

    Human skin is a primary target of oxidative stress from reactive oxygen species (ROS) generated from both extrinsic and intrinsic sources. Oxidative stress inhibits the production of collagen, the most abundant protein in skin, and thus contributes to connective tissue aging. Here we report that cysteine-rich protein 61 (CCN1), a negative regulator of collagen production, is markedly induced by ROS and mediates loss of type I collagen in human dermal fibroblasts. Conversely, antioxidant N-acetyl-L-cysteine significantly reduced CCN1 expression and prevented ROS-induced loss of type I collagen in both human dermal fibroblasts and human skin in vivo. ROS increased c-Jun, a critical member of transcription factor AP-1 complex, and increased c-Jun binding to the AP-1 site of the CCN1 promoter. Functional blocking of c-Jun significantly reduced CCN1 promoter and gene expression and thus prevented ROS-induced loss of type I collagen. Targeting the c-Jun/CCN1 axis may provide clinical benefit for connective tissue aging in human skin.

  5. Elevated cysteine-rich protein 61 (CCN1) promotes skin aging via upregulation of IL-1β in chronically sun-exposed human skin.

    PubMed

    Qin, Zhaoping; Okubo, Toru; Voorhees, John J; Fisher, Gary J; Quan, Taihao

    2014-02-01

    Chronic exposure of human skin to solar ultraviolet (UV) irradiation causes premature skin aging, which is characterized by reduced type I collagen production and increased fragmentation of the dermal collagenous extracellular matrix. This imbalance of collagen homeostasis is mediated, in part, by elevated expression of the matricellular protein cysteine-rich protein 61 (CCN1), in dermal fibroblasts, the primary collagen producing cell type in human skin. Here, we report that the actions of CCN1 are mediated by induction of interleukin 1β (IL-1β). CCN1 and IL-1β are strikingly induced by acute UV irradiation, and constitutively elevated in sun-exposed prematurely aged human skin. Elevated CCN1 rapidly induces IL-1β, inhibits type I collagen production, and upregulates matrix metalloproteinase-1, which degrades collagen fibrils. Blockade of IL-1β actions by IL-1 receptor antagonist largely prevents the deleterious effects of CCN1 on collagen homeostasis. Furthermore, knockdown of CCN1 significantly reduces induction of IL-1β by UV irradiation, and thereby partially prevents collagen loss. These data demonstrate that elevated CCN1promotes inflammaging and collagen loss via induction of IL-1β and thereby contributes to the pathophysiology of premature aging in chronically sun-exposed human skin.

  6. Molecular cloning, mapping to human chromosome 1 q21-q23, and cell binding characteristics of Spalpha, a new member of the scavenger receptor cysteine-rich (SRCR) family of proteins.

    PubMed

    Gebe, J A; Kiener, P A; Ring, H Z; Li, X; Francke, U; Aruffo, A

    1997-03-07

    CD5 and CD6, two type I cell surface antigens predominantly expressed by T cells and a subset of B cells, have been shown to function as accessory molecules capable of modulating T cell activation. Here we report the cloning of a cDNA encoding Spalpha, a secreted protein that is highly homologous to CD5 and CD6. Spalpha has the same domain organization as the extracellular region of CD5 and CD6 and is composed of three SRCR (scavenger receptor cysteine rich) domains. Chromosomal mapping by fluorescence in situ hybridization and radiation hybrid panel analysis indicated that the gene encoding Spalpha is located on the long arm of human chromosome 1 at q21-q23 within contig WC1.17. RNA transcripts encoding Spalpha were found in human bone marrow, spleen, lymph node, thymus, and fetal liver but not in non-lymphoid tissues. Cell binding studies with an Spalpha immunoglobulin (Spalpha-mIg) fusion protein indicated that Spalpha is capable of binding to peripheral monocytes but not to T or B cells. Spalpha-mIg was also found to bind to the monocyte precursor cell lines K-562 and weakly to THP-1 but not to U937. Spalpha-mIg also bound to the B cell line Raji and weakly to the T cell line HUT-78. These findings indicate that Spalpha, a novel secreted protein produced in lymphoid tissues, may regulate monocyte activation, function, and/or survival.

  7. Reversion-Inducing-Cysteine-Rich Protein With Kazal Motifs (RECK) Gene Single Nucleotide Polymorphism With Hepatocellular Carcinoma: A Case-Control Study.

    PubMed

    Bahgat, Dina M Rasheed; Shahin, Rasha Mohamad Hosny; Makar, Nada Nasr; Aziz, Ashraf Omar Abdel; Hunter, Shereen Shoukry

    2016-01-01

    The reversion-inducing-cysteine-rich protein with kazal motifs (RECK) gene is a transformation suppressor gene that can negatively regulate matrix metalloproteinases (MMPs) and inhibit tumor invasion, angiogenesis, and metastasis. So, the aim of this study was to analyze the effect of RECK gene rs 11788747 single nucleotide polymorphism (SNP) on hepatocellular carcinoma (HCC) susceptibility and its relation to various clinical and laboratory data of the patients. This is a case-control study including 200 HCC patients and 200 healthy controls. RECK rs 11788747 genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RECK rs 11788747 A/G and G/G genotypes frequencies were significantly higher in HCC patients compared to the healthy controls. The HCC patients possessing at least one polymorphic G allele were significantly at a higher risk of developing lymph nodes involvement and distant metastasis. This study revealed the role of RECK rs 11788747 SNP in HCC in Egyptian patients, which consequently might be used as a prognostic tool and could be added to its therapeutic strategies. © 2014 Wiley Periodicals, Inc.

  8. DMSA-Coated Iron Oxide Nanoparticles Greatly Affect the Expression of Genes Coding Cysteine-Rich Proteins by Their DMSA Coating.

    PubMed

    Zhang, Ling; Wang, Xin; Zou, Jinglu; Liu, Yingxun; Wang, Jinke

    2015-10-19

    The dimercaptosuccinic acid (DMSA) was widely used to coat iron oxide nanoparticles (FeNPs); however, its intracellular cytotoxicity remains to be adequately elucidated. This study analyzed the differentially expressed genes (DEGs) in four mammalian cells treated by a DMSA-coated magnetite FeNP at various doses at different times. The results revealed that about one-fourth of DEGs coded cysteine-rich proteins (CRPs) in all cells under each treatment, indicating that the nanoparticles greatly affected the expressions of CRP-coding genes. Additionally, about 26% of CRP-coding DEGs were enzyme genes in all cells, indicating that the nanoparticles greatly affected the expression of enzyme genes. Further experiments with the nanoparticles and a polyethylenimine (PEI)-coated magnetite FeNP revealed that the effect mainly resulted from DMSA carried into cells by the nanoparticles. This study thus first reported the cytotoxicity of DMSA at the gene transcription level as coating molecules of FeNPs. This study provides new insight into the molecular mechanism by which the DMSA-coated nanoparticles resulted in the transcriptional changes of many CRP-coding genes in cells. This study draws attention toward the intracellular cytotoxicity of DMSA as a coating molecule of nanoparticles, which has very low toxicity as an orally administered antidote due to its extracellular distribution.

  9. Association of the cysteine-rich secretory protein-3 (CRISP-3) and some of its polymorphisms with the quality of cryopreserved stallion semen.

    PubMed

    Usuga, Alexandra; Rojano, Benjamín A; Restrepo, Giovanni

    2017-08-31

    Contribution of seminal plasma proteins to semen freezability has been reported in several species, suggesting these proteins as genetic markers. The aim of this study was to evaluate the relationship between cysteine-rich secretory protein-3 (CRISP-3) and some of its single-nucleotide polymorphisms (SNPs) with post-thawing semen quality in stallions. DNA was obtained from 100 stallions, regions of interest were amplified by polymerase chain reaction and sequenced. Evaluated SNPs within the equine CRISP-3 gene were CRISP3c.+199A>G (SNP1), CRISP3c.+566C>A (SNP2), CRISP3c.+622G>A (SNP3) and CRISP3c.+716A>G (SNP4). CRISP-3 protein content in seminal plasma was determined by enzyme-linked immunosorbent assay. Semen from 30 stallions was cryopreserved and post-thaw motility, kinetics, abnormal morphology (AM), sperm vitality (SV) and membrane integrity (MI) were evaluated. Generalized linear models were fitted and means were compared using Tukey's test. Correlation and regression analyses were performed. For SNP1 and SNP3, the AA genotype had the highest results for motility and MI; for SNP2, the best results for motility and AM were obtained with the CC genotype. For SNP4, the GG genotype had the lowest results, except for MI. A high level of CRISP-3 protein in seminal plasma had the best results for motility, kinetics, SV and AM. In conclusion, there was a relationship between CRISP-3 genotype and seminal plasma protein and post-thawing semen quality in stallions.

  10. Participation of epididymal cysteine-rich secretory proteins in sperm-egg fusion and their potential use for male fertility regulation.

    PubMed

    Cohen, Debora J; Da Ros, Vanina G; Busso, Dolores; Ellerman, Diego A; Maldera, Julieta A; Goldweic, Nadia; Cuasnicú, Patricia S

    2007-07-01

    Rat protein DE is an androgen-dependent cysteine-rich secretory protein (CRISP) synthesized by proximal epididymal regions. DE, also known as CRISP-1, is localized on the equatorial segment of acrosome-reacted spermatozoa and participates in gamete fusion through binding to egg complementary sites. Immunization of rats with DE inhibits fertility and sperm fusion ability, suggesting that DE represents a good epididymal contraceptive target. Recombinant DE fragments and synthetic peptides revealed that DE binds to the egg via a 12-amino acid region of an evolutionarily conserved motif, Signature 2 (S2). The ability of other CRISP to bind to the rat egg was correlated with their S2 amino acid sequences. Although testicular protein Tpx-1 (CRISP-2) was capable of binding to rodent eggs, human epididymal AEG-related protein (ARP) and helothermine (from lizard saliva) were not. The S2 region presented only two substitutions in Tpx-1 and four in ARP and helothermine, compared with the DE S2, suggesting that this amino acid sequence was relevant for egg interaction. Studies with Tpx-1 and anti-Tpx-1 revealed the participation of this protein in gamete fusion through binding to complementary sites in the egg. In competition studies, DE reduced binding of Tpx-1 dose-dependently, indicating that both CRISP share the egg complementary sites. That anti-DE and anti-Tpx-1 inhibit sperm-egg fusion while recognizing only the corresponding proteins, suggests functional cooperation between these homologous CRISP to ensure fertilization success. These results increase our understanding of the molecular mechanisms of gamete fusion and contribute to the development of new and safer fertility regulating methods.

  11. Two COWP-like cysteine rich proteins from Eimeria nieschulzi (coccidia, apicomplexa) are expressed during sporulation and involved in the sporocyst wall formation.

    PubMed

    Jonscher, Ernst; Erdbeer, Alexander; Günther, Marie; Kurth, Michael

    2015-07-25

    The family of cysteine rich proteins of the oocyst wall (COWPs) originally described in Cryptosporidium can also be found in Toxoplasma gondii (TgOWPs) localised to the oocyst wall as well. Genome sequence analysis of Eimeria suggests that these proteins may also exist in this genus and led us to the assumption that these proteins may also play a role in oocyst wall formation. In this study, COWP-like encoding sequences had been identified in Eimeria nieschulzi. The predicted gene sequences were subsequently utilized in reporter gene assays to observe time of expression and localisation of the reporter protein in vivo. Both investigated proteins, EnOWP2 and EnOWP6, were expressed during sporulation. The EnOWP2-promoter driven mCherry was found in the cytoplasm and the EnOWP2, respectively EnOWP6, fused to mCherry was initially observed in the extracytoplasmatic space between sporoblast and oocyst wall. This, so far unnamed compartment was designated as circumplasm. Later, the mCherry reporter co-localised with the sporocyst wall of the sporulated oocysts. This observation had been confirmed by confocal microscopy, excystation experiments and IFA. Transcript analysis revealed the intron-exon structure of these genes and confirmed the expression of EnOWP2 and EnOWP6 during sporogony. Our results allow us to assume a role, of both investigated EnOWP proteins, in the sporocyst wall formation of E. nieschulzi. Data mining and sequence comparisons to T. gondii and other Eimeria species allow us to hypothesise a conserved process within the coccidia. A role in oocyst wall formation had not been observed in E. nieschulzi.

  12. Functional interaction of phospholipid hydroperoxide glutathione peroxidase with sperm mitochondrion-associated cysteine-rich protein discloses the adjacent cysteine motif as a new substrate of the selenoperoxidase.

    PubMed

    Maiorino, Matilde; Roveri, Antonella; Benazzi, Louise; Bosello, Valentina; Mauri, Pierluigi; Toppo, Stefano; Tosatto, Silvio C E; Ursini, Fulvio

    2005-11-18

    The mitochondrial capsule is a selenium- and disulfide-rich structure enchasing the outer mitochondrial membrane of mammalian spermatozoa. Among the proteins solubilized from the sperm mitochondrial capsule, we confirmed, by using a proteomic approach, the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPx) as a major component, and we also identified the sperm mitochondrion-associated cysteine-rich protein (SMCP) and fragments/aggregates of specific keratins that previously escaped detection (Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J., and Flohé, L. (1999) Science 285, 1393-1396). The evidence for a functional association between PHGPx, SMCP, and keratins is further supported by the identification of a sequence motif of regularly spaced Cys-Cys doublets common to SMCP and high sulfur keratin-associated proteins, involved in bundling hair shaft keratin by disulfide cross-linking. Following the oxidative polymerization of mitochondrial capsule proteins, catalyzed by PHGPx, two-dimensional redox electrophoresis analysis showed homo- and heteropolymers of SMCP and PHGPx, together with other minor components. Adjacent cysteine residues in SMCP peptides are oxidized to cystine by PHGPx. This unusual disulfide is known to drive, by reshuffling oxidative protein folding. On this basis we propose that oxidative polymerization of the mitochondrial capsule is primed by the formation of cystine on SMCP, followed by reshuffling. Occurrence of reshuffling is further supported by the calculated thermodynamic gain of the process. This study suggests a new mechanism where selenium catalysis drives the cross-linking of structural elements of the cytoskeleton via the oxidation of a keratin-associated protein.

  13. Genetic characterization of cysteine-rich type-b avenin-like protein coding genes in common wheat.

    PubMed

    Chen, X Y; Cao, X Y; Zhang, Y J; Islam, S; Zhang, J J; Yang, R C; Liu, J J; Li, G Y; Appels, R; Keeble-Gagnere, G; Ji, W Q; He, Z H; Ma, W J

    2016-08-09

    The wheat avenin-like proteins (ALP) are considered atypical gluten constituents and have shown positive effects on dough properties revealed using a transgenic approach. However, to date the genetic architecture of ALP genes is unclear, making it impossible to be utilized in wheat breeding. In the current study, three genes of type-b ALPs were identified and mapped to chromosomes 7AS, 4AL and 7DS. The coding gene sequence of both TaALP-7A and TaALP-7D was 855 bp long, encoding two identical homologous 284 amino acid long proteins. TaALP-4A was 858 bp long, encoding a 285 amino acid protein variant. Three alleles were identified for TaALP-7A and four for TaALP-4A. TaALP-7A alleles were of two types: type-1, which includes TaALP-7A1 andTaALP-7A2, encodes mature proteins, while type-2, represented byTaALP-7A3, contains a stop codon in the coding region and thus does not encode a mature protein. Dough quality testing of 102 wheat cultivars established a highly significant association of the type-1 TaALP-7A allele with better wheat processing quality. This allelic effects were confirmed among a range of commercial wheat cultivars. Our research makes the ALP be the first of such genetic variation source that can be readily utilized in wheat breeding.

  14. Genetic characterization of cysteine-rich type-b avenin-like protein coding genes in common wheat

    PubMed Central

    Chen, X. Y.; Cao, X. Y.; Zhang, Y. J.; Islam, S.; Zhang, J. J.; Yang, R. C.; Liu, J. J.; Li, G. Y.; Appels, R.; Keeble-Gagnere, G.; Ji, W. Q.; He, Z. H.; Ma, W. J.

    2016-01-01

    The wheat avenin-like proteins (ALP) are considered atypical gluten constituents and have shown positive effects on dough properties revealed using a transgenic approach. However, to date the genetic architecture of ALP genes is unclear, making it impossible to be utilized in wheat breeding. In the current study, three genes of type-b ALPs were identified and mapped to chromosomes 7AS, 4AL and 7DS. The coding gene sequence of both TaALP-7A and TaALP-7D was 855 bp long, encoding two identical homologous 284 amino acid long proteins. TaALP-4A was 858 bp long, encoding a 285 amino acid protein variant. Three alleles were identified for TaALP-7A and four for TaALP-4A. TaALP-7A alleles were of two types: type-1, which includes TaALP-7A1 andTaALP-7A2, encodes mature proteins, while type-2, represented byTaALP-7A3, contains a stop codon in the coding region and thus does not encode a mature protein. Dough quality testing of 102 wheat cultivars established a highly significant association of the type-1 TaALP-7A allele with better wheat processing quality. This allelic effects were confirmed among a range of commercial wheat cultivars. Our research makes the ALP be the first of such genetic variation source that can be readily utilized in wheat breeding. PMID:27503660

  15. Characterization of expression and stability of recombinant cystein-rich protein human MT1A from yeast.

    PubMed

    Jie, Li; Kaifeng, Shao; Dian, Yao; Lin, An; Binggen, Ru

    2005-08-01

    Metallothionein (MT) is the protein that has been shown to bind heavy metals, scavenge free radicals, protect DNA from radiation damage, and alleviate disease symptoms. However, only very limited success has been achieved in expression and production of active recombinant metallothionein. In this study, human metallothionein 1A (hMT1A) was transformed into yeast Pichia pastoris for expression with secretion of the protein into the medium. The expression system was optimized to obtain the targeted protein in active form at 335 mg per litre culture. hMT1A showed the character of extreme instability in the experiment. High concentration, aeration and heavy metal ions are the main factors affecting hMT1A stability.

  16. D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function

    PubMed Central

    Burtscher, Laura; Hajdu, Dorottya; Muñoz, Alberto; Gáspári, Zoltán; Read, Nick D.; Batta, Gyula; Marx, Florentine

    2017-01-01

    The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19). We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF. PMID:28072824

  17. The expression of cysteine-rich secretory protein 2 (CRISP2) and its specific regulator miR-27b in the spermatozoa of patients with asthenozoospermia.

    PubMed

    Zhou, Jun-Hao; Zhou, Qi-Zhao; Lyu, Xiao-Ming; Zhu, Ting; Chen, Zi-Jian; Chen, Ming-Kun; Xia, Hui; Wang, Chun-Yan; Qi, Tao; Li, Xin; Liu, Cun-Dong

    2015-01-01

    Cysteine-rich secretory protein 2 (CRISP2) is an important sperm protein and plays roles in spermatogenesis, modulation of flagellar motility, acrosome reaction, and gamete fusion. Clinical evidence shows a reduced CRISP2 expression in spermatozoa from asthenozoospermic patients, but the molecular mechanism underlying its reduction remains unknown. Herein, we carried out a study focusing on the CRISP2 reduction and its roles in asthenozoospermia. Initially, through analyzing CRISP2 expression and methylation on CRISP2 promoter activity in sperm, we observed a decreased expression of CRISP2 protein rather than its mRNA in the ejaculated spermatozoa from asthenozoospermic patients and no methylation in the CRISP2 promoter, suggesting CRISP2 expression may be regulated in the sperm at the posttranscriptional level. Subsequently, we found that microRNA 27b (miR-27b), predicted as a candidate regulator of CRISP2 using bioinformatics, was highly expressed in the ejaculated spermatozoa from asthenozoospermic patients. Luciferase reporter assay and transfection experiments disclosed that this microRNA could target CRISP2 by specifically binding its 3' untranslated region, suppressing CRISP2 expression. Extended clinical observation further confirmed a highly expressed miR-27b and its obviously negative correlation with CRISP2 protein expression in ejaculated spermatozoa samples from asthenozoospermic patients. Finally, we conducted a retrospective follow-up study to support that either high miR-27b expression or low CRISP2 protein expression was significantly associated with low sperm progressive motility, abnormal morphology, and infertility. Thus, this study provides the first preliminary insight into the mechanism leading to the reduced CRISP2 expression in asthenozoospermia, offering a potential therapeutic target for treating male infertility or for male contraception.

  18. Endometrial cysteine-rich secretory protein 3 is inhibited by human chorionic gonadotrophin, and is increased in the decidua of tubal ectopic pregnancy

    PubMed Central

    Horne, A.W.; Duncan, W.C.; King, A.E.; Burgess, S.; Lourenco, P.C.; Cornes, P.; Ghazal, P.; Williams, A.R.; Udby, L.; Critchley, H.O.D.

    2009-01-01

    Ectopic pregnancy (EP) remains a considerable cause of morbidity and occasional mortality. Currently, there is no reliable test to differentiate ectopic from intrauterine gestation. We have previously used array technology to demonstrate that differences in gene expression in decidualized endometrium from women with ectopic and intrauterine gestations could be used to identify candidate diagnostic biomarkers for EP. The aim of this study was to further investigate the decidual gene with the highest fold increase in EP, cysteine-rich secretory protein-3 (CRISP-3). Decidualized endometrium from gestation-matched women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6) and surgery for EP (n = 11) was subjected to quantitative RT–PCR, morphological assessment, immunohistochemistry and western blot analysis. Sera were analysed for progesterone and human chorionic gonadotrophin (hCG) levels. Immortalized endometrial epithelial cells were cultured with physiological concentrations of hCG. CRISP-3 mRNA and protein expression were greater in endometrium from ectopic when compared with intrauterine pregnancies (P < 0.05). CRISP-3 protein was localized to epithelium and granulocytes of endometrium. CRISP-3 serum concentrations were not different in women with ectopic compared with intrauterine pregnancies. CRISP-3 expression in endometrium was not related to the degree of decidualization or to serum progesterone levels. Endometrial CRISP-3 expression was inversely proportional to serum hCG concentrations (P < 0.001). Stimulation of endometrial epithelial cells with hCG in vitro caused a reduction in CRISP-3 expression (P < 0.01). The measurement of CRISP-3 in endometrium could provide an additional tool in the diagnosis of failing early pregnancy of unknown location. The absence of a local reduction in expression of CRISP-3 in decidualized endometrium of women with EP may be due to reduced exposure to hCG due to the ectopic

  19. Endometrial cysteine-rich secretory protein 3 is inhibited by human chorionic gonadotrophin, and is increased in the decidua of tubal ectopic pregnancy.

    PubMed

    Horne, A W; Duncan, W C; King, A E; Burgess, S; Lourenco, P C; Cornes, P; Ghazal, P; Williams, A R; Udby, L; Critchley, H O D

    2009-05-01

    Ectopic pregnancy (EP) remains a considerable cause of morbidity and occasional mortality. Currently, there is no reliable test to differentiate ectopic from intrauterine gestation. We have previously used array technology to demonstrate that differences in gene expression in decidualized endometrium from women with ectopic and intrauterine gestations could be used to identify candidate diagnostic biomarkers for EP. The aim of this study was to further investigate the decidual gene with the highest fold increase in EP, cysteine-rich secretory protein-3 (CRISP-3). Decidualized endometrium from gestation-matched women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6) and surgery for EP (n = 11) was subjected to quantitative RT-PCR, morphological assessment, immunohistochemistry and western blot analysis. Sera were analysed for progesterone and human chorionic gonadotrophin (hCG) levels. Immortalized endometrial epithelial cells were cultured with physiological concentrations of hCG. CRISP-3 mRNA and protein expression were greater in endometrium from ectopic when compared with intrauterine pregnancies (P < 0.05). CRISP-3 protein was localized to epithelium and granulocytes of endometrium. CRISP-3 serum concentrations were not different in women with ectopic compared with intrauterine pregnancies. CRISP-3 expression in endometrium was not related to the degree of decidualization or to serum progesterone levels. Endometrial CRISP-3 expression was inversely proportional to serum hCG concentrations (P < 0.001). Stimulation of endometrial epithelial cells with hCG in vitro caused a reduction in CRISP-3 expression (P < 0.01). The measurement of CRISP-3 in endometrium could provide an additional tool in the diagnosis of failing early pregnancy of unknown location. The absence of a local reduction in expression of CRISP-3 in decidualized endometrium of women with EP may be due to reduced exposure to hCG due to the ectopic

  20. Combined Treatment with Troglitazone and Lovastatin Inhibited Epidermal Growth Factor-Induced Migration through the Downregulation of Cysteine-Rich Protein 61 in Human Anaplastic Thyroid Cancer Cells

    PubMed Central

    Chin, Li-Han; Hsu, Sung-Po

    2015-01-01

    Our previous studies have demonstrated that epidermal growth factor (EGF) can induce cell migration through the induction of cysteine-rich protein 61 (Cyr61) in human anaplastic thyroid cancer (ATC) cells. The aim of the present study was to determine the inhibitory effects of combined treatment with the peroxisome proliferator-activated receptor-γ (PPARγ) ligand troglitazone and the cholesterol-lowering drug lovastatin at clinically achievable concentrations on ATC cell migration. Combined treatment with 5 μM troglitazone and 1 μM lovastatin exhibited no cytotoxicity but significantly inhibited EGF-induced migration, as determined using wound healing and Boyden chamber assays. Cotreatment with troglitazone and lovastatin altered the epithelial-to-mesenchymal-transition (EMT) -related marker gene expression of the cells; specifically, E-cadherin expression increased and vimentin expression decreased. In addition, cotreatment reduced the number of filopodia, which are believed to be involved in migration, and significantly inhibited EGF-induced Cyr61 mRNA and protein expression as well as Cyr61 secretion. Moreover, the phosphorylation levels of 2 crucial signal molecules for EGF-induced Cyr61 expression, the cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK), were decreased in cells cotreated with troglitazone and lovastatin. Performing a transient transfection assay revealed that the combined treatment significantly suppressed Cyr61 promoter activity. These results suggest that combined treatment with low doses of troglitazone and lovastatin effectively inhibits ATC cell migration and may serve as a novel therapeutic strategy for metastatic ATC. PMID:25742642

  1. Combined treatment with troglitazone and lovastatin inhibited epidermal growth factor-induced migration through the downregulation of cysteine-rich protein 61 in human anaplastic thyroid cancer cells.

    PubMed

    Chin, Li-Han; Hsu, Sung-Po; Zhong, Wen-Bin; Liang, Yu-Chih

    2015-01-01

    Our previous studies have demonstrated that epidermal growth factor (EGF) can induce cell migration through the induction of cysteine-rich protein 61 (Cyr61) in human anaplastic thyroid cancer (ATC) cells. The aim of the present study was to determine the inhibitory effects of combined treatment with the peroxisome proliferator-activated receptor-γ (PPARγ) ligand troglitazone and the cholesterol-lowering drug lovastatin at clinically achievable concentrations on ATC cell migration. Combined treatment with 5 μM troglitazone and 1 μM lovastatin exhibited no cytotoxicity but significantly inhibited EGF-induced migration, as determined using wound healing and Boyden chamber assays. Cotreatment with troglitazone and lovastatin altered the epithelial-to-mesenchymal-transition (EMT) -related marker gene expression of the cells; specifically, E-cadherin expression increased and vimentin expression decreased. In addition, cotreatment reduced the number of filopodia, which are believed to be involved in migration, and significantly inhibited EGF-induced Cyr61 mRNA and protein expression as well as Cyr61 secretion. Moreover, the phosphorylation levels of 2 crucial signal molecules for EGF-induced Cyr61 expression, the cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK), were decreased in cells cotreated with troglitazone and lovastatin. Performing a transient transfection assay revealed that the combined treatment significantly suppressed Cyr61 promoter activity. These results suggest that combined treatment with low doses of troglitazone and lovastatin effectively inhibits ATC cell migration and may serve as a novel therapeutic strategy for metastatic ATC.

  2. A Cysteine-Rich CCG Domain Contains a Novel [4Fe-4S] Cluster Binding Motif As Deduced from Studies with Subunit B of Heterodisulfide Reductase from Methanothermobacter marburgensis†

    PubMed Central

    Hamann, Nils; Mander, Gerd J.; Shokes, Jacob E.; Scott, Robert A.; Bennati, Marina; Hedderich, Reiner

    2013-01-01

    Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]3+ cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX31–39CCX35–36CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusual [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron–sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (gzyx= 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. 57Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with 57Fe hyperfine couplings very similar to that of CoM-HDR. CoM-33SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S3(O/N)1 geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to the Zn site. PMID:17929940

  3. A Cysteine-Rich CCG Domain Contains a Novel [4Fe-4S] Cluster Binding Motif As Deduced From Studies With Subunit B of Heterodisulfide Reductase From Methanothermobacter Marburgensis

    SciTech Connect

    Hamann, N.; Mander, G.J.; Shokes, J.E.; Scott, R.A.; Bennati, M.; Hedderich, R.

    2009-06-01

    Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]{sup 3+} cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX{sub 31-39}CCX{sub 35-36}CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusual [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (g{sub zyx} = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. {sup 57}Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with {sup 57}Fe hyperfine couplings very similar to that of CoM-HDR. CoM-{sup 33}SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S{sub 3}(O/N){sub 1} geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to the Zn

  4. The SH3 and cysteine-rich domain 3 (Stac3) gene is important to growth, fiber composition, and calcium release from the sarcoplasmic reticulum in postnatal skeletal muscle.

    PubMed

    Cong, Xiaofei; Doering, Jonathan; Mazala, Davi A G; Chin, Eva R; Grange, Robert W; Jiang, Honglin

    2016-01-01

    The SH3 and cysteine-rich domain 3 (Stac3) gene is specifically expressed in the skeletal muscle. Stac3 knockout mice die perinatally. In this study, we determined the potential role of Stac3 in postnatal skeletal muscle growth, fiber composition, and contraction by generating conditional Stac3 knockout mice. We disrupted the Stac3 gene in 4-week-old male mice using the Flp-FRT and tamoxifen-inducible Cre-loxP systems. RT-qPCR and western blotting analyses of the limb muscles of target mice indicated that nearly all Stac3 mRNA and more than 70 % of STAC3 protein were deleted 4 weeks after tamoxifen injection. Postnatal Stac3 deletion inhibited body and limb muscle mass gains. Histological staining and gene expression analyses revealed that postnatal Stac3 deletion decreased the size of myofibers and increased the percentage of myofibers containing centralized nuclei, with no effect on the total myofiber number. Grip strength and grip time tests indicated that postnatal Stac3 deletion decreased limb muscle strength in mice. Muscle contractile tests revealed that postnatal Stac3 deletion reduced electrostimulation-induced but not the ryanodine receptor agonist caffeine-induced maximal force output in the limb muscles. Calcium imaging analysis of single flexor digitorum brevis myofibers indicated that postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced calcium release from the sarcoplasmic reticulum. This study demonstrates that STAC3 is important to myofiber hypertrophy, myofiber-type composition, contraction, and excitation-induced calcium release from the sarcoplasmic reticulum in the postnatal skeletal muscle.

  5. AB035. The expression of cysteine-rich secretory protein 2 (CRISP2) and its specific regulator mir-27b in the spermatozoa of patients with asthenozoospermia

    PubMed Central

    Zhou, Jun-Hao; Zhou, Qi-Zhao; Lyu, Xiao-Ming; Zhu, Ting; Chen, Zi-Jian; Chen, Ming-Kun; Xia, Hui; Wang, Chun-Yan; Qi, Tao; Li, Xin; Liu, Cun-Dong

    2016-01-01

    Background Cysteine-rich secretory protein 2 (CRISP2) is an important sperm protein and plays roles in spermatogenesis, modulation of flagellar motility, acrosome reaction, and gamete fusion. Clinical evidence shows a reduced CRISP2 expression in spermatozoa from asthenozoospermic patients, but the molecular mechanism underlying its reduction remains unknown. Herein, we carried out a study focusing on the CRISP2 reduction and its roles in asthenozoospermia Methods Spermatozoa were isolated from 90 study subjects’ ejaculated semen samples. DNA methylation was evaluated using bisulfite-sequencing PCR and methylation-specific PCR. The CRISP2 mRNA and protein expression levels were examined in the ejaculated spermatozoa by qRT-PCR and Western blot respectively. miRNA expression was detected by qRT-PCR. The direct regulatory effect of miR-27b on CRISP2 was predicted computationally and validated via luciferase reporter assay and in vitro experiments in which miR-27b mimic or inhibitor was transfected into 293T cells. Respective correlations of miR-27b and CRISP2 protein expression with clinical features were analyzed using Spearman’s correlation coefficient. Results Initially, low expression of CRISP2 protein rather than its mRNA was observed in the ejaculated spermatozoa from asthenozoospermic patients relative to normozoospermic males. Meanwhile, methylation was not found in CRISP2 promoter. These data suggest a possible post-transcriptional regulation of CRISP2 in asthenozoospermia. Subsequently, bioinformatics prediction, luciferase reporter assay and miR-27b transfection experiments revealed that miR-27b could specifically target CRISP2 by binding its 3’-UTR, suppressing CRISP2 expression post-transcriptionally. Further evidence was provided by the clinical observation of a high miR-27b expression in the ejaculated spermatozoa from asthenozoospermic patients and a negative correlation between miR-27b and CRISP2 protein expression. Finally, a retrospective

  6. The Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain of Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection.

    PubMed

    Ma, Hongfang; Jiang, Longguang; Qiao, Songlin; Zhi, Yubao; Chen, Xin-Xin; Yang, Yanyan; Huang, Xiaojing; Huang, Mingdong; Li, Rui; Zhang, Gai-Ping

    2017-02-01

    Porcine reproductive and respiratory syndrome (PRRS) has become an economically critical factor in swine industry since its worldwide spread in the 1990s. Infection by its causative agent, PRRS virus (PRRSV), was proven to be mediated by an indispensable receptor, porcine CD163 (pCD163), and the fifth scavenger receptor cysteine-rich domain (SRCR5) is essential for virus infection. However, the structural details and specific residues of pCD163 SRCR5 involved in infection have not been defined yet. In this study, we prepared recombinant pCD163 SRCR5 in Drosophila melanogaster Schneider 2 (S2) cells and determined its crystal structure at a high resolution of 2.0 Å. This structure includes a markedly long loop region and shows a special electrostatic potential, and these are significantly different from those of other members of the scavenger receptor cysteine-rich superfamily (SRCR-SF). Subsequently, we carried out structure-based mutational studies to identify that the arginine residue at position 561 (Arg561) in the long loop region is important for PRRSV infection. Further, we showed Arg561 probably takes effect on the binding of pCD163 to PRRSV during virus invasion. Altogether the current work provides the first view of the CD163 SRCR domain, expands our knowledge of the invasion mechanism of PRRSV, and supports a molecular basis for prevention and control of the virus. PRRS has caused huge economic losses to pig farming. The syndrome is caused by PRRSV, and PRRSV infection has been shown to be mediated by host cell surface receptors. One of them, pCD163, is especially indispensable, and its SRCR5 domain has been further demonstrated to play a significant role in virus infection. However, its structural details and the residues involved in infection are unknown. In this study, we determined the crystal structure of pCD163 SRCR5 and then carried out site-directed mutational studies based on the crystal structure to elucidate which residue is important. Our

  7. Levels of plasma immunoglobulin G with specificity against the cysteine-rich interdomain regions of a semiconserved Plasmodium falciparum erythrocyte membrane protein 1, VAR4, predict protection against malarial anemia and febrile episodes.

    PubMed

    Lusingu, John P A; Jensen, Anja T R; Vestergaard, Lasse S; Minja, Daniel T; Dalgaard, Michael B; Gesase, Samwel; Mmbando, Bruno P; Kitua, Andrew Y; Lemnge, Martha M; Cavanagh, David; Hviid, Lars; Theander, Thor G

    2006-05-01

    Antibodies to variant surface antigen have been implicated as mediators of malaria immunity in studies measuring immunoglobulin G (IgG) binding to infected erythrocytes. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important target for these antibodies, but no study has directly linked the presence of PfEMP1 antibodies in children to protection. We measured plasma IgG levels to the cysteine-rich interdomain region 1alpha (CIDR1alpha) of VAR4 (VAR4-CIDR1alpha), a member of a semiconserved PfEMP1 subfamily, by enzyme-linked immunosorbent assay in 561 Tanzanian individuals, who were monitored clinically for 7 months. The participants resided in Mkokola (a high-transmission village where malaria is holoendemic) or Kwamasimba (a moderate-transmission village). For comparison, plasma IgG levels to two merozoite surface protein 1 (MSP1) constructs, MSP1-19 and MSP1 block 2, and a control CIDR1 domain were measured. VAR4-CIDR1alpha antibodies were acquired at an earlier age in Mkokola than in Kwamasimba, but after the age of 10 years the levels were comparable in the two villages. After controlling for age and other covariates, the risk of having anemia at enrollment was reduced in VAR4-CIDR1alpha responders for Mkokola (adjusted odds ratio [AOR], 0.49; 95% confidence interval [CI], 0.29 to 0.88; P = 0.016) and Kwamasimba (AOR, 0.33; 95% CI, 0.16 to 0.68; P = 0.003) villages. The risk of developing malaria fever was reduced among individuals with a measurable VAR4-CIDR1alpha response from Mkokola village (AOR, 0.51; 95% CI, 0.29 to 0.89; P = 0.018) but not in Kwamasimba. Antibody levels to the MSP1 constructs and the control CIDR1alpha domain were not associated with morbidity protection. These data strengthen the concept of developing vaccines based on PfEMP1.

  8. The cysteine-rich domain of the secreted proprotein convertases PC5A and PACE4 functions as a cell surface anchor and interacts with tissue inhibitors of metalloproteinases.

    PubMed

    Nour, Nadia; Mayer, Gaétan; Mort, John S; Salvas, Alexandre; Mbikay, Majambu; Morrison, Charlotte J; Overall, Christopher M; Seidah, Nabil G

    2005-11-01

    The proprotein convertases PC5, PACE4 and furin contain a C-terminal cysteine-rich domain (CRD) of unknown function. We demonstrate that the CRD confers to PC5A and PACE4 properties to bind tissue inhibitors of metalloproteinases (TIMPs) and the cell surface. Confocal microscopy and biochemical analyses revealed that the CRD is essential for cell surface tethering of PC5A and PACE4 and that it colocalizes and coimmunoprecipitates with the full-length and C-terminal domain of TIMP-2. Surface-bound PC5A in TIMP-2 null fibroblasts was only observed upon coexpression with TIMP-2. In COS-1 cells, plasma membrane-associated PC5A can be displaced by heparin, suramin, or heparinases I and III and by competition with excess exogenous TIMP-2. Furthermore, PC5A and TIMP-2 are shown to be colocalized over the surface of enterocytes in the mouse duodenum and jejunum, as well as in liver sinusoids. In conclusion, the CRD of PC5A and PACE4 functions as a cell surface anchor favoring the processing of their cognate surface-anchored substrates, including endothelial lipase.

  9. Transcriptome Analysis Revealed Highly Expressed Genes Encoding Secondary Metabolite Pathways and Small Cysteine-Rich Proteins in the Sclerotium of Lignosus rhinocerotis

    PubMed Central

    Yap, Hui-Yeng Y.; Chooi, Yit-Heng; Fung, Shin-Yee; Ng, Szu-Ting; Tan, Chon-Seng; Tan, Nget-Hong

    2015-01-01

    Lignosus rhinocerotis (Cooke) Ryvarden (tiger milk mushroom) has long been known for its nutritional and medicinal benefits among the local communities in Southeast Asia. However, the molecular and genetic basis of its medicinal and nutraceutical properties at transcriptional level have not been investigated. In this study, the transcriptome of L. rhinocerotis sclerotium, the part with medicinal value, was analyzed using high-throughput Illumina HiSeqTM platform with good sequencing quality and alignment results. A total of 3,673, 117, and 59,649 events of alternative splicing, novel transcripts, and SNP variation were found to enrich its current genome database. A large number of transcripts were expressed and involved in the processing of gene information and carbohydrate metabolism. A few highly expressed genes encoding the cysteine-rich cerato-platanin, hydrophobins, and sugar-binding lectins were identified and their possible roles in L. rhinocerotis were discussed. Genes encoding enzymes involved in the biosynthesis of glucans, six gene clusters encoding four terpene synthases and one each of non-ribosomal peptide synthetase and polyketide synthase, and 109 transcribed cytochrome P450 sequences were also identified in the transcriptome. The data from this study forms a valuable foundation for future research in the exploitation of this mushroom in pharmacological and industrial applications. PMID:26606395

  10. Synthesis, screening, and sequencing of cysteine-rich one-bead one-compound peptide libraries.

    PubMed

    Juskowiak, Gary L; McGee, Christopher J; Greaves, John; Van Vranken, David L

    2008-01-01

    Cysteine-rich peptides are valued as tags for biarsenical fluorophores and as environmentally important reagents for binding toxic heavy metals. Due to the inherent difficulties created by cysteine, the power of one-bead one-compound (OBOC) libraries has never been applied to the discovery of short cysteine-rich peptides. We have developed the first method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries. First, we synthesized a heavily biased cysteine-rich OBOC library, incorporating 50% cysteine at each position (Ac-X8-KM-TentaGel). Then, we developed conditions for cysteine alkylation, cyanogen bromide cleavage, and direct MS/MS sequencing of that library at the single bead level. The sequencing efficiency of this library was comparable to a traditional cysteine-free library. To validate screening of cysteine-rich OBOC libraries, we reacted a library with the biarsenical FlAsH and identified beads bearing the known biarsenical-binding motif (CCXXCC). These results enable OBOC libraries to be used in high-throughput discovery of cysteine-rich peptides for protein tagging, environmental remediation of metal contaminants, or cysteine-rich pharmaceuticals.

  11. Multi-environment QTL studies suggest a role for cysteine-rich protein kinase genes in quantitative resistance to blackleg disease in Brassica napus.

    PubMed

    Larkan, Nicholas J; Raman, Harsh; Lydiate, Derek J; Robinson, Stephen J; Yu, Fengqun; Barbulescu, Denise M; Raman, Rosy; Luckett, David J; Burton, Wayne; Wratten, Neil; Salisbury, Philip A; Rimmer, S Roger; Borhan, M Hossein

    2016-08-24

    Resistance to the blackleg disease of Brassica napus (canola/oilseed rape), caused by the hemibiotrophic fungal pathogen Leptosphaeria maculans, is determined by both race-specific resistance (R) genes and quantitative resistance loci (QTL), or adult-plant resistance (APR). While the introgression of R genes into breeding material is relatively simple, QTL are often detected sporadically, making them harder to capture in breeding programs. For the effective deployment of APR in crop varieties, resistance QTL need to have a reliable influence on phenotype in multiple environments and be well defined genetically to enable marker-assisted selection (MAS). Doubled-haploid populations produced from the susceptible B. napus variety Topas and APR varieties AG-Castle and AV-Sapphire were analysed for resistance to blackleg in two locations over 3 and 4 years, respectively. Three stable QTL were detected in each population, with two loci appearing to be common to both APR varieties. Physical delineation of three QTL regions was sufficient to identify candidate defense-related genes, including a cluster of cysteine-rich receptor-like kinases contained within a 49 gene QTL interval on chromosome A01. Individual L. maculans isolates were used to define the physical intervals for the race-specific R genes Rlm3 and Rlm4 and to identify QTL common to both field studies and the cotyledon resistance response. Through multi-environment QTL analysis we have identified and delineated four significant and stable QTL suitable for MAS of quantitative blackleg resistance in B. napus, and identified candidate genes which potentially play a role in quantitative defense responses to L. maculans.

  12. Substitution of a conserved cysteine-996 in a cysteine-rich motif of the laminin {alpha}2-chain in congenital muscular dystrophy with partial deficiency of the protein

    SciTech Connect

    Nissinen, M.; Xu Zhang; Tryggvason, K.

    1996-06-01

    Congenital muscular dystrophies (CMDs) are autosomal recessive muscle disorders of early onset. Approximately half of CMD patients present laminin {alpha}2-chain (merosin) deficiency in muscle biopsies, and the disease locus has been mapped to the region of the LAMA2 gene (6q22-23) in several families. Recently, two nonsense mutations in the laminin {alpha}2-chain gene were identified in CMD patients exhibiting complete deficiency of the laminin {alpha}2-chain in muscle biopsies. However, a subset of CMD patients with linkage to LAMA2 show only partial absence of the laminin {alpha}2-chain around muscle fibers, by immunocytochemical analysis. In the present study we have identified a homozygous missense mutation in the {alpha}2-chain gene of a consanguineous Turkish family with partial laminin {alpha}2-chain deficiency. The T{r_arrow}C transition at position 3035 in the cDNA sequence results in a Cys996{r_arrow}Arg substitution. The mutation that affects one of the conserved cysteine-rich repeats in the short arm of the laminin {alpha}2-chain should result in normal synthesis of the chain and in formation and secretion of a heterotrimeric laminin molecule. Muscular dysfunction is possibly caused either by abnormal disulfide cross-links and folding of the laminin repeat, leading to the disturbance of an as yet unknown binding function of the laminin {alpha}2-chain and to shorter half-life of the muscle-specific laminin-2 and laminin-4 isoforms, or by increased proteolytic sensitivity, leading to truncation of the short arm. 42 refs., 7 figs.

  13. Cysteine-Rich Peptide Family with Unusual Disulfide Connectivity from Jasminum sambac.

    PubMed

    Kumari, Geeta; Serra, Aida; Shin, Joon; Nguyen, Phuong Q T; Sze, Siu Kwan; Yoon, Ho Sup; Tam, James P

    2015-11-25

    Cysteine-rich peptides (CRPs) are natural products with privileged peptidyl structures that represent a potentially rich source of bioactive compounds. Here, the discovery and characterization of a novel plant CRP family, jasmintides from Jasminum sambac of the Oleaceae family, are described. Two 27-amino acid jasmintides (jS1 and jS2) were identified at the gene and protein levels. Disulfide bond mapping of jS1 by mass spectrometry and its confirmation by NMR spectroscopy revealed disulfide bond connectivity of C-1-C-5, C-2-C-4, and C-3-C-6, a cystine motif that has not been reported in plant CRPs. Structural determination showed that jS1 displays a well-defined structure framed by three short antiparallel β-sheets. Genomic analysis showed that jasmintides share a three-domain precursor arrangement with a C-terminal mature domain preceded by a long pro-domain of 46 residues and an intron cleavage site between the signal sequence and pro-domain. The compact cysteine-rich structure together with an N-terminal pyroglutamic acid residue confers jasmintides high resistance to heat and enzymatic degradation, including exopeptidase treatment. Collectively, these results reveal a new plant CRP structure with an unusual cystine connectivity, which could be useful as a scaffold for designing peptide drugs.

  14. Replacement of Porcine CD163 Scavenger Receptor Cysteine-Rich Domain 5 with a CD163-Like Homolog Confers Resistance of Pigs to Genotype 1 but Not Genotype 2 Porcine Reproductive and Respiratory Syndrome Virus.

    PubMed

    Wells, Kevin D; Bardot, Rachel; Whitworth, Kristin M; Trible, Benjamin R; Fang, Ying; Mileham, Alan; Kerrigan, Maureen A; Samuel, Melissa S; Prather, Randall S; Rowland, Raymond R R

    2017-01-15

    CD163 knockout (KO) pigs are resistant to infection with genotype 2 (type 2) porcine reproductive and respiratory syndrome virus (PRRSV). Furthermore, the substitution of CD163 scavenger receptor cysteine-rich (SRCR) domain 5 with a homolog of human CD163-like (hCD163L1) SRCR 8 domain confers resistance of transfected HEK cells to type 1 PRRSV. As a means to understand the role of domain 5 in PRRSV infection with both type 1 and type 2 viruses, pigs were genetically modified (GM) to possess one of the following genotypes: complete knockout (KO) of CD163, deletions within SRCR domain 5, or replacement (domain swap) of SRCR domain 5 with a synthesized exon encoding a homolog of hCD163L1 SRCR domain 8. Immunophenotyping of porcine alveolar macrophages (PAMs) showed that pigs with the KO or SRCR domain 5 deletion did not express CD163. When placed in culture, PAMs from pigs with the CD163 KO phenotype were completely resistant to a panel consisting of six type 1 and nine type 2 isolates. PAMs from pigs that possessed the hCD163L1 domain 8 homolog expressed CD163 and supported the replication of all type 2 isolates, but no type 1 viruses. Infection of CD163-modified pigs with representative type 1 and type 2 viruses confirmed the in vitro results. The results confirm that CD163 is the likely receptor for all PRRS viruses. Even though type 1 and type 2 viruses are considered phenotypically similar at several levels, there is a distinct difference between the viral genotypes in the recognition of CD163. Genetic modification of the CD163 gene creates the opportunity to develop production animals that are resistant to PRRS, the costliest viral disease to ever face the swine industry. The results create further opportunities to develop refinements in the modification of CD163 with the goal of making pigs refractory to infection while retaining important CD163 functions. Copyright © 2017 American Society for Microbiology.

  15. Replacement of Porcine CD163 Scavenger Receptor Cysteine-Rich Domain 5 with a CD163-Like Homolog Confers Resistance of Pigs to Genotype 1 but Not Genotype 2 Porcine Reproductive and Respiratory Syndrome Virus

    PubMed Central

    Wells, Kevin D.; Bardot, Rachel; Whitworth, Kristin M.; Trible, Benjamin R.; Fang, Ying; Mileham, Alan; Kerrigan, Maureen A.; Samuel, Melissa S.; Prather, Randall S.

    2016-01-01

    ABSTRACT CD163 knockout (KO) pigs are resistant to infection with genotype 2 (type 2) porcine reproductive and respiratory syndrome virus (PRRSV). Furthermore, the substitution of CD163 scavenger receptor cysteine-rich (SRCR) domain 5 with a homolog of human CD163-like (hCD163L1) SRCR 8 domain confers resistance of transfected HEK cells to type 1 PRRSV. As a means to understand the role of domain 5 in PRRSV infection with both type 1 and type 2 viruses, pigs were genetically modified (GM) to possess one of the following genotypes: complete knockout (KO) of CD163, deletions within SRCR domain 5, or replacement (domain swap) of SRCR domain 5 with a synthesized exon encoding a homolog of hCD163L1 SRCR domain 8. Immunophenotyping of porcine alveolar macrophages (PAMs) showed that pigs with the KO or SRCR domain 5 deletion did not express CD163. When placed in culture, PAMs from pigs with the CD163 KO phenotype were completely resistant to a panel consisting of six type 1 and nine type 2 isolates. PAMs from pigs that possessed the hCD163L1 domain 8 homolog expressed CD163 and supported the replication of all type 2 isolates, but no type 1 viruses. Infection of CD163-modified pigs with representative type 1 and type 2 viruses confirmed the in vitro results. The results confirm that CD163 is the likely receptor for all PRRS viruses. Even though type 1 and type 2 viruses are considered phenotypically similar at several levels, there is a distinct difference between the viral genotypes in the recognition of CD163. IMPORTANCE Genetic modification of the CD163 gene creates the opportunity to develop production animals that are resistant to PRRS, the costliest viral disease to ever face the swine industry. The results create further opportunities to develop refinements in the modification of CD163 with the goal of making pigs refractory to infection while retaining important CD163 functions. PMID:27847356

  16. Introgression of leginsulin, a cysteine-rich protein, and high-protein trait from an Asian soybean plant introduction genotype into a North American experimental soybean line

    USDA-ARS?s Scientific Manuscript database

    Soybean is an important protein source for both humans and animals. However, soybean proteins are relatively poor in the sulfur-containing amino acids, cysteine and methionine. Improving the content of endogenous proteins rich in sulfur containing amino acids could enhance the nutritive value of soy...

  17. In-silico prediction and modeling of the Entamoeba histolytica proteins: Serine-rich Entamoeba histolytica protein and 29 kDa Cysteine-rich protease.

    PubMed

    Manochitra, Kumar; Parija, Subhash Chandra

    2017-01-01

    Amoebiasis is the third most common parasitic cause of morbidity and mortality, particularly in countries with poor hygienic settings. There exists an ambiguity in the diagnosis of amoebiasis, and hence there arises a necessity for a better diagnostic approach. Serine-rich Entamoeba histolyticaprotein (SREHP), peroxiredoxin and Gal/GalNAc lectin are pivotal in E. histolyticavirulence and are extensively studied as diagnostic and vaccine targets. For elucidating the cellular function of these proteins, details regarding their respective quaternary structures are essential. However, studies in this aspect are scant. Hence, this study was carried out to predict the structure of these target proteins and characterize them structurally as well as functionally using appropriate in-silicomethods. The amino acid sequences of the proteins were retrieved from National Centre for Biotechnology Information database and aligned using ClustalW. Bioinformatic tools were employed in the secondary structure and tertiary structure prediction. The predicted structure was validated, and final refinement was carried out. The protein structures predicted by i-TASSER were found to be more accurate than Phyre2 based on the validation using SAVES server. The prediction suggests SREHP to be an extracellular protein, peroxiredoxin a peripheral membrane protein while Gal/GalNAc lectin was found to be a cell-wall protein. Signal peptides were found in the amino-acid sequences of SREHP and Gal/GalNAc lectin, whereas they were not present in the peroxiredoxin sequence. Gal/GalNAc lectin showed better antigenicity than the other two proteins studied. All the three proteins exhibited similarity in their structures and were mostly composed of loops. The structures of SREHP and peroxiredoxin were predicted successfully, while the structure of Gal/GalNAc lectin could not be predicted as it was a complex protein composed of sub-units. Also, this protein showed less similarity with the available

  18. Human immunodeficiency virus type 1 (HIV-1) transactivator of transcription through its intact core and cysteine-rich domains inhibits Wnt/β-catenin signaling in astrocytes: relevance to HIV neuropathogenesis.

    PubMed

    Henderson, Lisa J; Sharma, Amit; Monaco, Maria Chiara G; Major, Eugene O; Al-Harthi, Lena

    2012-11-14

    Wnt/β-catenin is a neuroprotective pathway regulating cell fate commitment in the CNS and many vital functions of neurons and glia. Its dysregulation is linked to a number of neurodegenerative diseases. Wnt/β-catenin is also a repressor of HIV transcription in multiple cell types, including astrocytes, which are dysregulated in HIV-associated neurocognitive disorder. Given that HIV proteins can overcome host restriction factors and that perturbations of Wnt/β-catenin signaling can compromise astrocyte function, we evaluated the impact of HIV transactivator of transcription (Tat) on Wnt/β-catenin signaling in astrocytes. HIV clade B Tat, in primary progenitor-derived astrocytes and U87MG cells, inhibited Wnt/β-catenin signaling as demonstrated by its inhibition of active β-catenin, TOPflash reporter activity, and Axin-2 (a downstream target of Wnt/β-catenin signaling). Point mutations in either the core region (K41A) or the cysteine-rich region (C30G) of Tat abrogated its ability to inhibit β-catenin signaling. Clade C Tat, which lacks the dicysteine motif, did not alter β-catenin signaling, confirming that the dicysteine motif is critical for Tat inhibition of β-catenin signaling. Tat coprecipitated with TCF-4 (a transcription factor that partners with β-catenin), suggesting a physical interaction between these two proteins. Furthermore, knockdown of β-catenin or TCF-4 enhanced docking of Tat at the TAR region of the HIV long terminal repeat. These findings highlight a bidirectional interference between Tat and Wnt/β-catenin that negatively impacts their cognate target genes. The consequences of this interaction include alleviation of Wnt/β-catenin-mediated suppression of HIV and possible astrocyte dysregulation contributing to HIV neuropathogenesis.

  19. A Cysteine-Rich Protein Kinase Associates with a Membrane Immune Complex and the Cysteine Residues Are Required for Cell Death1[OPEN

    PubMed Central

    Elmore, James M.; Creer, Athena Y.; Feng, Baomin; Franco, Jessica Y.; He, Ping; Phinney, Brett

    2017-01-01

    Membrane-localized proteins perceive and respond to biotic and abiotic stresses. We performed quantitative proteomics on plasma membrane-enriched samples from Arabidopsis (Arabidopsis thaliana) treated with bacterial flagellin. We identified multiple receptor-like protein kinases changing in abundance, including cysteine (Cys)-rich receptor-like kinases (CRKs) that are up-regulated upon the perception of flagellin. CRKs possess extracellular Cys-rich domains and constitute a gene family consisting of 46 members in Arabidopsis. The single transfer DNA insertion lines CRK28 and CRK29, two CRKs induced in response to flagellin perception, did not exhibit robust alterations in immune responses. In contrast, silencing of multiple bacterial flagellin-induced CRKs resulted in enhanced susceptibility to pathogenic Pseudomonas syringae, indicating functional redundancy in this large gene family. Enhanced expression of CRK28 in Arabidopsis increased disease resistance to P. syringae. Expression of CRK28 in Nicotiana benthamiana induced cell death, which required intact extracellular Cys residues and a conserved kinase active site. CRK28-mediated cell death required the common receptor-like protein kinase coreceptor BAK1. CRK28 associated with BAK1 as well as the activated FLAGELLIN-SENSING2 (FLS2) immune receptor complex. CRK28 self-associated as well as associated with the closely related CRK29. These data support a model where Arabidopsis CRKs are synthesized upon pathogen perception, associate with the FLS2 complex, and coordinately act to enhance plant immune responses. PMID:27852951

  20. The Ectocarpus IMMEDIATE UPRIGHT gene encodes a member of a novel family of cysteine-rich proteins with an unusual distribution across the eukaryotes.

    PubMed

    Macaisne, Nicolas; Liu, Fuli; Scornet, Delphine; Peters, Akira F; Lipinska, Agnieszka; Perrineau, Marie-Mathilde; Henry, Antoine; Strittmatter, Martina; Coelho, Susana M; Cock, J Mark

    2017-02-01

    The sporophyte generation of the brown alga Ectocarpus sp. exhibits an unusual pattern of development compared with the majority of brown algae. The first cell division is symmetrical and the apical-basal axis is established late in development. In the immediate upright (imm) mutant, the initial cell undergoes an asymmetric division to immediately establish the apical-basal axis. We provide evidence which suggests that this phenotype corresponds to the ancestral state of the sporophyte. The IMM gene encodes a protein of unknown function that contains a repeated motif also found in the EsV-1-7 gene of the Ectocarpus virus EsV-1. Brown algae possess large families of EsV-1-7 domain genes but these genes are rare in other stramenopiles, suggesting that the expansion of this family might have been linked with the emergence of multicellular complexity. EsV-1-7 domain genes have a patchy distribution across eukaryotic supergroups and occur in several viral genomes, suggesting possible horizontal transfer during eukaryote evolution. © 2017. Published by The Company of Biologists Ltd.

  1. The human analog of murine cystein rich protein 61 [correction of 16] is a 1alpha,25-dihydroxyvitamin D3 responsive immediate early gene in human fetal osteoblasts: regulation by cytokines, growth factors, and serum.

    PubMed

    Schütze, N; Lechner, A; Groll, C; Siggelkow, H; Hüfner, M; Köhrle, J; Jakob, F

    1998-04-01

    1Alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) is a potent mediator of differentiation and maintenance of specific functions of osteoblasts. To detect novel targets for 1,25-(OH)2D3 action, we applied differential display PCR to human fetal osteoblast-like cells and identified the human analog of murine cystein rich protein 61 (hCYR61) as a 1,25-(OH)2D3-responsive immediate early gene in differentiated fetal osteoblast-like cells. The murine gene CYR61 is important for cell-cell and cell-matrix interactions, and it belongs to an emerging gene family of cysteine-rich proteins. hCYR61 messenger RNA (mRNA) steady-state levels were stimulated 11-fold by 10 nM 1,25-(OH)2D3 by 1 h and declined to control levels by 4 h. This transient stimulation of hCYR61 mRNA was not inhibited by cycloheximide but was prevented by actinomycin D, indicating that the 1,25-(OH)2D3 effect involves transcriptional events and does not require de novo protein synthesis. hCYR61 mRNA stability was not influenced by 1,25(OH)2D3, whereas cycloheximide treatment stabilized hCYR61 mRNA. FCS, as well as growth factors and cytokines such as basic fibroblast growth factor, epidermal growth factor, tumor necrosis factor alpha, and interleukin-1, strongly elevated hCYR61 mRNA steady-state levels within 1 h. hCYR61 mRNA was expressed also in primary human osteoblasts and osteosarcoma cell lines. Using a commercial tissue blot, hCYR61 mRNA was only observed in skeletal muscle. The fast and transient response of hCYR 61 to 1,25-(OH)2D3, serum, growth factors, and cytokines suggests an important role of hCYR61 for osteoblast function and differentiation.

  2. Autoantigen 1 of the guinea pig sperm acrosome is the homologue of mouse Tpx-1 and human TPX1 and is a member of the cysteine-rich secretory protein (CRISP) family.

    PubMed

    Foster, J A; Gerton, G L

    1996-06-01

    We have cloned and sequenced cDNAs encoding autoantigen 1 (AA1), a testis-specific protein and the major autoantigen of the guinea pig sperm acrosome. The cDNA predicts a precursor protein of 244 amino acids including a 21 amino acid hydrophobic, secretory signal sequence. The mature polypeptide is predicted to have a molecular mass of 24,891 Daltons which agrees with the experimentally determined molecular weight of 25,000. Consistent with previous studies demonstrating that AA1 is not a glycoprotein, the predicted amino acid sequence contained no canonical sites for N-linked glycosylation. Comparison with other sequences showed that AA1 is the guinea pig homologue of the testis-specific protein Tpx-1 in mice and TPX1 in humans. AA1 also showed significant amino acid sequence homology with other cysteine-rich secretory proteins (CRISP's): rat and mouse acidic epididymal glycoproteins (AEG; also known as proteins D/E in rats) and helothermine, a toxin from the Mexican beaded lizard. In addition, AA1 had a lesser degree of homology with antigen 5 (vespid wasp venom), PR-1 (a plant pathogenesis related protein), and GliPR (a protein identified in human gliomas). Northern analysis of RNA from purified guinea pig spermatogenic cells showed that a 1.5 kb message was first detected in pachytene spermatocytes, was strongest in round spermatids, and was detected at a low level in condensing spermatids. Immunoblot analysis and metabolic labeling data of AA1 in spermatogenic cells showed that the protein was synthesized as early as the pachytene spermatocyte stage of spermatogenesis. Thus, the patterns of AA1 mRNA and protein expression during spermatogenesis are similar to the expression of other acrosomal mRNAs and proteins that are first detected meiotically.

  3. Crystallization and preliminary X-ray diffraction analyses of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels

    SciTech Connect

    Suzuki, Nobuhiro; Yamazaki, Yasuo; Fujimoto, Zui; Morita, Takashi; Mizuno, Hiroshi

    2005-08-01

    Crystals of pseudechetoxin and pseudecin, potent peptidic inhibitors of cyclic nucleotide-gated ion channels, have been prepared and X-ray diffraction data have been collected to 2.25 and 1.90 Å resolution, respectively. Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction of retinal and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins are structurally classified as cysteine-rich secretory proteins and exhibit structural features that are quite distinct from those of other known small peptidic channel blockers. This article describes the crystallization and preliminary X-ray diffraction analyses of these toxins. Crystals of PsTx belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 60.30, b = 61.59, c = 251.69 Å, and diffraction data were collected to 2.25 Å resolution. Crystals of Pdc also belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with similar unit-cell parameters a = 60.71, b = 61.67, c = 251.22 Å, and diffraction data were collected to 1.90 Å resolution.

  4. Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato

    PubMed Central

    Shcherbakova, Larisa A.; Odintsova, Tatyana I.; Stakheev, Alexander A.; Fravel, Deborah R.; Zavriev, Sergey K.

    2016-01-01

    The biocontrol effect of the non-pathogenic Fusarium oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL) has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP), which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues) and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed. PMID:26779237

  5. Higher sequence coverage and improved confidence in the identification of cysteine-rich proteins from the wool cuticle using combined chemical and enzymatic digestion.

    PubMed

    Koehn, Henning; Clerens, Stefan; Deb-Choudhury, Santanu; Morton, James D; Dyer, Jolon M; Plowman, Jeffrey E

    2009-12-01

    Keratin-associated proteins (KAPs) are important constituents of the wool cuticle, comprised of the endo-, exocuticle and a-layers, which contribute significantly to the fibre's molecular and mechanical characteristics. Relatively little is known about the distribution of specific KAPs across these layers, and correct protein identification of individual KAPs is difficult due to extensive homology and identity among individual KAPs. We here present evidence that, by specifically exploiting the high-cysteine content of KAPs in the wool cuticle, using 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage in combination with tryptic digestion, a larger number of KAPs can be identified than with standard trypsin-only digests. A total of 27 KAPs were identified, six of which could only be identified using NTCB. Furthermore, NTCB-mediated cleavage of cuticle proteins generated unique peptides critical for unambiguous identification of two KAPs, as well as significantly increasing the overall sequence coverage of most identified KAPs. Interestingly, some of the peptides found to be unique to particular KAPs could only be found in either the exo- or endocuticle. We conclude that for the analysis of high sulphur proteomes, specific targeting of cysteine residues using chemical agents such as NTCB can provide critical information for unambiguous protein identification.

  6. Cysteine-rich protein 61 (CCN1) and connective tissue growth factor (CCN2) at the crosshairs of ocular neovascular and fibrovascular disease therapy.

    PubMed

    Yan, Lulu; Chaqour, Brahim

    2013-12-01

    The vasculature forms a highly branched network investing every organ of vertebrate organisms. The retinal circulation, in particular, is supported by a central retinal artery branching into superficial arteries, which dive into the retina to form a dense network of capillaries in the deeper retinal layers. The function of the retina is highly dependent on the integrity and proper functioning of its vascular network and numerous ocular diseases including diabetic retinopathy, age-related macular degeneration and retinopathy of prematurity are caused by vascular abnormalities culminating in total and sometimes irreversible loss of vision. CCN1 and CCN2 are inducible extracellular matrix (ECM) proteins which play a major role in normal and aberrant formation of blood vessels as their expression is associated with developmental and pathological angiogenesis. Both CCN1 and CCN2 achieve disparate cell-type and context-dependent activities through modulation of the angiogenic and synthetic phenotype of vascular and mesenchymal cells respectively. At the molecular level, CCN1 and CCN2 may control capillary growth and vascular cell differentiation by altering the composition or function of the constitutive ECM proteins, potentiating or interfering with the activity of various ligands and/or their receptors, physically interfering with the ECM-cell surface interconnections, and/or reprogramming gene expression driving cells toward new phenotypes. As such, these proteins emerged as important prognostic markers and potential therapeutic targets in neovascular and fibrovascular diseases of the eye. The purpose of this review is to highlight our current knowledge and understanding of the most recent data linking CCN1 and CCN2 signaling to ocular neovascularization bolstering the potential value of targeting these proteins in a therapeutic context.

  7. Changes in rubisco, cysteine-rich proteins and antioxidant system of spinach (Spinacia oleracea L.) due to sulphur deficiency, cadmium stress and their combination.

    PubMed

    Bagheri, Rita; Ahmad, Javed; Bashir, Humayra; Iqbal, Muhammad; Qureshi, M Irfan

    2017-03-01

    Sulphur (S) deficiency, cadmium (Cd) toxicity and their combinations are of wide occurrence throughout agricultural lands. We assessed the impact of short-term (2 days) and long-term (4 days) applications of cadmium (40 μg/g soil) on spinach plants grown on sulphur-sufficient (300 μM SO4(2-)) and sulphur-deficient (30 μM SO4(2-)) soils. Compared with the control (+S and -Cd), oxidative stress was increased by S deficiency (-S and -Cd), cadmium (+S and +Cd) and their combination stress (-S and +Cd) in the order of (S deficiency) < (Cd stress) < (S deficiency and +Cd stress). SDS-PAGE profile of leaf proteins showed a high vulnerability of rubisco large subunit (RbcL) to S deficiency. Rubisco small subunit (RbcS) was particularly sensitive to Cd as well as dual stress (+Cd and -S) but increased with Cd in the presence of S. Cysteine content in low molecular weight proteins/peptide was also affected, showing a significant increase under cadmium treatment. Components of ascorbate-glutathione antioxidant system altered their levels, showing the maximum decline in ascorbate (ASA), dehydroascorbate (DHA), total ascorbate (ASA + DHA, hereafter TA), glutathione (GSH) and total glutathione (GSH + GSSG, hereafter TG) under S deficiency. However, total ascorbate and total glutathione increased, besides a marginal increase in their reduced and oxidized forms, when Cd was applied in the presence of sufficient S. Sulphur supply also helped in increasing the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT) under Cd stress. However, their activity suffered by S deficiency and by Cd stress during S deficiency. Each stress declined the contents of soluble protein and photosynthetic pigments; the highest decline in contents of protein and pigments occurred under S deficiency and dual stress respectively. The fresh and dry weights, although affected adversely by every stress, declined most under dual stress. It

  8. microRNA-200b and microRNA-200c promote colorectal cancer cell proliferation via targeting the reversion-inducing cysteine-rich protein with Kazal motifs.

    PubMed

    Pan, Yi; Liang, Hongwei; Chen, Weixu; Zhang, Hongjie; Wang, Nan; Wang, Feng; Zhang, Suyang; Liu, Yanqing; Zhao, Chihao; Yan, Xin; Zhang, Junfeng; Zhang, Chen-Yu; Gu, Hongwei; Zen, Ke; Chen, Xi

    2015-01-01

    MicroRNA-200b and microRNA-200c (miR-200b/c) are 2 of the most frequently upregulated oncomiRs in colorectal cancer cells. The role of miR-200b/c during colorectal tumorigenesis, however, remains unclear. In the present study, we report that miR-200b/c can promote colorectal cancer cell proliferation via targeting the reversion-inducing cysteine-rich protein with Kazal motifs (RECK). Firstly, bioinformatics analysis predicted RECK as a conserved target of miR-200b/c. By overexpressing or knocking down miR-200b/c in colorectal cancer cells, we experimentally validated that miR-200b/c are direct regulators of RECK. Secondly, an inverse correlation between the levels of miR-200b/c and RECK protein was found in human colorectal cancer tissues and cell lines. Thirdly, we demonstrated that repression of RECK by miR-200b/c consequently triggered SKP2 (S-phase kinase-associated protein 2) elevation and p27(Kip1) (also known as cyclin-dependent kinase inhibitor 1B) degradation in colorectal cancer cells, which eventually promotes cancer cell proliferation. Finally, promoting tumor cell growth by miR-200b/c-targeting RECK was also observed in the xenograft mouse model. Taken together, our results demonstrate that miR-200b/c play a critical role in promoting colorectal tumorigenesis through inhibiting RECK expression and subsequently triggering SKP2 elevation and p27(Kip1) degradation.

  9. The 5' UTR and 3' UTR of the sperm mitochondria-associated cysteine-rich protein mRNA regulate translation in spermatids by multiple mechanisms in transgenic mice.

    PubMed

    Hawthorne, Sabrina K; Busanelli, Rakhee R; Kleene, Kenneth C

    2006-09-01

    The Smcp mRNA encoding the sperm mitochondria-associated cysteine-rich protein is translationally repressed in round spermatids and translationally active in elongated spermatids. The patterns of transcription and translation of fusions of the Smcp promoter, the green fluorescent protein coding region (Gfp) and various combination of the Smcp and Gfp 5' UTR and 3' UTR have been studied in transgenic mice. 518 nt of Smcp 5' flanking region and 8 nt of 5' UTR drive transcription of mRNAs containing the Gfp coding region in early round spermatids at the same transcription start site as the natural Smcp gene. Transcripts containing both the Gfp 5' and 3' UTRs are translationally active in step 2 spermatids as detected by GFP fluorescence in squashes of living seminiferous tubules from adult testes, and the presence of polysomal mRNAs in sucrose gradient analyses of testes from 21-day-old prepubertal mice, which contain early round spermatids and lack elongated spermatids. By comparison, expression of GFP is delayed until steps 5 and 10, respectively, in transcripts containing the Smcp 5' UTR and Gfp 3' UTR and the Gfp 5' UTR and the Smcp 3' UTR. Sucrose gradient analysis of 21-day-old testes demonstrates that transcripts containing the Smcp 3' UTR exhibit a bimodal distribution in free-mRNPs and polysomes, indicating that the 3' UTR blocks the expression of GFP after the transcripts have entered the elongation phase, a mechanism that may involve microRNAs. The Smcp 5' UTR reduces the levels and size of polysomes in adult testis. In addition, the natural Smcp mRNA contains a positive control element that counteracts the inhibition of translation by the Smcp 5' UTR in adult testis, and the Smcp 3' UTR strongly localizes GFP fluorescence in step 10 spermatids.

  10. A multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like Arabidopsis lipid transfer proteins (LTPs) suggests diversified roles for these LTPs in plant growth and reproduction.

    PubMed

    Chae, Keun; Gonong, Benedict J; Kim, Seung-Chul; Kieslich, Chris A; Morikis, Dimitrios; Balasubramanian, Shruthi; Lord, Elizabeth M

    2010-10-01

    Lily stigma/style cysteine-rich adhesin (SCA), a plant lipid transfer protein (LTP) which is secreted into the extracellular matrix, functions in pollen tube guidance in fertilization. A gain-of-function mutant (ltp5-1) for Arabidopsis LTP5, an SCA-like molecule, was recently shown to display defects in sexual reproduction. In the current study, it is reported that ltp5-1 plants have dwarfed primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and display multibranching. These mutant phenotypes in vegetative growth are recessive. No abnormality was found in ltp5-1/+ plants. In a phylogenetic analysis of plant LTPs, SCA-like Arabidopsis LTPs were classified with conventional plant LTPs. Homology modelling-based electrostatic similarity index (ESI) clustering was used to show diversity in spatial distributions of electrostatic potentials of SCA-like LTPs, suggestive of their various roles in interaction in the extracellular matrix space. β-Glucuronidase (GUS) analysis showed that SCA-like Arabidopsis LTP genes are diversely present in various tissues. LTP4 was found specifically in the guard cells and LTP6 in trichomes as well as in other tissues. LTP1 levels were specifically abundant in the stigma, and both LTP3 and LTP6 in the ovules. LTP2 and LTP4 gene levels were up-regulated in whole seedlings with 20% polyethylene glycol (PEG) and 300 mM NaCl treatments, respectively. LTP5 was up-regulated in the hypocotyl with 3 d dark growth conditions. LTP6 was specifically expressed in the tip of the cotyledon under drought stress conditions. The results suggest that SCA-like Arabidopsis LTPs are multifunctional, with diversified roles in plant growth and reproduction.

  11. A multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like Arabidopsis lipid transfer proteins (LTPs) suggests diversified roles for these LTPs in plant growth and reproduction

    PubMed Central

    Chae, Keun; Gonong, Benedict J.; Kim, Seung-Chul; Kieslich, Chris A.; Morikis, Dimitrios; Balasubramanian, Shruthi; Lord, Elizabeth M.

    2010-01-01

    Lily stigma/style cysteine-rich adhesin (SCA), a plant lipid transfer protein (LTP) which is secreted into the extracellular matrix, functions in pollen tube guidance in fertilization. A gain-of-function mutant (ltp5-1) for Arabidopsis LTP5, an SCA-like molecule, was recently shown to display defects in sexual reproduction. In the current study, it is reported that ltp5-1 plants have dwarfed primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and display multibranching. These mutant phenotypes in vegetative growth are recessive. No abnormality was found in ltp5-1/+ plants. In a phylogenetic analysis of plant LTPs, SCA-like Arabidopsis LTPs were classified with conventional plant LTPs. Homology modelling-based electrostatic similarity index (ESI) clustering was used to show diversity in spatial distributions of electrostatic potentials of SCA-like LTPs, suggestive of their various roles in interaction in the extracellular matrix space. β-Glucuronidase (GUS) analysis showed that SCA-like Arabidopsis LTP genes are diversely present in various tissues. LTP4 was found specifically in the guard cells and LTP6 in trichomes as well as in other tissues. LTP1 levels were specifically abundant in the stigma, and both LTP3 and LTP6 in the ovules. LTP2 and LTP4 gene levels were up-regulated in whole seedlings with 20% polyethylene glycol (PEG) and 300 mM NaCl treatments, respectively. LTP5 was up-regulated in the hypocotyl with 3 d dark growth conditions. LTP6 was specifically expressed in the tip of the cotyledon under drought stress conditions. The results suggest that SCA-like Arabidopsis LTPs are multifunctional, with diversified roles in plant growth and reproduction. PMID:20667964

  12. AB087. The expression of cysteine-rich secretory protein 2 (CRISP2) and its specific regulator miR-27b in the spermatozoa of patients with asthenozoospermia

    PubMed Central

    Zhou, Junhao; Zhou, Qizhao; Lyu, Xiaoming; Zhu, Ting; Chen, Zijian; Chen, Mingkun; Xia, Hui; Wang, Chunyan; Qi, Tao; Li, Xin; Liu, Cundong

    2016-01-01

    Objective Cysteine-rich secretory protein 2 (CRISP2) is an important sperm protein and plays roles in spermatogenesis, modulation of flagellar motility, acrosome reaction, and gamete fusion. Clinical evidence shows a reduced CRISP2 expression in spermatozoa from asthenozoospermic patients, but the molecular mechanism underlying its reduction remains unknown. Herein, we carried out a study focusing on the CRISP2 reduction and its roles in asthenozoospermia. Methods Spermatozoa were isolated from 90 study subjects’ ejaculated semen samples. DNA methylation was evaluated using bisulfite-sequencing PCR and methylation-specific PCR. The CRISP2 mRNA and protein expression levels were examined in the ejaculated spermatozoa by qRT-PCR and Western blot respectively. MiRNA expression was detected by qRT-PCR. The direct regulatory effect of miR-27b on CRISP2 was predicted computationally and validated via luciferase reporter assay and in vitro experiments in which miR-27b mimic or inhibitor was transfected into 293T cells. Respective correlations of miR-27b and CRISP2 protein expression with clinical features were analyzed using Spearman’s correlation coefficient. Results Initially, low expression of CRISP2 protein rather than its mRNA was observed in the ejaculated spermatozoa from asthenozoospermic patients relative to normozoospermic males. Meanwhile, methylation was not found in CRISP2 promoter. These data suggest a possible post-transcriptional regulation of CRISP2 in asthenozoospermia. Subsequently, bioinformatics prediction, luciferase reporter assay and miR-27b transfection experiments revealed that miR-27b could specifically target CRISP2 by binding its 3’-UTR, suppressing CRISP2 expression post-transcriptionally. Further evidence was provided by the clinical observation of a high miR-27b expression in the ejaculated spermatozoa from asthenozoospermic patients and a negative correlation between miR-27b and CRISP2 protein expression. Finally, a retrospective

  13. Isolation and characterization of a novel wheat cysteine-rich receptor-like kinase gene induced by Rhizoctonia cerealis

    NASA Astrophysics Data System (ADS)

    Yang, Kun; Rong, Wei; Qi, Lin; Li, Jiarui; Wei, Xuening; Zhang, Zengyan

    2013-10-01

    Cysteine-rich receptor kinases (CRKs) belong to the receptor-like kinase family. Little is known about CRK genes in wheat. We isolated a wheat CRK gene TaCRK1 from Rhizoctonia cerealis-resistant wheat CI12633 based on a differentially expressed sequence identified by RNA-Sequencing (RNA-Seq) analysis. TaCRK1 was more highly expressed in CI12633 than in susceptible Wenmai 6. Transcription of TaCRK1 in wheat was induced in CI12633 after R. cerealis infection and exogenous abscisic acid (ABA) treatment. The deduced TaCRK1 protein contained a signal peptide, two DUF26 domains, a transmembrane domain, and a serine/threonine protein kinase domain. Transient expression of a green fluorescence protein fused with TaCRK1 in wheat and onion indicated that TaCRK1 may localize to plasma membranes. Characterization of TaCRK1 silencing induced by virus-mediated method in CI12633 showed that the downregulation of TaCRK1 transcript did not obviously impair resistance to R. cerealis. This study paves the way to further CRK research in wheat.

  14. Immunolocalization of keratin-associated beta-proteins (beta-keratins) in pad lamellae of geckos suggest that glycine-cysteine-rich proteins contribute to their flexibility and adhesiveness.

    PubMed

    Alibardi, Lorenzo

    2013-03-01

    The epidermis of digital pads in geckos comprises superficial microornamentation from the oberhautchen layer that form long setae allowing these lizards to climb vertical surfaces. The beta-layer is reduced in pad lamellae but persists up to the apical free margin. Setae are made of different proteins including keratin-associated beta-proteins, formerly indicated as beta-keratins. In order to identify specific setal proteins the present ultrastructural study on geckos pad lamellae analyzes the immunolocalization of three beta-proteins previously found in the epidermis and adhesive setae of the green anolis. A protein rich in glycine but poor in cysteine (HgG5-like) is absent or masked in gecko pad lamellae. Another protein rich in glycine and cysteine (HgGC3-like) is weakly present in setae, oberhautchen and beta-layer. A glycine and cysteine medium rich beta-protein (HgGC10-like) is present in the lower part of the beta-layer but is absent in the oberhautchen, setae, and mesos layer. The latter two proteins may form intermolecular bonds that contribute to the flexibility of the corneous material sustaining the setae. The pliable alpha-layer present beneath the thin beta-layer and in the hinge region of the pad lamellae also contains HgGC10-like proteins. Based on the possibility that some HgGC3-like or other cys-rich beta-proteins are charged in the setae it is suggested that their charges influence the mechanism of adhesion increasing the induction of dipoles on the substrate and enhancing attractive van der Waals forces. Copyright © 2013 Wiley Periodicals, Inc.

  15. A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus laevis Containing Proteinase-Inhibiting Activity.

    PubMed

    Wang, Yu-Wei; Tan, Ji-Min; Du, Can-Wei; Luan, Ning; Yan, Xiu-Wen; Lai, Ren; Lu, Qiu-Min

    2015-08-01

    Various bio-active substances in amphibian skins play important roles in survival of the amphibians. Many protease inhibitor peptides have been identified from amphibian skins, which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides, or to inhibit extracellular proteases produced by invading bacteria. However, there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America. In this work, a cDNA encoding a novel trypsin inhibitor-like (TIL) cysteine-rich peptide was identified from the skin cDNA library of L. laevis. The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence, IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC. The mature peptide was named LL-TIL. LL-TIL shares significant domain similarity with the peptides from the TIL supper family. Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested. Recombinant LL-TIL showed no antimicrobial activity, while it had trypsin-inhibiting activity with a Ki of 16.5178 μM. These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L. laevis. To the best of our knowledge, this is the first report of TIL peptide from frog skin.

  16. The cysteine rich necrotrophic effector SnTox1 produced by Stagonospora nodorum triggers susceptibility of wheat lines harboring Snn1

    USDA-ARS?s Scientific Manuscript database

    The gene encoding SnTox1, a necrotrophic effector from Stagonospora nodorum that causes necrosis of wheat lines expressing Snn1, has been verified by heterologous expression in Pichia pastoris. SnTox1 encodes a 117 amino acid cysteine rich protein with the first 17 amino acids predicted as a signal ...

  17. Dipeptide frequency/bias analysis identifies conserved sites of nonrandomness shared by cysteine-rich motifs.

    PubMed

    Campion, S R; Ameen, A S; Lai, L; King, J M; Munzenmaier, T N

    2001-08-15

    This report describes the application of a simple computational tool, AAPAIR.TAB, for the systematic analysis of the cysteine-rich EGF, Sushi, and Laminin motif/sequence families at the two-amino acid level. Automated dipeptide frequency/bias analysis detects preferences in the distribution of amino acids in established protein families, by determining which "ordered dipeptides" occur most frequently in comprehensive motif-specific sequence data sets. Graphic display of the dipeptide frequency/bias data revealed family-specific preferences for certain dipeptides, but more importantly detected a shared preference for employment of the ordered dipeptides Gly-Tyr (GY) and Gly-Phe (GF) in all three protein families. The dipeptide Asn-Gly (NG) also exhibited high-frequency and bias in the EGF and Sushi motif families, whereas Asn-Thr (NT) was distinguished in the Laminin family. Evaluation of the distribution of dipeptides identified by frequency/bias analysis subsequently revealed the highly restricted localization of the G(F/Y) and N(G/T) sequence elements at two separate sites of extreme conservation in the consensus sequence of all three sequence families. The similar employment of the high-frequency/bias dipeptides in three distinct protein sequence families was further correlated with the concurrence of these shared molecular determinants at similar positions within the distinctive scaffolds of three structurally divergent, but similarly employed, motif modules.

  18. Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis.

    PubMed

    Haag, Andreas F; Baloban, Mikhail; Sani, Monica; Kerscher, Bernhard; Pierre, Olivier; Farkas, Attila; Longhi, Renato; Boncompagni, Eric; Hérouart, Didier; Dall'angelo, Sergio; Kondorosi, Eva; Zanda, Matteo; Mergaert, Peter; Ferguson, Gail P

    2011-10-01

    Sinorhizobium meliloti differentiates into persisting, nitrogen-fixing bacteroids within root nodules of the legume Medicago truncatula. Nodule-specific cysteine-rich antimicrobial peptides (NCR AMPs) and the bacterial BacA protein are essential for bacteroid development. However, the bacterial factors central to the NCR AMP response and the in planta role of BacA are unknown. We investigated the hypothesis that BacA is critical for the bacterial response towards NCR AMPs. We found that BacA was not essential for NCR AMPs to induce features of S. meliloti bacteroids in vitro. Instead, BacA was critical to reduce the amount of NCR AMP-induced membrane permeabilization and bacterial killing in vitro. Within M. truncatula, both wild-type and BacA-deficient mutant bacteria were challenged with NCR AMPs, but this resulted in persistence of the wild-type bacteria and rapid cell death of the mutant bacteria. In contrast, BacA was dispensable for bacterial survival in an M. truncatula dnf1 mutant defective in NCR AMP transport to the bacterial compartment. Therefore, BacA is critical for the legume symbiosis by protecting S. meliloti against the bactericidal effects of NCR AMPs. Host AMPs are ubiquitous in nature and BacA proteins are essential for other chronic host infections by symbiotic and pathogenic bacteria. Hence, our findings suggest that BacA-mediated protection of bacteria against host AMPs is a critical stage in the establishment of different prolonged host infections.

  19. 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…

  20. 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…

  1. Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) from Mytilus galloprovincialis.

    PubMed

    Gerdol, Marco; Puillandre, Nicolas; De Moro, Gianluca; Guarnaccia, Corrado; Lucafò, Marianna; Benincasa, Monica; Zlatev, Ventislav; Manfrin, Chiara; Torboli, Valentina; Giulianini, Piero Giulio; Sava, Gianni; Venier, Paola; Pallavicini, Alberto

    2015-07-21

    We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling.

  2. Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) from Mytilus galloprovincialis

    PubMed Central

    Gerdol, Marco; Puillandre, Nicolas; Moro, Gianluca De; Guarnaccia, Corrado; Lucafò, Marianna; Benincasa, Monica; Zlatev, Ventislav; Manfrin, Chiara; Torboli, Valentina; Giulianini, Piero Giulio; Sava, Gianni; Venier, Paola; Pallavicini, Alberto

    2015-01-01

    We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling. PMID:26201648

  3. Identification of the erythrocyte binding domains of Plasmodium vivax and Plasmodium knowlesi proteins involved in erythrocyte invasion

    PubMed Central

    1994-01-01

    Plasmodium vivax and the related monkey malaria, P. knowlesi, require interaction with the Duffy blood group antigen, a receptor for a family of chemokines that includes interleukin 8, to invade human erythrocytes. One P. vivax and three P. knowlesi proteins that serve as erythrocyte binding ligands in such interactions share sequence homology. Expression of different regions of the P. vivax protein in COS7 cells identified a cysteine-rich domain that bound Duffy blood group-positive but not Duffy blood group-negative human erythrocytes. The homologous domain of the P. knowlesi proteins also bound erythrocytes, but had different specificities. The P. vivax and P. knowlesi binding domains lie in one of two regions of homology with the P. falciparum sialic acid binding protein, another erythrocyte binding ligand, indicating conservation of the domain for erythrocyte binding in evolutionarily distant malaria species. The binding domains of these malaria ligands represent potential vaccine candidates and targets for receptor-blockade therapy. PMID:8046329

  4. Cysteine-rich receptor-like kinase CRK5 as a regulator of growth, development, and ultraviolet radiation responses in Arabidopsis thaliana.

    PubMed

    Burdiak, Paweł; Rusaczonek, Anna; Witoń, Damian; Głów, Dawid; Karpiński, Stanisław

    2015-06-01

    In plants, receptor-like protein kinases play essential roles in signal transduction by recognizing extracellular stimuli and activating the downstream signalling pathways. Cysteine-rich receptor-like kinases (CRKs) constitute a large subfamily of receptor-like protein kinases, with 44 members in Arabidopsis thaliana. They are distinguished by the novel C-X8-C-X2-C motif (DUF26) in the extracellular domains. One of them, CRK5, is an important component of the biochemical machinery involved in the regulation of essential physiological processes. Functional characterization of crk5 mutant plants showed their clear phenotype, manifested by impaired stomatal conductance and accelerated senescence. This phenotype correlated with accumulation of reactive oxygen species, higher foliar levels of ethylene and salicylic acid, and increased transcript abundance for genes associated with signalling pathways corresponding to these hormones. Moreover, the crk5 plants displayed enhanced cell death and oxidative damage in response to ultraviolet radiation. Complementation of CRK5 mutation managed to recover the wild-type phenotype, indicating an essential role of this gene in the regulation of growth, development, and acclimatory responses. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  5. Sac phosphatase domain proteins.

    PubMed Central

    Hughes, W E; Cooke, F T; Parker, P J

    2000-01-01

    Advances in our understanding of the roles of phosphatidylinositol phosphates in controlling cellular functions such as endocytosis, exocytosis and the actin cytoskeleton have included new insights into the phosphatases that are responsible for the interconversion of these lipids. One of these is an entirely novel class of phosphatase domain found in a number of well characterized proteins. Proteins containing this Sac phosphatase domain include the yeast Saccharomyces cerevisiae proteins Sac1p and Fig4p. The Sac phosphatase domain is also found within the mammalian phosphoinositide 5-phosphatase synaptojanin and the yeast synaptojanin homologues Inp51p, Inp52p and Inp53p. These proteins therefore contain both Sac phosphatase and 5-phosphatase domains. This review describes the Sac phosphatase domain-containing proteins and their actions, with particular reference to the genetic and biochemical insights provided by study of the yeast Saccharomyces cerevisiae. PMID:10947947

  6. Enhanced Arabidopsis pattern-triggered immunity by overexpression of cysteine-rich receptor-like kinases

    PubMed Central

    Yeh, Yu-Hung; Chang, Yu-Hsien; Huang, Pin-Yao; Huang, Jing-Bo; Zimmerli, Laurent

    2015-01-01

    Upon recognition of microbe-associated molecular patterns (MAMPs) such as the bacterial flagellin (or the derived peptide flg22) by pattern-recognition receptors (PRRs) such as the FLAGELLIN SENSING2 (FLS2), plants activate the pattern-triggered immunity (PTI) response. The L-type lectin receptor kinase-VI.2 (LecRK-VI.2) is a positive regulator of Arabidopsis thaliana PTI. Cysteine-rich receptor-like kinases (CRKs) possess two copies of the C-X8-C-X2-C (DUF26) motif in their extracellular domains and are thought to be involved in plant stress resistance, but data about CRK functions are scarce. Here, we show that Arabidopsis overexpressing the LecRK-VI.2-responsive CRK4, CRK6, and CRK36 demonstrated an enhanced PTI response and were resistant to virulent bacteria Pseudomonas syringae pv. tomato DC3000. Notably, the flg22-triggered oxidative burst was primed in CRK4, CRK6, and CRK36 transgenics and up-regulation of the PTI-responsive gene FLG22-INDUCED RECEPTOR-LIKE 1 (FRK1) was potentiated upon flg22 treatment in CRK4 and CRK6 overexpression lines or constitutively increased by CRK36 overexpression. PTI-mediated callose deposition was not affected by overexpression of CRK4 and CRK6, while CRK36 overexpression lines demonstrated constitutive accumulation of callose. In addition, Pst DC3000-mediated stomatal reopening was blocked in CRK4 and CRK36 overexpression lines, while overexpression of CRK6 induced constitutive stomatal closure suggesting a strengthening of stomatal immunity. Finally, bimolecular fluorescence complementation and co-immunoprecipitation analyses in Arabidopsis protoplasts suggested that the plasma membrane localized CRK4, CRK6, and CRK36 associate with the PRR FLS2. Association with FLS2 and the observation that overexpression of CRK4, CRK6, and CRK36 boosts specific PTI outputs and resistance to bacteria suggest a role for these CRKs in Arabidopsis innate immunity. PMID:26029224

  7. Protection of Sinorhizobium against Host Cysteine-Rich Antimicrobial Peptides Is Critical for Symbiosis

    PubMed Central

    Sani, Monica; Kerscher, Bernhard; Pierre, Olivier; Farkas, Attila; Longhi, Renato; Boncompagni, Eric; Hérouart, Didier; Dall’Angelo, Sergio; Kondorosi, Eva; Zanda, Matteo; Mergaert, Peter; Ferguson, Gail P.

    2011-01-01

    Sinorhizobium meliloti differentiates into persisting, nitrogen-fixing bacteroids within root nodules of the legume Medicago truncatula. Nodule-specific cysteine-rich antimicrobial peptides (NCR AMPs) and the bacterial BacA protein are essential for bacteroid development. However, the bacterial factors central to the NCR AMP response and the in planta role of BacA are unknown. We investigated the hypothesis that BacA is critical for the bacterial response towards NCR AMPs. We found that BacA was not essential for NCR AMPs to induce features of S. meliloti bacteroids in vitro. Instead, BacA was critical to reduce the amount of NCR AMP-induced membrane permeabilization and bacterial killing in vitro. Within M. truncatula, both wild-type and BacA-deficient mutant bacteria were challenged with NCR AMPs, but this resulted in persistence of the wild-type bacteria and rapid cell death of the mutant bacteria. In contrast, BacA was dispensable for bacterial survival in an M. truncatula dnf1 mutant defective in NCR AMP transport to the bacterial compartment. Therefore, BacA is critical for the legume symbiosis by protecting S. meliloti against the bactericidal effects of NCR AMPs. Host AMPs are ubiquitous in nature and BacA proteins are essential for other chronic host infections by symbiotic and pathogenic bacteria. Hence, our findings suggest that BacA-mediated protection of bacteria against host AMPs is a critical stage in the establishment of different prolonged host infections. PMID:21990963

  8. Functional analysis of a novel cysteine-rich antimicrobial peptide from the salivary glands of the tick Rhipicephalus haemaphysaloides.

    PubMed

    Zhang, Houshuang; Yang, Siqi; Gong, Haiyan; Cao, Jie; Zhou, Yongzhi; Zhou, Jinlin

    2015-10-01

    Ticks encounter various microbes while sucking blood from an infected host and carrying these pathogens in themselves. Ticks can then transmit these pathogens to vertebrate hosts. The immune system of ticks can be stimulated to produce many bioactive molecules during feeding and pathogen invasion. Antimicrobial peptides (AMPs) are key effector molecules of a tick's immune response, as they can kill invading pathogenic microorganisms. In this study, we identified a novel cysteine-rich AMP, designated Rhamp1, in the salivary glands of unfed and fed female ticks (Rhipicephalus haemaphysaloides). Rhamp1 is encoded by a gene with an open reading frame of 333 bp, which in turn encodes a peptide of 12 kDa with a 22 amino acid residue signal peptide. The Rhamp1 protein had a pI of 8.6 and contained six conserved cysteine residues at the C-terminus. Rhamp1 shared 43% amino acid identity with a secreted cysteine-rich protein of another tick species, Ixodes scapularis. We cloned the Rhamp1 gene and attempted to express a recombinant protein using prokaryotic and eukaryotic systems, to determine its biological significance. Recombinant Rhamp1 was successfully expressed in both systems, yielding a glutathione S-transferase (GST)-tagged protein (36 kDa) from the prokaryotic system, and a polyhistidine-tagged Rhamp1 protein (14 kDa) from the eukaryotic system. Rhamp1 inhibited the activities of chymotrypsin (16%) and elastase (22%) and exerted low hemolytic activity. It also inhibited the growth of Gram-negative bacteria, including Pseudomonas aeruginosa (49%), Salmonella typhimurium (50%), and Escherichia coli (52%). Our findings suggest that Rhamp1 is a novel AMP in R. haemaphysaloides with the ability to inhibit proteinase activity.

  9. The Arabidopsis thaliana cysteine-rich receptor-like kinases CRK6 and CRK7 protect against apoplastic oxidative stress.

    PubMed

    Idänheimo, Niina; Gauthier, Adrien; Salojärvi, Jarkko; Siligato, Riccardo; Brosché, Mikael; Kollist, Hannes; Mähönen, Ari Pekka; Kangasjärvi, Jaakko; Wrzaczek, Michael

    2014-03-07

    Receptor-like kinases are important regulators of many different processes in plants. Despite their large number only a few have been functionally characterized. One of the largest subgroups of receptor-like kinases in Arabidopsis is the cysteine-rich receptor like kinases (CRKs). High sequence similarity among the CRKs has been suggested as major cause for functional redundancy. The genomic localization of CRK genes in back-to-back repeats has made their characterization through mutant analysis unpractical. Expression profiling has linked the CRKs with reactive oxygen species, important signaling molecules in plants. Here we have investigated the role of two CRKs, CRK6 and CRK7, and analyzed their role in extracellular ROS signaling. CRK6 and CRK7 are active protein kinases with differential preference for divalent cations. Our results suggest that CRK7 is involved in mediating the responses to extracellular but not chloroplastic ROS production.

  10. Structure of a two-CAP-domain protein from the human hookworm parasite Necator americanus

    SciTech Connect

    Asojo, Oluwatoyin A.

    2011-05-01

    The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite N. americanus refined to a resolution limit of 2.2 Å is presented. Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structure was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins.

  11. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  12. Cellulose binding domain proteins

    SciTech Connect

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  13. Three-dimensional interplay among the ligand-binding domains of the urokinase-plasminogen-activator-receptor-associated protein, Endo180

    PubMed Central

    Rivera-Calzada, Angel; Robertson, David; MacFadyen, John R.; Boskovic, Jasminka; Isacke, Clare M.; Llorca, Oscar

    2003-01-01

    Endo180, also known as the urokinase plasminogen activator receptor (uPAR)-associated protein (uPARAP), is one of the four members of the mannose receptor family, and is implicated in extracellular-matrix remodelling through its interactions with collagens, sugars and uPAR. The extracellular portion of Endo180 contains an amino-terminal cysteine-rich domain, a single fibronectin type II domain and eight C-type lectin-like domains. We have purified a soluble version of Endo180 and analysed it by single-particle electron microscopy to obtain a three-dimensional structure of the N-terminal part of the protein at a resolution of 17 Å and reveal, for the first time, the interactions between non-adjacent domains in the mannose receptor family. We show that for Endo180, the cysteine-rich domain contacts the second C-type lectin-like domain, thus providing structural insight into how modulation of its several ligand interactions may regulate Endo180 receptor function. PMID:12856000

  14. A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide.

    PubMed

    Clement, Herlinda; Flores, Vianey; Diego-Garcia, Elia; Corrales-Garcia, Ligia; Villegas, Elba; Corzo, Gerardo

    2015-01-01

    The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin. The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps. It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a(+). Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer. Both expression systems pQE40 and pET28a(+) expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitro conditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand

  15. A high-throughput peptidomic strategy to decipher the molecular diversity of cyclic cysteine-rich peptides

    PubMed Central

    Serra, Aida; Hemu, Xinya; Nguyen, Giang K. T.; Nguyen, Ngan T. K.; Sze, Siu Kwan; Tam, James P.

    2016-01-01

    Cyclotides are plant cyclic cysteine-rich peptides (CRPs). The cyclic nature is reported to be gene-determined with a precursor containing a cyclization-competent domain which contains an essential C-terminal Asn/Asp (Asx) processing signal recognized by a cyclase. Linear forms of cyclotides are rare and are likely uncyclizable because they lack this essential C-terminal Asx signal (uncyclotide). Here we show that in the cyclotide-producing plant Clitoria ternatea, both cyclic and acyclic products, collectively named cliotides, can be bioprocessed from the same cyclization-competent precursor. Using an improved peptidomic strategy coupled with the novel Asx-specific endopeptidase butelase 2 to linearize cliotides at a biosynthetic ligation site for transcriptomic analysis, we characterized 272 cliotides derived from 38 genes. Several types of post-translational modifications of the processed cyclotides were observed, including deamidation, oxidation, hydroxylation, dehydration, glycosylation, methylation, and truncation. Taken together, our results suggest that cyclotide biosynthesis involves ‘fuzzy’ processing of precursors into both cyclic and linear forms as well as post-translational modifications to achieve molecular diversity, which is a commonly found trait of natural product biosynthesis. PMID:26965458

  16. A high-throughput peptidomic strategy to decipher the molecular diversity of cyclic cysteine-rich peptides.

    PubMed

    Serra, Aida; Hemu, Xinya; Nguyen, Giang K T; Nguyen, Ngan T K; Sze, Siu Kwan; Tam, James P

    2016-03-11

    Cyclotides are plant cyclic cysteine-rich peptides (CRPs). The cyclic nature is reported to be gene-determined with a precursor containing a cyclization-competent domain which contains an essential C-terminal Asn/Asp (Asx) processing signal recognized by a cyclase. Linear forms of cyclotides are rare and are likely uncyclizable because they lack this essential C-terminal Asx signal (uncyclotide). Here we show that in the cyclotide-producing plant Clitoria ternatea, both cyclic and acyclic products, collectively named cliotides, can be bioprocessed from the same cyclization-competent precursor. Using an improved peptidomic strategy coupled with the novel Asx-specific endopeptidase butelase 2 to linearize cliotides at a biosynthetic ligation site for transcriptomic analysis, we characterized 272 cliotides derived from 38 genes. Several types of post-translational modifications of the processed cyclotides were observed, including deamidation, oxidation, hydroxylation, dehydration, glycosylation, methylation, and truncation. Taken together, our results suggest that cyclotide biosynthesis involves 'fuzzy' processing of precursors into both cyclic and linear forms as well as post-translational modifications to achieve molecular diversity, which is a commonly found trait of natural product biosynthesis.

  17. Glioma Specific Extracellular Missense Mutations in the First Cysteine Rich Region of Epidermal Growth Factor Receptor (EGFR) Initiate Ligand Independent Activation.

    PubMed

    Ymer, Susie I; Greenall, Sameer A; Cvrljevic, Anna; Cao, Diana X; Donoghue, Jacqui F; Epa, V Chandana; Scott, Andrew M; Adams, Timothy E; Johns, Terrance G

    2011-04-18

    The epidermal growth factor receptor (EGFR) is overexpressed or mutated in glioma. Recently, a series of missense mutations in the extracellular domain (ECD) of EGFR were reported in glioma patients. Some of these mutations clustered within a cysteine-rich region of the EGFR targeted by the therapeutic antibody mAb806. This region is only exposed when EGFR activates and appears to locally misfold during activation. We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors. Both mutants were autophosphorylated in the absence of ligand and enhanced cell survival and anchorage-independent and xenograft growth. The ECD truncation that produces the de2-7EGFR (or EGFRvIII), the most common EGFR mutation in glioma, generates a free cysteine in this same region. Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation. Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR. These ECD mutations confirm that the cysteine-rich region of EGFR around the mAb806 epitope has a significant role in receptor activation.

  18. Glioma Specific Extracellular Missense Mutations in the First Cysteine Rich Region of Epidermal Growth Factor Receptor (EGFR) Initiate Ligand Independent Activation

    PubMed Central

    Ymer, Susie I.; Greenall, Sameer A.; Cvrljevic, Anna; Cao, Diana X.; Donoghue, Jacqui F.; Epa, V. Chandana; Scott, Andrew M.; Adams, Timothy E.; Johns, Terrance G.

    2011-01-01

    The epidermal growth factor receptor (EGFR) is overexpressed or mutated in glioma. Recently, a series of missense mutations in the extracellular domain (ECD) of EGFR were reported in glioma patients. Some of these mutations clustered within a cysteine-rich region of the EGFR targeted by the therapeutic antibody mAb806. This region is only exposed when EGFR activates and appears to locally misfold during activation. We expressed two of these mutations (R324L and E330K) in NR6 mouse fibroblasts, as they do not express any EGFR-related receptors. Both mutants were autophosphorylated in the absence of ligand and enhanced cell survival and anchorage-independent and xenograft growth. The ECD truncation that produces the de2-7EGFR (or EGFRvIII), the most common EGFR mutation in glioma, generates a free cysteine in this same region. Using a technique optimized for detecting disulfide-bonded dimers, we definitively demonstrated that the de2-7EGFR is robustly dimerized and that ablation of the free cysteine prevents dimerization and activation. Modeling of the R324L mutation suggests it may cause transient breaking of disulfide bonds, leading to similar disulfide-bonded dimers as seen for the de2-7EGFR. These ECD mutations confirm that the cysteine-rich region of EGFR around the mAb806 epitope has a significant role in receptor activation. PMID:24212795

  19. Protein domain connectivity and essentiality

    NASA Astrophysics Data System (ADS)

    da F. Costa, L.; Rodrigues, F. A.; Travieso, G.

    2006-10-01

    Protein-protein interactions can be properly modeled as scale-free complex networks, while the lethality of proteins has been correlated with the node degrees, therefore defining a lethality-centrality rule. In this work the authors revisit this relevant problem by focusing attention not on proteins as a whole, but on their functional domains, which are ultimately responsible for their binding potential. Four networks are considered: the original protein-protein interaction network, its randomized version, and two domain networks assuming different lethality hypotheses. By using formal statistical analysis, they show that the correlation between connectivity and essentiality is higher for domains than for proteins.

  20. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    SciTech Connect

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. ); Flores, B.M. ); Hagen, F.S. )

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

  1. The inhibition of calcium carbonate crystal growth by the cysteine-rich Mdm2 peptide.

    PubMed

    Dalas, E; Chalias, A; Gatos, D; Barlos, K

    2006-08-15

    The crystal growth of calcite, the most stable calcium carbonate polymorph, in the presence of the cysteine-rich Mdm2 peptide (containing 48 amino acids in the ring finger configuration), has been investigated by the constant composition technique. Crystallization took place exclusively on well-characterized calcite crystals in solutions supersaturated only with respect to this calcium carbonate salt. The kinetic results indicated a surface diffusion spiral growth mechanism. The presence of the Mdm2 peptide inhibited the crystal growth of calcite by 22-58% in the concentration range tested, through adsorption onto the active growth sites of the calcite crystal surface. The kinetic results favored a Langmuir-type adsorption model, and the value of the calculated affinity constant was k(aff)=147x10(4) dm(3)mol(-1), a(ads)=0.29.

  2. A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica.

    PubMed

    Zhong, Jian; Wang, Wenhong; Yang, Xiaomei; Yan, Xiuwen; Liu, Rui

    2013-01-01

    Antimicrobial peptides (AMPs) are important components of the innate immunity. Many antimicrobial peptides have been found from marine mollusks. Little information about AMPs of mollusks living on land is available. A novel cysteine-rich antimicrobial peptide (mytimacin-AF) belonging to the peptide family of mytimacins was purified and characterized from the mucus of the snail of Achatina fulica. Its cDNA was also cloned from the cDNA library. Mytimacin-AF is composed of 80 amino acid residues including 10 cysteines. Mytimacin-AF showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria and the fungus Candida albicans. Among tested microorganisms, it exerted strongest antimicrobial activity against Staphylococcus aureus with a minimal peptide concentration (MIC) of 1.9 μg/ml. Mytimacin-AF had little hemolytic activity against human blood red cells. The current work confirmed the presence of mytimacin-like antimicrobial peptide in land-living mollusks.

  3. Decreased expression of the cardiac LIM domain protein MLP in chronic human heart failure.

    PubMed

    Zolk, O; Caroni, P; Böhm, M

    2000-06-13

    The cardiac LIM domain protein MLP, a member of the cysteine-rich protein family, is an essential regulator of cardiac muscle development. Mice with a disruption of the MLP gene resemble the morphological and clinical picture of dilated cardiomyopathy and heart failure in humans. We investigated whether altered MLP expression is significant for the pathogenesis of human heart failure. Immunohistochemistry and in situ hybridization confirmed the expression of MLP protein and mRNA in human cardiomyocytes. Western blot analysis revealed that the MLP peptide was present in the contractile protein fraction but not in the cytosolic or membrane fraction and that the binding of MLP to myofibrils required functional zinc finger domains. MLP immunoreactivity was decreased approximately 50% (P<0.05) in the left ventricular myocardium of patients with chronic heart failure due to dilated or ischemic cardiomyopathy compared with non-failing donor hearts. MLP mRNA expression, as assessed by Northern blot experiments, was not significantly different between failing and non-failing control hearts, which suggests that decreased MLP synthesis or increased MLP protein turnover, rather than a decreased number of RNA transcripts, may play a role. Because MLP may promote myofibril assembly, the down-regulation of this adapter protein might play an essential role in myofibril derangement or impaired myofibril rearrangement in the failing human myocardium.

  4. Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton

    PubMed Central

    1992-01-01

    Interaction with extracellular matrix can trigger a variety of responses by cells including changes in specific gene expression and cell differentiation. The mechanism by which cell surface events are coupled to the transcriptional machinery is not understood, however, proteins localized at sites of cell-substratum contact are likely to function as signal transducers. We have recently purified and characterized a low abundance adhesion plaque protein called zyxin (Crawford, A. W., and M. C. Beckerle. 1991. J. Biol. Chem. 266:5847- 5853; Crawford, A. W., J. W. Michelsen, and M. C. Beckerle. 1992. J. Cell Biol. 116:1381-1393). We have now isolated and sequenced zyxin cDNA and we report here that zyxin exhibits an unusual proline-rich NH2- terminus followed by three tandemly arrayed LIM domains. LIM domains have previously been identified in proteins that play important roles in transcriptional regulation and cellular differentiation. LIM domains have been proposed to coordinate metal ions and we have demonstrated by atomic absorption spectroscopy that purified zyxin binds zinc, a result consistent with the idea that zyxin has zinc fingers. In addition, we have discovered that zyxin interacts in vitro with a 23-kD protein that also exhibits LIM domains. Microsequence analysis has revealed that the 23-kD protein (or cCRP) is the chicken homologue of the human cysteine- rich protein (hCRP). By double-label indirect immunofluorescence, we found that zyxin and cCRP are extensively colocalized in chicken embryo fibroblasts, consistent with the idea that they interact in vivo. We conclude that LIM domains are zinc-binding sequences that may be involved in protein-protein interactions. The demonstration that two cytoskeletal proteins, zyxin and cCRP, share a sequence motif with proteins important for transcriptional regulation raises the possibility that zyxin and cCRP are components of a signal transduction pathway that mediates adhesion-stimulated changes in gene

  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. The LIM motif defines a specific zinc-binding protein domain.

    PubMed

    Michelsen, J W; Schmeichel, K L; Beckerle, M C; Winge, D R

    1993-05-15

    The cysteine-rich protein (CRP) contains two copies of the LIM sequence motif, CX2CX17HX2CX2CX2CX17-CX2C, that was first identified in the homeodomain proteins Lin-11, Is1-1, and Mec-3. The abundance and spacing of the cysteine residues in the LIM motif are reminiscent of a metal-binding domain. We examined the metal-binding properties of CRP isolated from chicken smooth muscle (cCRP) and from a bacterial expression system and observed that cCRP is a specific Zn-binding metalloprotein. Four Zn(II) ions are maximally bound to cCRP, consistent with the idea that each LIM domain coordinates two metal ions. From spectroscopic studies of Co(II)- and 113Cd(II)-substituted cCRP, we determined that each metal ion is tetrahedrally coordinated with cysteinyl sulfurs dominating the ligand types. One metal site within each LIM motif has tetrathiolate (S4) coordination, the second site may either be S4 or S3N1. The LIM motif represents another example of a specific Zn-binding protein sequence.

  7. Alternative splicing for members of human mosaic domain superfamilies. I. The CH and LIM domains containing group of proteins.

    PubMed

    Friedberg, Felix

    2009-05-01

    In this paper we examine (restricted to homo sapiens) the products resulting from gene duplication and the subsequent alternative splicing for the members of a multidomain group of proteins which possess the evolutionary conserved calponin homology CH domain, i.e. an "actin binding domain", as a singlet and which, in addition, contain the conserved cysteine rich double Zn finger possessing Lim domain, also as a singlet. Seven genes, resulting from gene duplications, were identified that code for seven group members for which pre-mRNAs appear to have undergone multiple alternative splicing: Mical 1, 2 and 3 are located on chromosomes 6q21, 11p15 and 22q11, respectively. The LMO7 gene is present on chromosome 13q22 and the LIMCH1 gene on chromosome 4p13. Micall1 is mapped to chromosome 22q13 and Micall2 to chromosome 7p22. Translated Gen/Bank ESTs suggest the existence of multiple products alternatively spliced from the pre-mRNAs encoded by these genes. Characteristic indicators of such splicing among the proteins derived from one gene must include containment of some common extensive 100% identical regions. In some instances only one exon might be partly or completely eliminated. Sometimes alternative splicing is also associated with an increased frequency of creation of an exon or part of an exon from an intron. Not only coding regions for the body of the protein but also for its N- or -C ends could be affected by the splicing. If created forms are merely beginning at different starting points but remain identical in sequence thereafter, their existence as products of alternate splicing must be questioned. In the splicings, described in this paper, multiple isoforms rather than a single isoform appear as products during the gene expression.

  8. Cysteine-rich low molecular weight antimicrobial peptides from Brevibacillus and related genera for biotechnological applications.

    PubMed

    Baindara, Piyush; Kapoor, Anoop; Korpole, Suresh; Grover, Vishakha

    2017-06-01

    The production of natural antimicrobial peptides (AMPs) is an innate immunity trait of all life forms including eukaryotes and prokaryotes. While these AMPs are usually called as defensins in eukaryotes, they are known as bacteriocins in prokaryotes. Bacteriocins are more diverse AMPs considering their varied composition and posttranslational modifications. Accordingly, this review is focused on cysteine-rich AMPs resembling eukaryotic defensins such as laterosporulin from Brevibacillus spp. and associated peptides secreted by the members of related genera. In fact, structural studies of laterosporulin showed the pattern typically observed in human defensins and therefore, should be considered as bacterial defensin. Although the biosynthesis mechanism of bacterial defensins displayed high similarities, variations in amino acid composition and structure provided the molecular basis for a better understanding of their properties. They are reported to inhibit Gram-positive, Gram-negative, non-multiplying and human pathogenic bacteria. The extreme stability is due to the presence of intra-molecular disulfide bonds in prokaryotic defensins and reveals their potential clinical and food preservation applications. Notably, they are also reported to have potential anticancer properties. Therefore, this review is focused on multitude of diverse applications of bacterial defensins, exploring the possible correlations between their structural, functional and possible biotechnological applications.

  9. Induction of cysteine-rich motor neuron 1 mRNA expression in vascular endothelial cells.

    PubMed

    Nakashima, Yukiko; Takahashi, Satoru

    2014-08-22

    Cysteine-rich motor neuron 1 (CRIM1) is expressed in vascular endothelial cells and plays a crucial role in angiogenesis. In this study, we investigated the expression of CRIM1 mRNA in human umbilical vein endothelial cells (HUVECs). CRIM1 mRNA levels were not altered in vascular endothelial growth factor (VEGF)-stimulated monolayer HUVECs or in cells in collagen gels without VEGF. In contrast, the expression of CRIM1 mRNA was elevated in VEGF-stimulated cells in collagen gels. The increase in CRIM1 mRNA expression was observed even at 2h when HUVECs did not form tubular structures in collagen gels. Extracellular signal-regulated kinase (Erk) 1/2, Akt and focal adhesion kinase (FAK) were activated by VEGF in HUVECs. The VEGF-induced expression of CRIM1 mRNA was significantly abrogated by PD98059 or PF562271, but was not affected by LY294002. These results demonstrate that CRIM1 is an early response gene in the presence of both angiogenic stimulation (VEGF) and environmental (extracellular matrix) factors, and Erk and FAK might be involved in the upregulation of CRIM1 mRNA expression in vascular endothelial cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Cysteine-rich peptides (CRPs) mediate diverse aspects of cell-cell communication in plant reproduction and development.

    PubMed

    Marshall, Eleanor; Costa, Liliana M; Gutierrez-Marcos, Jose

    2011-03-01

    Cell-cell communication in plants is essential for the correct co-ordination of reproduction, growth, and development. Studies to dissect this mode of communication have previously focussed primarily on the action of plant hormones as mediators of intercellular signalling. In animals, peptide signalling is a well-documented intercellular communication system, however, relatively little is known about this system in plants. In recent years, numerous reports have emerged about small, secreted peptides controlling different aspects of plant reproduction. Interestingly, most of these peptides are cysteine-rich, and there is convincing evidence suggesting multiple roles for related cysteine-rich peptides (CRPs) as signalling factors in developmental patterning as well as during plant pathogen responses and symbiosis. In this review, we discuss how CRPs are emerging as key signalling factors in regulating multiple aspects of vegetative growth and reproductive development in plants.

  11. Robustness of Helicobacter pylori Infection Conferred by Context-Variable Redundancy among Cysteine-Rich Paralogs

    PubMed Central

    Putty, Kalyani; Marcus, Sarah A.; Mittl, Peer R. E.; Bogadi, Lindsey E.; Hunter, Allison M.; Arur, Swathi; Berg, Douglas E.; Sethu, Palaniappan; Kalia, Awdhesh

    2013-01-01

    Deletion of single genes from expanded gene families in bacterial genomes often does not elicit a phenotype thus implying redundancy or functional non-essentiality of paralogous genes. The molecular mechanisms that facilitate evolutionary maintenance of such paralogs despite selective pressures against redundancy remain mostly unexplored. Here, we investigate the evolutionary, genetic, and functional interaction between the Helicobacter pylori cysteine-rich paralogs hcpG and hcpC in the context of H. pylori infection of cultured mammalian cells. We find that in natural H. pylori populations both hcpG and hcpC are maintained by positive selection in a dual genetic relationship that switches from complete redundancy during early infection, whereby ΔhcpC or ΔhcpG mutants themselves show no growth defect but a significant growth defect is seen in the ΔhcpC,ΔhcpG double mutant, to quantitative redundancy during late infection wherein the growth defect of the ΔhcpC mutant is exacerbated in the ΔhcpC,ΔhcpG double mutant although the ΔhcpG mutant itself shows no defect. Moreover, during early infection both hcpG and hcpC are essential for optimal translocation of the H. pylori HspB/GroEL chaperone, but during middle-to-late infection hcpC alone is necessary and sufficient for HspB/GroEL translocation thereby revealing the lack of functional compensation among paralogs. We propose that evolution of context-dependent differences in the nature of genetic redundancy, and function, between hcpG and hcpC may facilitate their maintenance in H. pylori genomes, and confer robustness to H. pylori growth during infection of cultured mammalian cells. PMID:23555707

  12. Diversity in protein domain superfamilies

    PubMed Central

    Das, Sayoni; Dawson, Natalie L; Orengo, Christine A

    2015-01-01

    Whilst ∼93% of domain superfamilies appear to be relatively structurally and functionally conserved based on the available data from the CATH-Gene3D domain classification resource, the remainder are much more diverse. In this review, we consider how domains in some of the most ubiquitous and promiscuous superfamilies have evolved, in particular the plasticity in their functional sites and surfaces which expands the repertoire of molecules they interact with and actions performed on them. To what extent can we identify a core function for these superfamilies which would allow us to develop a ‘domain grammar of function’ whereby a protein's biological role can be proposed from its constituent domains? Clearly the first step is to understand the extent to which these components vary and how changes in their molecular make-up modifies function. PMID:26451979

  13. Structure of Protein Having Inhibitory Disintegrin and Leukotriene Scavenging Functions Contained in Single Domain

    SciTech Connect

    Xu, Xueqing; Francischetti, Ivo M.B.; Lai, Ren; Ribeiro, José M.C.; Andersen, John F.

    2012-08-10

    The antihemostatic/antiangiogenic protein tablysin-15 is a member of the CAP (cysteine-rich secretory, antigen 5, and pathogenesis-related 1 protein) superfamily and has been shown to bind the integrins {alpha}{sub IIb}{beta}{sub 3} and {alpha}{sub V}{beta}{sub 3} by means of an Arg-Gly-Asp (RGD) tripeptide sequence. Here we describe the x-ray crystal structure of tablysin-15 and show that the RGD motif is located in a novel structural context. The motif itself is contained in a type II {beta}-turn structure that is similar in its conformation to the RGD sequence of the cyclic pentapeptide cilengitide when bound to integrin {alpha}V{beta}3. The CAP domain also contains a hydrophobic channel that appears to bind a fatty acid molecule in the crystal structure after purification from Escherichia coli. After delipidation of the protein, tablysin-15 was found to bind proinflammatory cysteinyl leukotrienes with submicromolar affinities. The structure of the leukotriene E{sub 4}-tablysin-15 complex shows that the ligand binds with the nonfunctionalized end of the fatty acid chain buried in the hydrophobic pocket, whereas the carboxylate end of the ligand binds forms hydrogen bond/salt bridge interactions with polar side chains at the channel entrance. Therefore, tablysin-15 functions as an inhibitor of integrin function and as an anti-inflammatory scavenger of eicosanoids.

  14. Structural studies of human glioma pathogenesis-related protein 1.

    PubMed

    Asojo, Oluwatoyin A; Koski, Raymond A; Bonafé, Nathalie

    2011-10-01

    Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn2+ complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn2+ similarly to snake-venom CRISPs, which are involved in Zn2+-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.

  15. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, O.; Yosef, K.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1998-02-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  16. Cellulose binding domain fusion proteins

    SciTech Connect

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  17. Extending Protein Domain Boundary Predictors to Detect Discontinuous Domains

    PubMed Central

    Xue, Zhidong; Jang, Richard; Govindarajoo, Brandon; Huang, Yichu; Wang, Yan

    2015-01-01

    A variety of protein domain predictors were developed to predict protein domain boundaries in recent years, but most of them cannot predict discontinuous domains. Considering nearly 40% of multidomain proteins contain one or more discontinuous domains, we have developed DomEx to enable domain boundary predictors to detect discontinuous domains by assembling the continuous domain segments. Discontinuous domains are predicted by matching the sequence profile of concatenated continuous domain segments with the profiles from a single-domain library derived from SCOP and CATH, and Pfam. Then the matches are filtered by similarity to library templates, a symmetric index score and a profile-profile alignment score. DomEx recalled 32.3% discontinuous domains with 86.5% precision when tested on 97 non-homologous protein chains containing 58 continuous and 99 discontinuous domains, in which the predicted domain segments are within ±20 residues of the boundary definitions in CATH 3.5. Compared with our recently developed predictor, ThreaDom, which is the state-of-the-art tool to detect discontinuous-domains, DomEx recalled 26.7% discontinuous domains with 72.7% precision in a benchmark with 29 discontinuous-domain chains, where ThreaDom failed to predict any discontinuous domains. Furthermore, combined with ThreaDom, the method ranked number one among 10 predictors. The source code and datasets are available at https://github.com/xuezhidong/DomEx. PMID:26502173

  18. Increased Understanding of the Biochemistry and Biosynthesis of MUC2 and Other Gel-Forming Mucins Through the Recombinant Expression of Their Protein Domains

    PubMed Central

    Ambort, Daniel; Thomsson, Elisabeth; Johansson, Malin E. V.; Hansson, Gunnar C.

    2016-01-01

    The gel-forming mucins are large and heavily O-glycosylated proteins which build up mucus gels. The recombinant production of full-length gel-forming mucins has not been possible to date. In order to study mucin biosynthesis and biochemistry, we and others have taken the alternative approach of constructing different recombinant proteins consisting of one or several domains of these large proteins and expressing them separately in different cell lines. Using this approach, we have determined that MUC2, the intestinal gel-forming mucin, dimerizes via its C-terminal cysteine-knot domain and also trimerizes via one of the N-terminal von Willebrand D domains. Both of these interactions are disulfide bond mediated. Via this assembly, a molecular network is built by which the mucus gel is formed. Here we discuss not only the functional understanding obtained from studies of the recombinant proteins, but also highlight the difficulties encountered when these proteins were produced recombinantly. We often found an accumulation of the proteins in the ER and consequently no secretion. This was especially apparent when the cysteine-rich domains of the N- and C-terminal parts of the mucins were expressed. Other proteins that we constructed were either not secreted or not expressed at all. Despite these problems, the knowledge of mucin biosynthesis and assembly has advanced considerably through the studies of these recombinant proteins. PMID:23359125

  19. 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.

  20. Stability of domain structures in multi-domain proteins

    PubMed Central

    Bhaskara, Ramachandra M.; Srinivasan, Narayanaswamy

    2011-01-01

    Multi-domain proteins have many advantages with respect to stability and folding inside cells. Here we attempt to understand the intricate relationship between the domain-domain interactions and the stability of domains in isolation. We provide quantitative treatment and proof for prevailing intuitive ideas on the strategies employed by nature to stabilize otherwise unstable domains. We find that domains incapable of independent stability are stabilized by favourable interactions with tethered domains in the multi-domain context. Stability of such folds to exist independently is optimized by evolution. Specific residue mutations in the sites equivalent to inter-domain interface enhance the overall solvation, thereby stabilizing these domain folds independently. A few naturally occurring variants at these sites alter communication between domains and affect stability leading to disease manifestation. Our analysis provides safe guidelines for mutagenesis which have attractive applications in obtaining stable fragments and domain constructs essential for structural studies by crystallography and NMR. PMID:22355559

  1. Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10

    PubMed Central

    1991-01-01

    Human umbilical vein endothelial (HUVE) cells have been previously reported to express the genes for the A and B chains of PDGF and to secrete PDGF-related factors into culture media. Antihuman PDGF IgG affinity chromatography was used to purify PDGF-related activity from HUVE cell-conditioned media. Immunoblot analysis of the affinity- purified proteins with anti-PDGF IgG and antibodies specific for the A or B chain peptides of PDGF combined with chemotactic and mitogenic assays revealed that the major PDGF immunorelated molecule secreted by HUVE cells is a monomer of approximately 36-38 kD and that less than 10% of the purified biologically active molecules are PDGF A or B chain peptides. Screening of an HUVE cell cDNA library in the expression vector lambda gtl 1 with the anti-PDGF antibody resulted in the cloning and sequencing of a cDNA with an open reading frame encoding a 38-kD cysteine-rich secreted protein which we show to be the major PDGF- related mitogen secreted by human vascular endothelial cells. The protein has a 45% overall homology to the translation product of the v- src-induced CEF-10 mRNA from chick embryo fibroblasts. We have termed this new mitogen connective tissue growth factor. PMID:1654338

  2. Myticin, a novel cysteine-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel Mytilus galloprovincialis.

    PubMed

    Mitta, G; Hubert, F; Noël, T; Roch, P

    1999-10-01

    We report here the isolation of two isoforms of a novel cysteine-rich peptide from haemocytes (isoform A of 4.438 Da and B of 4.562 Da) and plasma (isoform A) of the mussel, Mytilus galloprovincialis. The two molecules display antibacterial activity against gram-positive bacteria, whereas only isoform B is active against the fungus Fusarium oxysporum and a gram-negative bacteria Escherichia coli D31. Complete peptide sequences were determined by a combination of Edman degradation, mass spectrometry and cDNA cloning using a haemocyte cDNA library. The mature molecules, named myticins, comprise 40 residues with four intramolecular disulfide bridges and a cysteine array in the primary structure different to that of the previously characterized cysteine-rich antimicrobial peptides. Sequence analysis of the cloned cDNAs revealed that myticin precursors consist of 96 amino acids with a putative signal peptide of 20 amino acids, the antimicrobial peptide sequence and a 36-residue C-terminal extension. This structure suggests that myticins are synthesized as preproproteins and then processed by various proteolytic events before storage of the active peptide in the haemocytes. Myticin precursors are expressed mainly in the haemocytes as revealed by Northern blot analysis.

  3. Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide.

    PubMed

    Haag, Andreas F; Kerscher, Bernhard; Dall'Angelo, Sergio; Sani, Monica; Longhi, Renato; Baloban, Mikhail; Wilson, Heather M; Mergaert, Peter; Zanda, Matteo; Ferguson, Gail P

    2012-03-30

    The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S-S) bridges. However, the natural configuration of NCR AMP S-S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S-S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S-S bridges from cysteines 1-2, 3-4 to 1-3, 2-4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.

  4. Inverse expression of cystein-rich 61 (Cyr61/CCN1) and connective tissue growth factor (CTGF/CCN2) in borderline tumors and carcinomas of the ovary.

    PubMed

    Bartel, Frank; Balschun, Katharina; Gradhand, Elise; Strauss, Hans G; Dittmer, Jürgen; Hauptmann, Steffen

    2012-09-01

    Members of the CCN [cystein-rich 61 (Cyr61)/connective tissue growth factor (CTGF)/nephroblastoma (NOV)] protein family are involved in the regulation of cellular proliferation, apoptosis, and migration and are also assumed to play a role in carcinogenesis. Therefore, we performed a retrospective study to investigate the immunohistochemical expression of both Cyr61 and CTGF in 92 borderline tumors (BOTs) and 107 invasive carcinomas of the ovary (IOCs). To determine their diagnostic and prognostic value, we correlated protein expression with clinicopathologic factors including overall and disease-free survival. Cyr61 and CTGF were found to be inversely expressed in both BOTs and IOCs, with a stronger expression of Cyr61 in IOCs. Moreover, Cyr61 was found to be preferentially expressed in high-grade serous carcinomas, whereas CTGF was found more frequently in low-grade serous carcinomas. Weak Cyr61 levels correlated with both low estrogen receptor and p53 expression (P=0.038, P=0.04, respectively). However, no association was observed between CTGF, estrogen receptor, and p53 expression levels in IOCs. Regarding prognosis, Cyr61 was found to be of no value, but the loss of CTGF was found to be associated with a poor prognosis in multivariate analysis of overall (relative risk 2.8; P=0.050) and disease-free (relative risk 2.3; P=0.031) survival. Cyr61 and CTGF are inversely expressed in BOTs and IOCs, and loss of CTGF independently indicates poor prognosis in IOCs.

  5. Exhaustive enumeration of protein domain families.

    PubMed

    Heger, Andreas; Holm, Liisa

    2003-05-02

    Domains are considered as the basic units of protein folding, evolution, and function. Decomposing each protein into modular domains is thus a basic prerequisite for accurate functional classification of biological molecules. Here, we present ADDA, an automatic algorithm for domain decomposition and clustering of all protein domain families. We use alignments derived from an all-on-all sequence comparison to define domains within protein sequences based on a global maximum likelihood model. In all, 90% of domain boundaries are predicted within 10% of domain size when compared with the manual domain definitions given in the SCOP database. A representative database of 249,264 protein sequences were decomposed into 450,462 domains. These domains were clustered on the basis of sequence similarities into 33,879 domain families containing at least two members with less than 40% sequence identity. Validation against family definitions in the manually curated databases SCOP and PFAM indicates almost perfect unification of various large domain families while contamination by unrelated sequences remains at a low level. The global survey of protein-domain space by ADDA confirms that most large and universal domain families are already described in PFAM and/or SMART. However, a survey of the complete set of mobile modules leads to the identification of 1479 new interesting domain families which shuffle around in multi-domain proteins. The data are publicly available at ftp://ftp.ebi.ac.uk/pub/contrib/heger/adda.

  6. Inferring Domain-Domain Interactions from Protein-Protein Interactions with Formal Concept Analysis

    PubMed Central

    Khor, Susan

    2014-01-01

    Identifying reliable domain-domain interactions will increase our ability to predict novel protein-protein interactions, to unravel interactions in protein complexes, and thus gain more information about the function and behavior of genes. One of the challenges of identifying reliable domain-domain interactions is domain promiscuity. Promiscuous domains are domains that can occur in many domain architectures and are therefore found in many proteins. This becomes a problem for a method where the score of a domain-pair is the ratio between observed and expected frequencies because the protein-protein interaction network is sparse. As such, many protein-pairs will be non-interacting and domain-pairs with promiscuous domains will be penalized. This domain promiscuity challenge to the problem of inferring reliable domain-domain interactions from protein-protein interactions has been recognized, and a number of work-arounds have been proposed. This paper reports on an application of Formal Concept Analysis to this problem. It is found that the relationship between formal concepts provides a natural way for rare domains to elevate the rank of promiscuous domain-pairs and enrich highly ranked domain-pairs with reliable domain-domain interactions. This piggybacking of promiscuous domain-pairs onto less promiscuous domain-pairs is possible only with concept lattices whose attribute-labels are not reduced and is enhanced by the presence of proteins that comprise both promiscuous and rare domains. PMID:24586450

  7. Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein

    PubMed Central

    Darwiche, Rabih; Kelleher, Alan; Hudspeth, Elissa M.; Schneiter, Roger; Asojo, Oluwatoyin A.

    2016-01-01

    The production, crystal structure, and functional characterization of the C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of pathogen-related yeast protein-1 (Pry1) from Saccharomyces cerevisiae is presented. The CAP domain of Pry1 (Pry1CAP) is functional in vivo as its expression restores cholesterol export to yeast mutants lacking endogenous Pry1 and Pry2. Recombinant Pry1CAP forms dimers in solution, is sufficient for in vitro cholesterol binding, and has comparable binding properties as full-length Pry1. Two crystal structures of Pry1CAP are reported, one with Mg2+ coordinated to the conserved CAP tetrad (His208, Glu215, Glu233 and His250) in spacegroup I41 and the other without divalent cations in spacegroup P6122. The latter structure contains four 1,4-dioxane molecules from the crystallization solution, one of which sits in the cholesterol binding site. Both structures reveal that the divalent cation and cholesterol binding sites are connected upon dimerization, providing a structural basis for the observed Mg2+-dependent sterol binding by Pry1. PMID:27344972

  8. Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein

    NASA Astrophysics Data System (ADS)

    Darwiche, Rabih; Kelleher, Alan; Hudspeth, Elissa M.; Schneiter, Roger; Asojo, Oluwatoyin A.

    2016-06-01

    The production, crystal structure, and functional characterization of the C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of pathogen-related yeast protein-1 (Pry1) from Saccharomyces cerevisiae is presented. The CAP domain of Pry1 (Pry1CAP) is functional in vivo as its expression restores cholesterol export to yeast mutants lacking endogenous Pry1 and Pry2. Recombinant Pry1CAP forms dimers in solution, is sufficient for in vitro cholesterol binding, and has comparable binding properties as full-length Pry1. Two crystal structures of Pry1CAP are reported, one with Mg2+ coordinated to the conserved CAP tetrad (His208, Glu215, Glu233 and His250) in spacegroup I41 and the other without divalent cations in spacegroup P6122. The latter structure contains four 1,4-dioxane molecules from the crystallization solution, one of which sits in the cholesterol binding site. Both structures reveal that the divalent cation and cholesterol binding sites are connected upon dimerization, providing a structural basis for the observed Mg2+-dependent sterol binding by Pry1.

  9. Direct Association of Sprouty-related Protein with an EVH1 Domain (SPRED) 1 or SPRED2 with DYRK1A Modifies Substrate/Kinase Interactions*

    PubMed Central

    Li, Dan; Jackson, Rebecca A.; Yusoff, Permeen; Guy, Graeme R.

    2010-01-01

    The mammalian SPRED (Sprouty-related protein with an EVH1 domain) proteins include a family of three members, SPRED1–3. Currently, little is known about their biochemistry. The best described, SPRED1, has been shown to inhibit the Ras/ERK pathway downstream of Ras. All three SPREDs have a cysteine-rich domain (CRD) that has high homology to the CRD of the Sprouty family of proteins, several of which are also Ras/ERK inhibitors. In the belief that binding partners would clarify SPRED function, we assayed for their associated proteins. Here, we describe the direct and endogenous interaction of SPRED1 and SPRED2 with the novel kinase, DYRK1A. DYRK1A has become the subject of recent research focus as it plays a central role in Caenorhabditis elegans oocyte maturation and egg activation, and there is strong evidence that it could be involved in Down syndrome in humans. Both SPRED1 and SPRED2 inhibit the ability of DYRK1A to phosphorylate its substrates, Tau and STAT3. This inhibition occurs via an interaction of the CRD of the SPREDs with the kinase domain of DYRK1A. DYRK1A substrates must bind to the kinase to enable phosphorylation, and SPRED proteins compete for the same binding site to modify this process. Our accumulated evidence indicates that the SPRED proteins are likely physiological modifiers of DYRK1A. PMID:20736167

  10. ADM-1, a protein with metalloprotease- and disintegrin-like domains, is expressed in syncytial organs, sperm, and sheath cells of sensory organs in Caenorhabditis elegans.

    PubMed Central

    Podbilewicz, B

    1996-01-01

    A search was carried out for homologues of possible fusogenic proteins to study their function in a genetically tractable animal. The isolation, molecular, and cellular characterization of the Caenorhabditis elegans adm-1 gene (a disintegrin and metalloprotease domain) are described. A glycoprotein analogous to viral fusion proteins has been identified on the surface of guinea pig sperm (PH-30/fertilin) and is implicated in sperm-egg fusion. adm-1 is the first reported invertebrate gene related to PH-30 and a family of proteins containing snake venom disintegrin- and metalloprotease-like domains. ADM-1 shows a domain organization identical to PH-30. It contains prepro, metalloprotease, disintegrin, cysteine rich with putative fusion peptide, epidermal growth factor-like repeat, transmembrane, and cytoplasmic domains. Antibodies which recognize ADM-1 protein in immunoblots were generated. Using immunofluorescence and in situ hybridization, the products of adm-1 have been detected in specific cells during different stages of development. The localization of ADM-1 to the plasma membrane of embryonic cells and to the sheath cells of sensory organs suggests a function in cell adhesion. ADM-1 expression in the hypodermis, pharynx, vulva, and mature sperm is consistent with a putative role in somatic and gamete cell fusions. Images PMID:8970152

  11. Protein structural domains: definition and prediction.

    PubMed

    Ezkurdia, Iakes; Tress, Michael L

    2011-11-01

    Recognition and prediction of structural domains in proteins is an important part of structure and function prediction. This unit lists the range of tools available for domain prediction, and describes sequence and structural analysis tools that complement domain prediction methods. Also detailed are the basic domain prediction steps, along with suggested strategies for different protein sequences and potential pitfalls in domain boundary prediction. The difficult problem of domain orientation prediction is also discussed. All the resources necessary for domain boundary prediction are accessible via publicly available Web servers and databases and do not require computational expertise.

  12. Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion

    SciTech Connect

    Petit, Chad M.; Chouljenko, Vladimir N.; Iyer, Arun; Colgrove, Robin; Farzan, Michael; Knipe, David M.; Kousoulas, K.G. . E-mail: vtgusk@lsu.edu

    2007-04-10

    The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion was reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of {sup 3}H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion.

  13. Innate immunity. Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis.

    PubMed

    Charlet, M; Chernysh, S; Philippe, H; Hetru, C; Hoffmann, J A; Bulet, P

    1996-09-06

    We have isolated from the blood of immune-challenged and untreated mussels (Mytilus edulis) antibacterial and antifungal peptides. We have characterized two isoforms of a novel 34-residue, cysteine-rich, peptide with potent bactericidal activity and partially characterized a novel 6.2-kDa antifungal peptide containing 12 cysteines. We report the presence of two members of the insect defensin family of antibacterial peptides and provide a phylogenetic analysis that indicates that mollusc and arthropod defensins have a common ancestry. Our data argue that circulating antimicrobial peptides represent an ancient host defense mechanism that predated the separation between molluscs and arthropods at the root of the Cambrian, about 545 million years ago.

  14. Solution structure of the focal adhesion adaptor PINCH LIM1 domain and characterization of its interaction with the integrin-linked kinase ankyrin repeat domain.

    PubMed

    Velyvis, A; Yang, Y; Wu, C; Qin, J

    2001-02-16

    PINCH is a recently identified adaptor protein that comprises an array of five LIM domains. PINCH functions through LIM-mediated protein-protein interactions that are involved in cell adhesion, growth, and differentiation. The LIM1 domain of PINCH interacts with integrin-linked kinase (ILK), thereby mediating focal adhesions via a specific integrin/ILK signaling pathway. We have solved the NMR structure of the PINCH LIM1 domain and characterized its binding to ILK. LIM1 contains two contiguous zinc fingers of the CCHC and CCCH types and adopts a global fold similar to that of functionally distinct LIM domains from cysteine-rich protein and cysteine-rich intestinal protein families with CCHC and CCCC zinc finger types. Gel-filtration and NMR experiments demonstrated a 1:1 complex between PINCH LIM1 and the ankyrin repeat domain of ILK. A chemical shift mapping experiment identified regions in PINCH LIM1 that are important for interaction with ILK. Comparison of surface features between PINCH LIM1 and other functionally different LIM domains indicated that the LIM motif might have a highly variable mode in recognizing various target proteins.

  15. The structure of the extracellular domain of the jumping translocation breakpoint protein reveals a variation of the midkine fold.

    PubMed

    Rousseau, Francois; Pan, Borlan; Fairbrother, Wayne J; Bazan, J Fernando; Lingel, Andreas

    2012-01-06

    Jumping Translocation Breakpoint (JTB) is an orphan receptor that is conserved from nematodes to humans and whose gene expression in humans is strikingly upregulated in diverse types of cancers. Translocations occur frequently at the hJTB genomic locus, leading to multiple copies of a truncated JTB gene, which potentially encodes a soluble secreted ectodomain. In addition, JTB and its orthologs likely represent a unique and ancient protein family since homologs could not be identified by direct sequence comparison. In the present study, we have determined the NMR solution structure of the N-terminal ectodomain of human JTB, showing that its fold architecture is a new variant of a three-β-strand antiparallel β-meander. The JTB structure has a distant relationship to the midkine/pleiotrophin fold, particularly in the conservation of distinctive disulfide bridge patterns. The structure of this newly characterized small cysteine-rich domain suggests potential involvement of JTB in interactions with proteins or extracellular matrix and may help to uncover the elusive biological functions of this protein. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Enhanced protein domain discovery using taxonomy

    PubMed Central

    Coin, Lachlan; Bateman, Alex; Durbin, Richard

    2004-01-01

    Background It is well known that different species have different protein domain repertoires, and indeed that some protein domains are kingdom specific. This information has not yet been incorporated into statistical methods for finding domains in sequences of amino acids. Results We show that by incorporating our understanding of the taxonomic distribution of specific protein domains, we can enhance domain recognition in protein sequences. We identify 4447 new instances of Pfam domains in the SP-TREMBL database using this technique, equivalent to the coverage increase given by the last 8.3% of Pfam families and to a 0.7% increase in the number of domain predictions. We use PSI-BLAST to cross-validate our new predictions. We also benchmark our approach using a SCOP test set of proteins of known structure, and demonstrate improvements relative to standard Hidden Markov model techniques. Conclusions Explicitly including knowledge about the taxonomic distribution of protein domains can enhance protein domain recognition. Our method can also incorporate other context-specific domain distributions – such as domain co-occurrence and protein localisation. PMID:15137915

  17. Mapping the Protein Domain Structures of the Respiratory Mucins: a mucin proteome coverage study

    PubMed Central

    Cao, Rui; Wang, T. Tiffany; DeMaria, Genevieve; Sheehan, John K.; Kesimer, Mehmet

    2012-01-01

    Mucin genes encode a family of the largest expressed proteins in the human genome. The proteins are highly substituted with O-linked oligosaccharides which greatly restrict access to the peptide backbones. The genomic organization of the N-terminal, O-glycosylated, and C-terminal regions of most of the mucins has been established and is available in the sequence databases. However, much less is known about the fate of their exposed protein regions after translation and secretion, and, to date, detailed proteomic studies complementary to the genomic studies are rather limited. Using mucins isolated from cultured human airway epithelial cell secretions, trypsin digestion and mass spectrometry, we investigated the proteome coverage of the mucins responsible for the maintenance and protection of the airway epithelia. Excluding the heavily glycosylated mucin domains, up to 85% coverage of the N-terminal region of the gel forming mucins MUC5B and MUC5AC was achieved, and up to 60% of the C-terminal regions were covered, suggesting that more N- and sparsely O-glycosylated regions as well as possible other modifications are available at the C-terminus. All possible peptides from the cysteine-rich regions that interrupt the heavily glycosylated mucin domains were identified. Interestingly, 43 cleavage sites from ten different domains of MUC5B and MUC5AC were identified, which possessed a non-tryptic cleavage site on the N-terminal end of the peptide, indicating potential exposure to proteolytic and/or “spontaneous cleavages”. Some of these non-tryptic cleavages may be important for proper maturation of the molecule, before and/or after secretion. Most of the peptides identified from MUC16 were from the SEA region. Surprisingly, three peptides were clearly identified from its heavily glycosylated regions. Up to 25% coverage of MUC4 was achieved covering seven different domains of the molecule. All peptides from the MUC1 cytoplasmic domain were detected along with the

  18. Multiple graph regularized protein domain ranking

    PubMed Central

    2012-01-01

    Background Protein domain ranking is a fundamental task in structural biology. Most protein domain ranking methods rely on the pairwise comparison of protein domains while neglecting the global manifold structure of the protein domain database. Recently, graph regularized ranking that exploits the global structure of the graph defined by the pairwise similarities has been proposed. However, the existing graph regularized ranking methods are very sensitive to the choice of the graph model and parameters, and this remains a difficult problem for most of the protein domain ranking methods. Results To tackle this problem, we have developed the Multiple Graph regularized Ranking algorithm, MultiG-Rank. Instead of using a single graph to regularize the ranking scores, MultiG-Rank approximates the intrinsic manifold of protein domain distribution by combining multiple initial graphs for the regularization. Graph weights are learned with ranking scores jointly and automatically, by alternately minimizing an objective function in an iterative algorithm. Experimental results on a subset of the ASTRAL SCOP protein domain database demonstrate that MultiG-Rank achieves a better ranking performance than single graph regularized ranking methods and pairwise similarity based ranking methods. Conclusion The problem of graph model and parameter selection in graph regularized protein domain ranking can be solved effectively by combining multiple graphs. This aspect of generalization introduces a new frontier in applying multiple graphs to solving protein domain ranking applications. PMID:23157331

  19. 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.

  20. Structural studies of human glioma pathogenesis-related protein 1

    SciTech Connect

    Asojo, Oluwatoyin A.; Koski, Raymond A.; Bonafé, Nathalie

    2011-10-01

    Structural analysis of a truncated soluble domain of human glioma pathogenesis-related protein 1, a membrane protein implicated in the proliferation of aggressive brain cancer, is presented. Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn{sup 2+} complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn{sup 2+} similarly to snake-venom CRISPs, which are involved in Zn{sup 2+}-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.

  1. A novel cysteine-rich neurotrophic factor in Aplysia facilitates growth, MAPK activation, and long-term synaptic facilitation.

    PubMed

    Pu, Lu; Kopec, Ashley M; Boyle, Heather D; Carew, Thomas J

    2014-04-01

    Neurotrophins are critically involved in developmental processes such as neuronal cell survival, growth, and differentiation, as well as in adult synaptic plasticity contributing to learning and memory. Our previous studies examining neurotrophins and memory formation in Aplysia showed that a TrkB ligand is required for MAPK activation, long-term synaptic facilitation (LTF), and long-term memory (LTM) for sensitization. These studies indicate that neurotrophin-like molecules in Aplysia can act as key elements in a functionally conserved TrkB signaling pathway. Here we report that we have cloned and characterized a novel neurotrophic factor, Aplysia cysteine-rich neurotrophic factor (apCRNF), which shares classical structural and functional characteristics with mammalian neurotrophins. We show that apCRNF (1) is highly enriched in the CNS, (2) enhances neurite elongation and branching, (3) interacts with mammalian TrkB and p75(NTR), (4) is released from Aplysia CNS in an activity-dependent fashion, (5) facilitates MAPK activation in a tyrosine kinase dependent manner in response to sensitizing stimuli, and (6) facilitates the induction of LTF. These results show that apCRNF is a native neurotrophic factor in Aplysia that can engage the molecular and synaptic mechanisms underlying memory formation.

  2. Molecular cloning and characterization of lustrin A, a matrix protein from shell and pearl nacre of Haliotis rufescens.

    PubMed

    Shen, X; Belcher, A M; Hansma, P K; Stucky, G D; Morse, D E

    1997-12-19

    A specialized extracellular matrix of proteins and polysaccharides controls the morphology and packing of calcium carbonate crystals and becomes occluded within the mineralized composite during formation of the molluscan shell and pearl. We have cloned and characterized the cDNA coding for Lustrin A, a newly described matrix protein from the nacreous layer of the shell and pearl produced by the abalone, Haliotis rufescens, a marine gastropod mollusc. The full-length cDNA is 4,439 base pairs (bp) long and contains an open reading frame coding for 1,428 amino acids. The deduced amino acid sequence reveals a highly modular structure with a high proportion of Ser (16%), Pro (14%), Gly (13%), and Cys (9%). The protein contains ten highly conserved cysteine-rich domains interspersed by eight proline-rich domains; a glycine- and serine-rich domain lies between the two cysteine-rich domains nearest the C terminus, and these are followed by a basic domain and a C-terminal domain that is highly similar to known protease inhibitors. The glycine- and serine-rich domain and at least one of the proline-rich domains show sequence similarity to proteins of two extracellular matrix superfamilies (one of which also is involved in the mineralized matrixes of bone, dentin, and avian eggshell). The arrangement of alternating cysteine-rich domains and proline-rich domains is strikingly similar to that found in frustulins, the proteins that are integral to the silicified cell wall of diatoms. Its modular structure suggests that Lustrin A is a multifunctional protein, whereas the occurrence of related sequences suggest it is a member of a multiprotein family.

  3. A novel cysteine-rich antifungal peptide ToAMP4 from Taraxacum officinale Wigg. flowers.

    PubMed

    Astafieva, A A; Rogozhin, Eugene A; Andreev, Yaroslav A; Odintsova, T I; Kozlov, S A; Grishin, Eugene V; Egorov, Tsezi A

    2013-09-01

    A novel peptide named ToAMP4 was isolated from Taraxacum officinale Wigg. flowers by a combination of acetic acid extraction and different types of chromatography: affinity, size-exclusion, and RP-HPLC. The amino acid sequence of ToAMP4 was determined by automated Edman degradation. The peptide is basic, consists of 41 amino acids, and incorporates three disulphide bonds. Due to the unusual cysteine spacing pattern, ToAMP4 does not belong to any known plant AMP family, but classifies together with two other antimicrobial peptides ToAMP1 and ToAMP2 previously isolated from the dandelion flowers. To study the biological activity of ToAMP4, it was successfully produced in a prokaryotic expression system as a fusion protein with thioredoxin. The recombinant peptide was shown to be identical to the native ToAMP4 by chromatographic behavior, molecular mass, and N-terminal amino acid sequence. The peptide displays broad-spectrum antifungal activity against important phytopathogens. Two ToAMP4-mediated inhibition strategies depending on the fungus were demonstrated. The results obtained add to our knowledge on the structural and functional diversity of AMPs in plants. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  4. A thermodynamic definition of protein domains

    PubMed Central

    Porter, Lauren L.; Rose, George D.

    2012-01-01

    Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics. PMID:22635268

  5. A thermodynamic definition of protein domains.

    PubMed

    Porter, Lauren L; Rose, George D

    2012-06-12

    Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.

  6. The architecture of the protein domain universe.

    PubMed

    Dokholyan, Nikolay V

    2005-03-14

    Understanding the design of the universe of protein structures may provide insights into protein evolution. We study the architecture of the protein domain universe, which has been found to poses peculiar scale-free properties. We examine the origin of these scale-free properties of the graph of protein domain structures (PDUG) and determine that that the PDUG is not modular, i.e. it does not consist of modules with uniform properties. Instead, we find the PDUG to be self-similar at all scales. We further characterize the PDUG architecture by studying the properties of the hub nodes that are responsible for the scale-free connectivity of the PDUG. We introduce a measure of the betweenness centrality of protein domains in the PDUG and find a power-law distribution of the betweenness centrality values. The scale-free distribution of hubs in the protein universe suggests that a set of specific statistical mechanics models, such as the self-organized criticality model, can potentially identify the principal driving forces of protein evolution. We also find a gatekeeper protein domain, removal of which partitions the largest cluster into two large sub-clusters. We suggest that the loss of such gatekeeper protein domains in the course of evolution is responsible for the creation of new fold families.

  7. Inhibition of the proliferation and acceleration of migration of vascular endothelial cells by increased cysteine-rich motor neuron 1

    SciTech Connect

    Nakashima, Yukiko; Morimoto, Mayuka; Toda, Ken-ichi; Shinya, Tomohiro; Sato, Keizo; Takahashi, Satoru

    2015-07-03

    Cysteine-rich motor neuron 1 (CRIM1) is upregulated only in extracellular matrix gels by angiogenic factors such as vascular endothelial growth factor (VEGF). It then plays a critical role in the tube formation of endothelial cells. In the present study, we investigated the effects of increased CRIM1 on other endothelial functions such as proliferation and migration. Knock down of CRIM1 had no effect on VEGF-induced proliferation or migration of human umbilical vein endothelial cells (HUVECs), indicating that basal CRIM1 is not involved in the proliferation or migration of endothelial cells. Stable CRIM1-overexpressing endothelial F-2 cells, termed CR1 and CR2, were constructed, because it was difficult to prepare monolayer HUVECs that expressed high levels of CRIM1. Proliferation was reduced and migration was accelerated in both CR1 and CR2 cells, compared with normal F-2 cells. Furthermore, the transient overexpression of CRIM1 resulted in decreased proliferation and increased migration of bovine aortic endothelial cells. In contrast, neither proliferation nor migration of COS-7 cells were changed by the overexpression of CRIM1. These results demonstrate that increased CRIM1 reduces the proliferation and accelerates the migration of endothelial cells. These CRIM1 effects might contribute to tube formation of endothelial cells. CRIM1 induced by angiogenic factors may serve as a regulator in endothelial cells to switch from proliferating cells to morphological differentiation. - Highlights: • CRIM1 was upregulated only in tubular endothelial cells, but not in monolayers. • Increased CRIM1 reduced the proliferation of endothelial cells. • Increased CRIM1 accelerated the migration of endothelial cells. • Increased CRIM1 had no effect on the proliferation or migration of COS-7 cells.

  8. A New, High Yield, Rapid, and Cost-Effective Protocol to Deprotection of Cysteine-Rich Conopeptide, Omega-Conotoxin MVIIA.

    PubMed

    Eisapoor, Seyed Sahand; Jamili, Shahla; Shahbazzadeh, Delavar; Ghavam Mostafavi, Pargol; Pooshang Bagheri, Kamran

    2016-05-01

    Almost all conopeptides purified from Conus venoms are cysteine-rich peptides. Among them, omega-conotoxin MVIIA, FDA approved peptide drug (Prialt(®)), selected as a cysteine-rich model that its protection from oxidation is critical during solid phase synthesis. Deprotection of cysteines is a crucial step after peptide synthesis. The current study aimed to set up a new highly efficient deprotection protocol for omega-conotoxin MVIIA. Deprotection was performed based on mercury acetate with significant major modification. The protocol accomplished based on the best molar ratio of peptide/mercury/2-ME that adjusted to 0.2 mm/3 mm/10 mm (50 μg/1 mg/10 μL). The yield and purity of omega-conotoxin MVIIA obtained at 93 and 95%, respectively. The total time of protocol shortened to 90 min instead of 6-20 h in routine methods. In this study, omega-conotoxin MVIIA was recovered in high yield and in the shortest time. Despite of other known protocols, molar ratio adjusted to minimum amount. In conclusion, this protocol would be suggested to cost-effective deprotection of thiol groups for similar cysteine-rich peptides.

  9. Domain structure of Lassa virus L protein.

    PubMed

    Brunotte, Linda; Lelke, Michaela; Hass, Meike; Kleinsteuber, Katja; Becker-Ziaja, Beate; Günther, Stephan

    2011-01-01

    The 200-kDa L protein of arenaviruses plays a central role in viral genome replication and transcription. This study aimed at providing evidence for the domain structure of L protein by combining bioinformatics with a stepwise mutagenesis approach using the Lassa virus minireplicon system. Potential interdomain linkers were predicted using various algorithms. The prediction was challenged by insertion of flexible sequences into the predicted linkers. Insertion of 5 or 10 amino acid residues was tolerated at seven sites (S407, G446, G467, G774, G939, S1952, and V2074 in Lassa virus AV). At two of these sites, G467 and G939, L protein could be split into an N-terminal and a C-terminal part, which were able to trans-complement each other and reconstitute a functional complex upon coexpression. Coimmunoprecipitation studies revealed physical interaction between the N- and C-terminal domains, irrespective of whether L protein was split at G467 or G939. In confocal immunofluorescence microscopy, the N-terminal domains showed a dot-like, sometimes perinuclear, cytoplasmic distribution similar to that of full-length L protein, while the C-terminal domains were homogenously distributed in cytoplasm. The latter were redistributed into the dot-like structures upon coexpression with the corresponding N-terminal domain. In conclusion, this study demonstrates two interdomain linkers in Lassa virus L protein, at G467 and G939, suggesting that L protein is composed of at least three structural domains spanning residues 1 to 467, 467 to 939, and 939 to 2220. The first domain seems to mediate accumulation of L protein into cytoplasmic dot-like structures.

  10. Molecular cloning and characterization of two novel genes from hexaploid wheat that encode double PR-1 domains coupled with a receptor-like protein kinase.

    PubMed

    Lu, Shunwen; Faris, Justin D; Edwards, Michael C

    2017-04-01

    Hexaploid wheat (Triticum aestivum L.) contains at least 23 TaPr-1 genes encoding the group 1 pathogenesis-related (PR-1) proteins as identified in our previous work. Here, we report the cloning and characterization of TaPr-1-rk1 and TaPr-1-rk2, two novel genes closely related to the wheat PR-1 family. The two TaPr-1-rk genes are located on homoeologous chromosomes 3D and 3A, respectively, and each contains a large open reading frame (7385 or 6060 bp) that is interrupted by seven introns and subjected to alternative splicing (AS) with five or six isoforms of mRNA transcripts. The deduced full-length TaPR-1-RK1 and TaPR-1-RK2 proteins (95% identity) contain two repeat PR-1 domains, the second of which is fused via a transmembrane helix to a serine/threonine kinase catalytic (STKc) domain characteristic of receptor-like protein kinases. Phylogenetic analysis indicated that the two PR-1 domains of the TaPR-1-RK proteins form sister clades with their homologues identified in other monocot plants and are well separated from stand-alone PR-1 proteins, whereas the STKc domains may have originated from cysteine-rich receptor-like kinases (CRKs). Reverse-transcriptase-PCR analysis revealed that the TaPr-1-rk genes are predominantly expressed in wheat leaves and their expression levels are elevated in response to pathogen attack, such as infection by barley stripe mosaic virus (BSMV), and also to stress conditions, most obviously, to soil salinity. This is the first report of PR-1-CRK hybrid proteins in wheat. The data may shed new insights into the function/evolutionary origin of the PR-1 family and the STKc-mediated defense/stress response pathways in plants.

  11. Discovering interacting domains and motifs in protein-protein interactions.

    PubMed

    Hugo, Willy; Sung, Wing-Kin; Ng, See-Kiong

    2013-01-01

    Many important biological processes, such as the signaling pathways, require protein-protein interactions (PPIs) that are designed for fast response to stimuli. These interactions are usually transient, easily formed, and disrupted, yet specific. Many of these transient interactions involve the binding of a protein domain to a short stretch (3-10) of amino acid residues, which can be characterized by a sequence pattern, i.e., a short linear motif (SLiM). We call these interacting domains and motifs domain-SLiM interactions. Existing methods have focused on discovering SLiMs in the interacting proteins' sequence data. With the recent increase in protein structures, we have a new opportunity to detect SLiMs directly from the proteins' 3D structures instead of their linear sequences. In this chapter, we describe a computational method called SLiMDIet to directly detect SLiMs on domain interfaces extracted from 3D structures of PPIs. SLiMDIet comprises two steps: (1) interaction interfaces belonging to the same domain are extracted and grouped together using structural clustering and (2) the extracted interaction interfaces in each cluster are structurally aligned to extract the corresponding SLiM. Using SLiMDIet, de novo SLiMs interacting with protein domains can be computationally detected from structurally clustered domain-SLiM interactions for PFAM domains which have available 3D structures in the PDB database.

  12. Proteins and cholesterol-rich domains.

    PubMed

    Epand, Richard M

    2008-01-01

    Biological membranes are composed of many molecular species of lipids and proteins. These molecules do not mix ideally. In the plane of the membrane components are segregated into domains that are enriched in certain lipids and proteins. Cholesterol is a membrane lipid that is not uniformly distributed in the membrane. Proteins play an important role in determining cholesterol distribution. Certain types of protein lipidation are known to cause the lipoprotein to sequester with cholesterol and to stabilize cholesterol-rich domains. However, proteins that are excluded from such domains also contribute to the redistribution of cholesterol. One of the motifs that favor interaction with cholesterol is the CRAC motif. The role of the CRAC motif of the gp41 fusogenic protein of HIV is discussed. The distribution of the multianionic lipid, phosphatidylinositol(4,5)bis-phosphate (PtnIns(4,5)P2), is also not uniform in cell membranes. This lipid has several functions in the cell, including a morphological role in determining the sites of attachment of the actin cytoskeleton to the plasma membrane. PtnIns(4,5)P2 is sequestered by proteins having clusters of cationic residues in their sequence. Certain proteins containing cationic clusters also contain moieties such as myristoylation or a CRAC segment that would also endow them with the ability to sequester to a cholesterol-rich domain. These proteins interact with PtnIns(4,5)P2 in a cholesterol-dependent manner forming domains that are enriched in both cholesterol and in PtnIns(4,5)P2 but can also be distinct from liquid-ordered raft-like domains.

  13. Model peptide studies of sequence regions in the elastomeric biomineralization protein, Lustrin A. I. The C-domain consensus-PG-, -NVNCT-motif.

    PubMed

    Zhang, Bo; Wustman, Brandon A; Morse, Daniel; Evans, John Spencer

    2002-05-01

    The lustrin superfamily represents a unique group of biomineralization proteins localized between layered aragonite mineral plates (i.e., nacre layer) in mollusk shell. Recent atomic force microscopy (AFM) pulling studies have demonstrated that the lustrin-containing organic nacre layer in the abalone, Haliotis rufescens, exhibits a typical sawtooth force-extension curve with hysteretic recovery. This force extension behavior is reminiscent of reversible unfolding and refolding in elastomeric proteins such as titin and tenascin. Since secondary structure plays an important role in force-induced protein unfolding and refolding, the question is, What secondary structure(s) exist within the major domains of Lustrin A? Using a model peptide (FPGKNVNCTSGE) representing the 12-residue consensus sequence found near the N-termini of the first eight cysteine-rich domains (C-domains) within the Lustrin A protein, we employed CD, NMR spectroscopy, and simulated annealing/minimization to determine the secondary structure preferences for this sequence. At pH 7.4, we find that the 12-mer sequence adopts a loop conformation, consisting of a "bend" or "turn" involving residues G3-K4 and N7-C8-T9, with extended conformations arising at F1-G3; K4-V6; T9-S10-G11 in the sequence. Minor pH-dependent conformational effects were noted for this peptide; however, there is no evidence for a salt-bridge interaction between the K4 and E12 side chains. The presence of a loop conformation within the highly conserved -PG-, -NVNCT- sequence of C1-C8 domains may have important structural and mechanistic implications for the Lustrin A protein with regard to elastic behavior.

  14. 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.

  15. ECOD: An Evolutionary Classification of Protein Domains

    PubMed Central

    Kinch, Lisa N.; Pei, Jimin; Shi, Shuoyong; Kim, Bong-Hyun; Grishin, Nick V.

    2014-01-01

    Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or “fold”). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies. PMID:25474468

  16. ECOD: an evolutionary classification of protein domains.

    PubMed

    Cheng, Hua; Schaeffer, R Dustin; Liao, Yuxing; Kinch, Lisa N; Pei, Jimin; Shi, Shuoyong; Kim, Bong-Hyun; Grishin, Nick V

    2014-12-01

    Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or "fold"). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies.

  17. cDNA cloning of a mouse mammary epithelial cell surface protein reveals the existence of epidermal growth factor-like domains linked to factor VIII-like sequences

    SciTech Connect

    Stubbs, J.D.; Bui, A. San Francisco State Univ., CA ); Lekutis, C.; Singer, K.L.; Srinivasan, U.; Parry, G. ); Yuzuki, D. )

    1990-11-01

    A 2.1-kilobase cDNA coding for a surface protein of mammary epithelial cells has been isolated from a mouse mammary gland {lambda}gt11 cDNA library. Sequence analysis of this cDNA reveals an open reading frame of 1,389 base pairs that defines a protein with a molecular mass of 51.5 dKa. Structural analysis of the predicted sequence identifies two putative functional domains of the protein: (i) an N-terminal cysteine-rich region that is similar to epidermal growth factor-like domains of Drosophila Notch-1 protein and (ii) a large segment of the sequence that exhibited 54.5% identify with C-terminal domains of human coagulation factors VIII and V. These similarities in structure are used to predict the possible functions of the protein and its means of interaction with the cell surface. mRNA expression was detectable in mammary tissue from nonpregnant animals but was maximal in the lactating gland. In cultured cells, mRNA levels also correlated with the degree of cellular differentiation.

  18. Identifying structural domains of proteins using clustering

    PubMed Central

    2012-01-01

    Background Protein structures are comprised of modular elements known as domains. These units are used and re-used over and over in nature, and usually serve some particular function in the structure. Thus it is useful to be able to break up a protein of interest into its component domains, prior to similarity searching for example. Numerous computational methods exist for doing so, but most operate only on a single protein chain and many are limited to making a series of cuts to the sequence, while domains can and do span multiple chains. Results This study presents a novel clustering-based approach to domain identification, which works equally well on individual chains or entire complexes. The method is simple and fast, taking only a few milliseconds to run, and works by clustering either vectors representing secondary structure elements, or buried alpha-carbon positions, using average-linkage clustering. Each resulting cluster corresponds to a domain of the structure. The method is competitive with others, achieving 70% agreement with SCOP on a large non-redundant data set, and 80% on a set more heavily weighted in multi-domain proteins on which both SCOP and CATH agree. Conclusions It is encouraging that a basic method such as this performs nearly as well or better than some far more complex approaches. This suggests that protein domains are indeed for the most part simply compact regions of structure with a higher density of buried contacts within themselves than between each other. By representing the structure as a set of points or vectors in space, it allows us to break free of any artificial limitations that other approaches may depend upon. PMID:23116496

  19. Inferring Evolutionary Scenarios for Protein Domain Compositions

    NASA Astrophysics Data System (ADS)

    Wiedenhoeft, John; Krause, Roland; Eulenstein, Oliver

    Essential cellular processes are controlled by functional interactions of protein domains, which can be inferred from their evolutionary histories. Methods to reconstruct these histories are challenged by the complexity of reconstructing macroevolutionary events. In this work we model these events using a novel network-like structure that represents the evolution of domain combinations, called plexus. We describe an algorithm to find a plexus that represents the evolution of a given collection of domain histories as phylogenetic trees with the minimum number of macroevolutionary events, and demonstrate its effectiveness in practice.

  20. Genetic variability and natural selection at the ligand domain of the Duffy binding protein in brazilian Plasmodium vivax populations

    PubMed Central

    2010-01-01

    Background Plasmodium vivax malaria is a major public health challenge in Latin America, Asia and Oceania, with 130-435 million clinical cases per year worldwide. Invasion of host blood cells by P. vivax mainly depends on a type I membrane protein called Duffy binding protein (PvDBP). The erythrocyte-binding motif of PvDBP is a 170 amino-acid stretch located in its cysteine-rich region II (PvDBPII), which is the most variable segment of the protein. Methods To test whether diversifying natural selection has shaped the nucleotide diversity of PvDBPII in Brazilian populations, this region was sequenced in 122 isolates from six different geographic areas. A Bayesian method was applied to test for the action of natural selection under a population genetic model that incorporates recombination. The analysis was integrated with a structural model of PvDBPII, and T- and B-cell epitopes were localized on the 3-D structure. Results The results suggest that: (i) recombination plays an important role in determining the haplotype structure of PvDBPII, and (ii) PvDBPII appears to contain neutrally evolving codons as well as codons evolving under natural selection. Diversifying selection preferentially acts on sites identified as epitopes, particularly on amino acid residues 417, 419, and 424, which show strong linkage disequilibrium. Conclusions This study shows that some polymorphisms of PvDBPII are present near the erythrocyte-binding domain and might serve to elude antibodies that inhibit cell invasion. Therefore, these polymorphisms should be taken into account when designing vaccines aimed at eliciting antibodies to inhibit erythrocyte invasion. PMID:21092207

  1. Folding mechanism of a multiple independently-folding domain protein: double B domain of protein A.

    PubMed

    Arora, Pooja; Hammes, Gordon G; Oas, Terrence G

    2006-10-10

    The antibody binding properties of staphylococcal protein A (SpA) can be attributed to the presence of five highly homologous domains (E, D, A, B, and C). Although the folding of the B domain of protein A (BdpA) is well-characterized, the folding behavior of this domain in the context of full-length SpA in the cell remains unexplored. The sequence of the B domain is 89 and 91% identical to those of domains A and C, respectively. We have fused B domain sequences (BBdpA) as a close approximation of the A-B or B-C portion of SpA. Circular dichroism and fluorescence-detected denaturation curves of BBdpA are experimentally indistinguishable from those of BdpA. The rate constants for folding and unfolding from NMR line shape analysis for the single- and double-domain proteins are the same within experimental uncertainties (+/-20%). These results support the designation of SpA as a multiple independently-folding domain (MIFD) protein. We develop a mathematical model that describes the folding thermodynamics and kinetics of MIFD proteins. The model depicts MIFD protein folding and unfolding as a parallel network and explicitly calculates the flux through all parallel pathways. These fluxes are combined to give a complete description of the global thermodynamics and kinetics of the folding and unfolding of MIFD proteins. The global rates for complete folding and unfolding of a MIFD protein and those of the individual domains depend on the stability of the protein. We show that the global unfolding rate of a MIFD protein may be many orders of magnitude slower than that of the constituent domains.

  2. The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling.

    PubMed

    Chintala, Hemabindu; Krupska, Izabela; Yan, Lulu; Lau, Lester; Grant, Maria; Chaqour, Brahim

    2015-07-01

    Physiological angiogenesis depends on the highly coordinated actions of multiple angiogenic regulators. CCN1 is a secreted cysteine-rich and integrin-binding matricellular protein required for proper cardiovascular development. However, our understanding of the cellular origins and activities of this molecule is incomplete. Here, we show that CCN1 is predominantly expressed in angiogenic endothelial cells (ECs) at the leading front of actively growing vessels in the mouse retina. Endothelial deletion of CCN1 in mice using a Cre-Lox system is associated with EC hyperplasia, loss of pericyte coverage and formation of dense retinal vascular networks lacking the normal hierarchical arrangement of arterioles, capillaries and venules. CCN1 is a product of an immediate-early gene that is transcriptionally induced in ECs in response to stimulation by vascular endothelial growth factor (VEGF). We found that CCN1 activity is integrated with VEGF receptor 2 (VEGF-R2) activation and downstream signaling pathways required for tubular network formation. CCN1-integrin binding increased the expression of and association between Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) and VEGF-R2, which leads to rapid dephosphorylation of VEGF-R2 tyrosine, thus preventing EC hyperproliferation. Predictably, CCN1 further brings receptors/signaling molecules into proximity that are otherwise spatially separated. Furthermore, CCN1 induces integrin-dependent Notch activation in cultured ECs, and its targeted gene inactivation in vivo alters Notch-dependent vascular specification and remodeling, suggesting that functional levels of Notch signaling requires CCN1 activity. These data highlight novel functions of CCN1 as a naturally optimized molecule, fine-controlling key processes in physiological angiogenesis and safeguarding against aberrant angiogenic responses.

  3. The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling

    PubMed Central

    Chintala, Hemabindu; Krupska, Izabela; Yan, Lulu; Lau, Lester; Grant, Maria; Chaqour, Brahim

    2015-01-01

    Physiological angiogenesis depends on the highly coordinated actions of multiple angiogenic regulators. CCN1 is a secreted cysteine-rich and integrin-binding matricellular protein required for proper cardiovascular development. However, our understanding of the cellular origins and activities of this molecule is incomplete. Here, we show that CCN1 is predominantly expressed in angiogenic endothelial cells (ECs) at the leading front of actively growing vessels in the mouse retina. Endothelial deletion of CCN1 in mice using a Cre-Lox system is associated with EC hyperplasia, loss of pericyte coverage and formation of dense retinal vascular networks lacking the normal hierarchical arrangement of arterioles, capillaries and venules. CCN1 is a product of an immediate-early gene that is transcriptionally induced in ECs in response to stimulation by vascular endothelial growth factor (VEGF). We found that CCN1 activity is integrated with VEGF receptor 2 (VEGF-R2) activation and downstream signaling pathways required for tubular network formation. CCN1-integrin binding increased the expression of and association between Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) and VEGF-R2, which leads to rapid dephosphorylation of VEGF-R2 tyrosine, thus preventing EC hyperproliferation. Predictably, CCN1 further brings receptors/signaling molecules into proximity that are otherwise spatially separated. Furthermore, CCN1 induces integrin-dependent Notch activation in cultured ECs, and its targeted gene inactivation in vivo alters Notch-dependent vascular specification and remodeling, suggesting that functional levels of Notch signaling requires CCN1 activity. These data highlight novel functions of CCN1 as a naturally optimized molecule, fine-controlling key processes in physiological angiogenesis and safeguarding against aberrant angiogenic responses. PMID:26002917

  4. A small cysteine-rich protein from the Asian soybean rust fungus, Phakopsora pachyrhizi, suppresses plant immunity

    USDA-ARS?s Scientific Manuscript database

    The Asian soybean rust fungus, Phakopsora pachyrhizi, is an obligate pathogen capable of causing explosive disease epidemics that drastically reduce the yield of soybean (Glycine max). Currently, the molecular mechanisms by which P. pachyrhizi and other rust fungi cause disease are poorly understood...

  5. BAR domain proteins regulate Rho GTPase signaling

    PubMed Central

    Aspenström, Pontus

    2014-01-01

    BAR proteins comprise a heterogeneous group of multi-domain proteins with diverse biological functions. The common denominator is the Bin-Amphiphysin-Rvs (BAR) domain that not only confers targeting to lipid bilayers, but also provides scaffolding to mold lipid membranes into concave or convex surfaces. This function of BAR proteins is an important determinant in the dynamic reconstruction of membrane vesicles, as well as of the plasma membrane. Several BAR proteins function as linkers between cytoskeletal regulation and membrane dynamics. These links are provided by direct interactions between BAR proteins and actin-nucleation-promoting factors of the Wiskott-Aldrich syndrome protein family and the Diaphanous-related formins. The Rho GTPases are key factors for orchestration of this intricate interplay. This review describes how BAR proteins regulate the activity of Rho GTPases, as well as how Rho GTPases regulate the function of BAR proteins. This mutual collaboration is a central factor in the regulation of vital cellular processes, such as cell migration, cytokinesis, intracellular transport, endocytosis, and exocytosis. PMID:25483303

  6. Linking in domain-swapped protein dimers

    PubMed Central

    Baiesi, Marco; Orlandini, Enzo; Trovato, Antonio; Seno, Flavio

    2016-01-01

    The presence of knots has been observed in a small fraction of single-domain proteins and related to their thermodynamic and kinetic properties. The exchanging of identical structural elements, typical of domain-swapped proteins, makes such dimers suitable candidates to validate the possibility that mutual entanglement between chains may play a similar role for protein complexes. We suggest that such entanglement is captured by the linking number. This represents, for two closed curves, the number of times that each curve winds around the other. We show that closing the curves is not necessary, as a novel parameter G′, termed Gaussian entanglement, is strongly correlated with the linking number. Based on 110 non redundant domain-swapped dimers, our analysis evidences a high fraction of chains with a significant intertwining, that is with |G′| > 1. We report that Nature promotes configurations with negative mutual entanglement and surprisingly, it seems to suppress intertwining in long protein dimers. Supported by numerical simulations of dimer dissociation, our results provide a novel topology-based classification of protein-swapped dimers together with some preliminary evidence of its impact on their physical and biological properties. PMID:27659606

  7. Protein transduction domain delivery of therapeutic macromolecules.

    PubMed

    van den Berg, Arjen; Dowdy, Steven F

    2011-12-01

    Owing to their unprecedented selectivity, specific activity and potential for 1000+ fold amplification of signal, macromolecules, such as peptides, catalytic protein domains, complete proteins, and oligonucleotides, offer great potential as therapeutic molecules. However, therapeutic use of macromolecules is limited by their poor penetration in tissues and their inability to cross the cellular membrane. The discovery of small cationic peptides that cross the membrane, called Protein Transduction Domains (PTDs) or Cell Penetrating Peptides (CPPs), in the late 1980s opened the door to cellular delivery of large, bioactive molecules. Now, PTDs are widely used as research tools, and impressively, multiple clinical trials are testing PTD-mediated delivery of macromolecular drug conjugates in patients with a variety of diseases. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. hSP, the domain-duplicated homolog of pS2 protein, is co-expressed with pS2 in stomach but not in breast carcinoma.

    PubMed Central

    Tomasetto, C; Rio, M C; Gautier, C; Wolf, C; Hareuveni, M; Chambon, P; Lathe, R

    1990-01-01

    Approximately 50% of human breast tumors secrete a small cysteine-rich protein, pS2, of unknown function. pS2 protein was recently found to be homologous to a porcine protein with hormonogastric activity, pancreatic spasmolytic polypeptide (PSP), in which the 5-cysteine domain present in pS2 is tandemly duplicated. We have characterized cDNA species encoding PSP and its human and mouse counterparts, hSP and mSP. We show that hSP and pS2 are separately encoded in the genome, and that the two proteins are co-expressed in normal stomach epithelium. However, expression of hSP was not detected in breast tumors. Computer analysis revealed that the pattern of conserved cysteine residues in hSP and pS2, the P domain, is present at the N termini of two other mammalian proteins, intestinal sucrase-isomaltase and lysosomal alpha-glucosidase. Images Fig. 3. Fig. 3. Fig. 5. Fig. 7. Fig. 8. Fig. 9. PMID:2303034

  9. Joining RDC data from flexible protein domains

    NASA Astrophysics Data System (ADS)

    Sgheri, Luca

    2010-11-01

    We study the inverse problem of determining the conformational freedom of two protein domains from residual dipolar coupling (RDC) measurements. For each paramagnetic ion attached to one of the domains we obtain a magnetic susceptibility tensor χ from the RDC of couples of atoms of that domain, and a mean paramagnetic susceptibility tensor {\\bar{\\chi }} from the RDC of couples of atoms of the other domain. The latter is an integral average of rotations of χ which depends on the conformational freedom of the two domains. In this paper we consider the case when we have data from paramagnetic ions attached separately to each of the domains. We prove that in this case not all the elements of χ and {\\bar{\\chi }} are independent. We derive the mathematical equations for the compatibility of the measurements and show how these relations can be used in the presence of noisy data to determine a compatible set of χ and {\\bar{\\chi }} with an unconstrained minimization. If available, information about the shape of the noise can be included in the target function. We show that in this case the compatible set obtained has a reduced error with respect to the noisy data.

  10. 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.

  11. Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand

    NASA Astrophysics Data System (ADS)

    Zhang, Xianjun; Zhao, Fei; Wu, Yiran; Yang, Jun; Han, Gye Won; Zhao, Suwen; Ishchenko, Andrii; Ye, Lintao; Lin, Xi; Ding, Kang; Dharmarajan, Venkatasubramanian; Griffin, Patrick R.; Gati, Cornelius; Nelson, Garrett; Hunter, Mark S.; Hanson, Michael A.; Cherezov, Vadim; Stevens, Raymond C.; Tan, Wenfu; Tao, Houchao; Xu, Fei

    2017-05-01

    The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.

  12. The Evolutionary History of Protein Domains Viewed by Species Phylogeny

    PubMed Central

    Yang, Song; Bourne, Philip E.

    2009-01-01

    Background Protein structural domains are evolutionary units whose relationships can be detected over long evolutionary distances. The evolutionary history of protein domains, including the origin of protein domains, the identification of domain loss, transfer, duplication and combination with other domains to form new proteins, and the formation of the entire protein domain repertoire, are of great interest. Methodology/Principal Findings A methodology is presented for providing a parsimonious domain history based on gain, loss, vertical and horizontal transfer derived from the complete genomic domain assignments of 1015 organisms across the tree of life. When mapped to species trees the evolutionary history of domains and domain combinations is revealed, and the general evolutionary trend of domain and combination is analyzed. Conclusions/Significance We show that this approach provides a powerful tool to study how new proteins and functions emerged and to study such processes as horizontal gene transfer among more distant species. PMID:20041107

  13. 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.

  14. Phylogenetic Analysis of Brassica rapa MATH-Domain Proteins

    PubMed Central

    Zhao, Liming; Huang, Yong; Hu, Yan; He, Xiaoli; Shen, Wenhui; Liu, Chunlin; Ruan, Ying

    2013-01-01

    The MATH (meprin and TRAF-C homology) domain is a fold of seven anti-parallel β-helices involved in protein-protein interaction. Here, we report the identification and characterization of 90 MATH-domain proteins from the Brassica rapa genome. By sequence analysis together with MATH-domain proteins from other species, the B. rapa MATH-domain proteins can be grouped into 6 classes. Class-I protein has one or several MATH domains without any other recognizable domain; Class-II protein contains a MATH domain together with a conserved BTB (Broad Complex, Tramtrack, and Bric-a-Brac ) domain; Class-III protein belongs to the MATH/Filament domain family; Class-IV protein contains a MATH domain frequently combined with some other domains; Class-V protein has a relative long sequence but contains only one MATH domain; Class-VI protein is characterized by the presence of Peptidase and UBQ (Ubiquitinylation) domains together with one MATH domain. As part of our study regarding seed development of B. rapa, six genes are screened by SSH (Suppression Subtractive Hybridization) and their expression levels are analyzed in combination with seed developmental stages, and expression patterns suggested that Bra001786, Bra03578 and Bra036572 may be seed development specific genes, while Bra001787, Bra020541 and Bra040904 may be involved in seed and flower organ development. This study provides the first characterization of the MATH domain proteins in B. rapa PMID:24179444

  15. Evaluating, Comparing, and Interpreting Protein Domain Hierarchies

    PubMed Central

    2014-01-01

    Abstract Arranging protein domain sequences hierarchically into evolutionarily divergent subgroups is important for investigating evolutionary history, for speeding up web-based similarity searches, for identifying sequence determinants of protein function, and for genome annotation. However, whether or not a particular hierarchy is optimal is often unclear, and independently constructed hierarchies for the same domain can often differ significantly. This article describes methods for statistically evaluating specific aspects of a hierarchy, for probing the criteria underlying its construction and for direct comparisons between hierarchies. Information theoretical notions are used to quantify the contributions of specific hierarchical features to the underlying statistical model. Such features include subhierarchies, sequence subgroups, individual sequences, and subgroup-associated signature patterns. Underlying properties are graphically displayed in plots of each specific feature's contributions, in heat maps of pattern residue conservation, in “contrast alignments,” and through cross-mapping of subgroups between hierarchies. Together, these approaches provide a deeper understanding of protein domain functional divergence, reveal uncertainties caused by inconsistent patterns of sequence conservation, and help resolve conflicts between competing hierarchies. PMID:24559108

  16. Independent Structural Domains in Paramyxovirus Polymerase Protein*

    PubMed Central

    Dochow, Melanie; Krumm, Stefanie A.; Crowe, James E.; Moore, Martin L.; Plemper, Richard K.

    2012-01-01

    All enzymatic activities required for genomic replication and transcription of nonsegmented negative strand RNA viruses (or Mononegavirales) are believed to be concentrated in the viral polymerase (L) protein. However, our insight into the organization of these different enzymatic activities into a bioactive tertiary structure remains rudimentary. Fragments of Mononegavirales polymerases analyzed to date cannot restore bioactivity through trans-complementation, unlike the related L proteins of segmented NSVs. We investigated the domain organization of phylogenetically diverse Paramyxovirus L proteins derived from measles virus (MeV), Nipah virus (NiV), and respiratory syncytial virus (RSV). Through a comprehensive in silico and experimental analysis of domain intersections, we defined MeV L position 615 as an interdomain candidate in addition to the previously reported residue 1708. Only position 1708 of MeV and the homologous positions in NiV and RSV L also tolerated the insertion of epitope tags. Splitting of MeV L at residue 1708 created fragments that were unable to physically interact and trans-complement, but strikingly, these activities were reconstituted by the addition of dimerization tags to the fragments. Equivalently split fragments of NiV, RSV, and MeV L oligomerized with comparable efficiency in all homo- and heterotypic combinations, but only the homotypic pairs were able to trans-complement. These results demonstrate that synthesis as a single polypeptide is not required for the Mononegavirales polymerases to adopt a proper tertiary conformation. Paramyxovirus polymerases are composed of at least two truly independent folding domains that lack a traditional interface but require molecular compatibility for bioactivity. The functional probing of the L domain architecture through trans-complementation is anticipated to be applicable to all Mononegavirales polymerases. PMID:22215662

  17. Synthetic mimetics of protein secondary structure domains

    PubMed Central

    Ross, Nathan T.; Katt, William P.; Hamilton, Andrew D.

    2010-01-01

    Proteins modulate the majority of all biological functions and are primarily composed of highly organized secondary structural elements such as helices, turns and sheets. Many of these functions are affected by a small number of key protein–protein contacts, often involving one or more of these well-defined structural elements. Given the ubiquitous nature of these protein recognition domains, their mimicry by peptidic and non-peptidic scaffolds has become a major focus of contemporary research. This review examines several key advances in secondary structure mimicry over the past several years, particularly focusing upon scaffolds that show not only promising projection of functional groups, but also a proven effect in biological systems. PMID:20123744

  18. 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.

  19. Plant SAM-Domain Proteins Start to Reveal Their Roles.

    PubMed

    Denay, Grégoire; Vachon, Gilles; Dumas, Renaud; Zubieta, Chloe; Parcy, François

    2017-08-01

    Proteins often act in complexes assembled via protein-protein interaction domains. The sterile alpha motif (SAM) domain is one of the most prominent interaction domains in animals and is present in proteins of diverse functions. This domain allows head-to-tail closed oligomerisation or polymer formation resulting in homo- and/or heterocomplexes that have been shown to be important for proper protein localisation and function. In plants this domain is also present but has been poorly studied except for recent studies on the LEAFY floral regulator and the tRNA import component (TRIC)1/2 proteins. Here we catalogue SAM domain-containing proteins from arabidopsis (Arabidopsis thaliana), compare plant and other eukaryotic SAM domains, and perform homology modelling to probe plant SAM domain interaction capabilities. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Evolution of Protein Domain Repeats in Metazoa

    PubMed Central

    Schüler, Andreas; Bornberg-Bauer, Erich

    2016-01-01

    Repeats are ubiquitous elements of proteins and they play important roles for cellular function and during evolution. Repeats are, however, also notoriously difficult to capture computationally and large scale studies so far had difficulties in linking genetic causes, structural properties and evolutionary trajectories of protein repeats. Here we apply recently developed methods for repeat detection and analysis to a large dataset comprising over hundred metazoan genomes. We find that repeats in larger protein families experience generally very few insertions or deletions (indels) of repeat units but there is also a significant fraction of noteworthy volatile outliers with very high indel rates. Analysis of structural data indicates that repeats with an open structure and independently folding units are more volatile and more likely to be intrinsically disordered. Such disordered repeats are also significantly enriched in sites with a high functional potential such as linear motifs. Furthermore, the most volatile repeats have a high sequence similarity between their units. Since many volatile repeats also show signs of recombination, we conclude they are often shaped by concerted evolution. Intriguingly, many of these conserved yet volatile repeats are involved in host-pathogen interactions where they might foster fast but subtle adaptation in biological arms races. Key Words: protein evolution, domain rearrangements, protein repeats, concerted evolution. PMID:27671125

  1. Stabilization of the cysteine-rich conotoxin MrIA by using a 1,2,3-triazole as a disulfide bond mimetic.

    PubMed

    Gori, Alessandro; Wang, Ching-I A; Harvey, Peta J; Rosengren, K Johan; Bhola, Rebecca F; Gelmi, Maria L; Longhi, Renato; Christie, Macdonald J; Lewis, Richard J; Alewood, Paul F; Brust, Andreas

    2015-01-19

    The design of disulfide bond mimetics is an important strategy for optimising cysteine-rich peptides in drug development. Mimetics of the drug lead conotoxin MrIA, in which one disulfide bond is selectively replaced of by a 1,4-disubstituted-1,2,3-triazole bridge, are described. Sequential copper-catalyzed azide-alkyne cycloaddition (CuAAC; click reaction) followed by disulfide formation resulted in the regioselective syntheses of triazole-disulfide hybrid MrIA analogues. Mimetics with a triazole replacing the Cys4-Cys13 disulfide bond retained tertiary structure and full in vitro and in vivo activity as norepinephrine reuptake inhibitors. Importantly, these mimetics are resistant to reduction in the presence of glutathione, thus resulting in improved plasma stability and increased suitability for drug development. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Extra domains in secondary transport carriers and channel proteins.

    PubMed

    Barabote, Ravi D; Tamang, Dorjee G; Abeywardena, Shannon N; Fallah, Neda S; Fu, Jeffrey Yu Chung; Lio, Jeffrey K; Mirhosseini, Pegah; Pezeshk, Ronnie; Podell, Sheila; Salampessy, Marnae L; Thever, Mark D; Saier, Milton H

    2006-10-01

    "Extra" domains in members of the families of secondary transport carrier and channel proteins provide secondary functions that expand, amplify or restrict the functional nature of these proteins. Domains in secondary carriers include TrkA and SPX domains in DASS family members, DedA domains in TRAP-T family members (both of the IT superfamily), Kazal-2 and PDZ domains in OAT family members (of the MF superfamily), USP, IIA(Fru) and TrkA domains in ABT family members (of the APC superfamily), ricin domains in OST family members, and TrkA domains in AAE family members. Some transporters contain highly hydrophilic domains consisting of multiple repeat units that can also be found in proteins of dissimilar function. Similarly, transmembrane alpha-helical channel-forming proteins contain unique, conserved, hydrophilic domains, most of which are not found in carriers. In some cases the functions of these domains are known. They may be ligand binding domains, phosphorylation domains, signal transduction domains, protein/protein interaction domains or complex carbohydrate-binding domains. These domains mediate regulation, subunit interactions, or subcellular targeting. Phylogenetic analyses show that while some of these domains are restricted to closely related proteins derived from specific organismal types, others are nearly ubiquitous within a particular family of transporters and occur in a tremendous diversity of organisms. The former probably became associated with the transporters late in the evolutionary process; the latter probably became associated with the carriers much earlier. These domains can be located at either end of the transporter or in a central region, depending on the domain and transporter family. These studies provide useful information about the evolution of extra domains in channels and secondary carriers and provide novel clues concerning function.

  3. The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation*

    PubMed Central

    Ho, Tina; Watt, Brenda; Spruce, Lynn A.; Seeholzer, Steven H.; Marks, Michael S.

    2016-01-01

    The formation of functional amyloid must be carefully regulated to prevent the accumulation of potentially toxic products. Premelanosome protein (PMEL) forms non-toxic functional amyloid fibrils that assemble into sheets upon which melanins ultimately are deposited within the melanosomes of pigment cells. PMEL is synthesized in the endoplasmic reticulum but forms amyloid only within post-Golgi melanosome precursors; thus, PMEL must traverse the secretory pathway in a non-amyloid form. Here, we identified two pre-amyloid PMEL intermediates that likely regulate the timing of fibril formation. Analyses by non-reducing SDS-PAGE, size exclusion chromatography, and sedimentation velocity revealed two native high Mr disulfide-bonded species that contain Golgi-modified forms of PMEL. These species correspond to disulfide bond-containing dimeric and monomeric PMEL isoforms that contain no other proteins as judged by two-dimensional PAGE of metabolically labeled/immunoprecipitated PMEL and by mass spectrometry of affinity-purified complexes. Metabolic pulse-chase analyses, small molecule inhibitor treatments, and evaluation of site-directed mutants suggest that the PMEL dimer forms around the time of endoplasmic reticulum exit and is resolved by disulfide bond rearrangement into a monomeric form within the late Golgi or a post-Golgi compartment. Mutagenesis of individual cysteine residues within the non-amyloid cysteine-rich Kringle-like domain stabilizes the disulfide-bonded dimer and impairs fibril formation as determined by electron microscopy. Our data show that the Kringle-like domain facilitates the resolution of disulfide-bonded PMEL dimers and promotes PMEL functional amyloid formation, thereby suggesting that PMEL dimers must be resolved to monomers to generate functional amyloid fibrils. PMID:26694611

  4. Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain.

    PubMed

    Bocian-Ostrzycka, Katarzyna M; Łasica, Anna M; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J; Drabik, Karolina; Dobosz, Aneta M; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K

    2015-01-01

    Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation - periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity.

  5. Functional and evolutionary analyses of Helicobacter pylori HP0231 (DsbK) protein with strong oxidative and chaperone activity characterized by a highly diverged dimerization domain

    PubMed Central

    Bocian-Ostrzycka, Katarzyna M.; Łasica, Anna M.; Dunin-Horkawicz, Stanisław; Grzeszczuk, Magdalena J.; Drabik, Karolina; Dobosz, Aneta M.; Godlewska, Renata; Nowak, Elżbieta; Collet, Jean-Francois; Jagusztyn-Krynicka, Elżbieta K.

    2015-01-01

    Helicobacter pylori does not encode the classical DsbA/DsbB oxidoreductases that are crucial for oxidative folding of extracytoplasmic proteins. Instead, this microorganism encodes an untypical two proteins playing a role in disulfide bond formation – periplasmic HP0231, which structure resembles that of EcDsbC/DsbG, and its redox partner, a membrane protein HpDsbI (HP0595) with a β-propeller structure. The aim of presented work was to assess relations between HP0231 structure and function. We showed that HP0231 is most closely related evolutionarily to the catalytic domain of DsbG, even though it possesses a catalytic motif typical for canonical DsbA proteins. Similarly, the highly diverged N-terminal dimerization domain is homologous to the dimerization domain of DsbG. To better understand the functioning of this atypical oxidoreductase, we examined its activity using in vivo and in vitro experiments. We found that HP0231 exhibits oxidizing and chaperone activities but no isomerizing activity, even though H. pylori does not contain a classical DsbC. We also show that HP0231 is not involved in the introduction of disulfide bonds into HcpC (Helicobacter cysteine-rich protein C), a protein involved in the modulation of the H. pylori interaction with its host. Additionally, we also constructed a truncated version of HP0231 lacking the dimerization domain, denoted HP0231m, and showed that it acts in Escherichia coli cells in a DsbB-dependent manner. In contrast, HP0231m and classical monomeric EcDsbA (E. coli DsbA protein) were both unable to complement the lack of HP0231 in H. pylori cells, though they exist in oxidized forms. HP0231m is inactive in the insulin reduction assay and possesses high chaperone activity, in contrast to EcDsbA. In conclusion, HP0231 combines oxidative functions characteristic of DsbA proteins and chaperone activity characteristic of DsbC/DsbG, and it lacks isomerization activity. PMID:26500620

  6. The expanding superfamily of gelsolin homology domain proteins.

    PubMed

    Ghoshdastider, Umesh; Popp, David; Burtnick, Leslie D; Robinson, Robert C

    2013-11-01

    The gelsolin homology (GH) domain has been found to date exclusively in actin-binding proteins. In humans, three copies of the domain are present in CapG, five copies in supervillin, and six copies each in adseverin, gelsolin, flightless I and the villins: villin, advillin and villin-like protein. Caenorhabditis elegans contains a four-GH-domain protein, GSNL-1. These architectures are predicted to have arisen from gene triplication followed by gene duplication to result in the six-domain protein. The subsequent loss of one, two or three domains produced the five-, four-, and three-domain proteins, respectively. Here we conducted BLAST and hidden Markov based searches of UniProt and NCBI databases to identify novel gelsolin domain containing proteins. The variety in architectures suggests that the GH domain has been tested in many molecular constructions during evolution. Of particular note is flightless-like I protein (FLIIL1) from Entamoeba histolytica, which combines a leucine rich repeats (LRR) domain, seven GH domains, and a headpiece domain, thus combining many of the features of flightless I with those of villin or supervillin. As such, the GH domain superfamily appears to have developed along complex routes. The distribution of these proteins was analyzed in the 343 completely sequenced genomes, mapped onto the tree of life, and phylogenetic trees of the proteins were constructed to gain insight into their evolution. © 2013 Wiley Periodicals, Inc. Copyright © 2013 Wiley Periodicals, Inc.

  7. Stochastic single-molecule dynamics of synaptic membrane protein domains

    NASA Astrophysics Data System (ADS)

    Kahraman, Osman; Li, Yiwei; Haselwandter, Christoph A.

    2016-09-01

    Motivated by single-molecule experiments on synaptic membrane protein domains, we use a stochastic lattice model to study protein reaction and diffusion processes in crowded membranes. We find that the stochastic reaction-diffusion dynamics of synaptic proteins provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the single-molecule trajectories observed for synaptic proteins, and spatially inhomogeneous protein lifetimes at the cell membrane. Our results suggest that central aspects of the single-molecule and collective dynamics observed for membrane protein domains can be understood in terms of stochastic reaction-diffusion processes at the cell membrane.

  8. Protein domain assignment from the recurrence of locally similar structures

    PubMed Central

    Tai, Chin-Hsien; Sam, Vichetra; Gibrat, Jean-Francois; Garnier, Jean; Munson, Peter J.

    2010-01-01

    Domains are basic units of protein structure and essential for exploring protein fold space and structure evolution. With the structural genomics initiative, the number of protein structures in the Protein Databank (PDB) is increasing dramatically and domain assignments need to be done automatically. Most existing structural domain assignment programs define domains using the compactness of the domains and/or the number and strength of intra-domain versus inter-domain contacts. Here we present a different approach based on the recurrence of locally similar structural pieces (LSSPs) found by one-against-all structure comparisons with a dataset of 6,373 protein chains from the PDB. Residues of the query protein are clustered using LSSPs via three different procedures to define domains. This approach gives results that are comparable to several existing programs that use geometrical and other structural information explicitly. Remarkably, most of the proteins that contribute the LSSPs defining a domain do not themselves contain the domain of interest. This study shows that domains can be defined by a collection of relatively small locally similar structural pieces containing, on average, four secondary structure elements. In addition, it indicates that domains are indeed made of recurrent small structural pieces that are used to build protein structures of many different folds as suggested by recent studies. PMID:21287617

  9. Comparative analysis of ATRX, a chromatin remodeling protein.

    PubMed

    Park, Daniel J; Pask, Andrew J; Huynh, Kim; Renfree, Marilyn B; Harley, Vincent R; Graves, Jennifer A Marshall

    2004-09-15

    The ATRX protein, associated with X-linked alpha-thalassaemia, mental retardation and developmental abnormalities including genital dysgenesis, has been proposed to function as a global transcriptional regulator within a multi-protein complex. However, an understanding of the composition and mechanics of this machinery has remained elusive. We applied inter-specific comparative analysis to identify conserved elements which may be involved in regulating the conformation of chromatin. As part of this study, we cloned and sequenced the entire translatable coding region (7.4 kb) of the ATRX gene from a model marsupial (tammar wallaby, Macropus eugenii). We identify an ATRX ancestral core, conserved between plants, fish and mammals, comprising the cysteine-rich and SWI2/SNF2 helicase-like regions and protein interaction domains. Our data are consistent with the model of the cysteine-rich region as a DNA-binding zinc finger adjacent to a protein-binding (plant homeodomain-like) domain. Alignment of vertebrate ATRX sequences highlights other conserved elements, including a negatively charged mammalian sequence which we propose to be involved in binding of positively charged histone tails.

  10. The disintegrin domain of ADAM9: a ligand for multiple β1 renal integrins

    PubMed Central

    2004-01-01

    Renal tubular epithelial cells in all nephron segments express a distinct member of the metalloprotease-disintegrin family, ADAM9 (a disintegrin and metalloprotease 9), in a punctate basolateral distribution co-localized to the β1 integrin chain [Mahimkar, Baricos, Visaya, Pollock and Lovett (2000) J. Am. Soc. Nephrol. 11, 595–603]. Discrete segments of the nephron express several defined β1 integrins, suggesting that ADAM9 interacts with multiple renal integrins and thereby regulates epithelial cell–matrix interactions. Intact ADAM9 and a series of deletion constructs sequentially lacking the metalloprotease domain and the disintegrin domain were assembled as chimaeras with a C-terminal GFP (green fluorescent protein) tag. Stable expression of the ADAM9/GFP protein on the surface of HEK-293 cells (human embryonic kidney 293 cells) significantly decreased adhesion to types I and IV collagen, vitronectin and laminin, but had little effect on adhesion to fibronectin. Expression of the disintegrin/cysteine-rich/GFP construct yielded a similar, but more marked pattern of decreased adhesion. Expression of the cysteine-rich/GFP construct had no effect on adhesion, indicating that the disintegrin domain was responsible for the competitive inhibition of cell–matrix binding. To define the specific renal tubular β1 integrins interacting with the ADAM9 disintegrin domain, a recombinant GST (glutathione S-transferase)-disintegrin protein was used as a substrate in adhesion assays in the presence or absence of specific integrin-blocking antibodies. Inclusion of antibodies to α1, α3, α6, αv and β1 blocked adhesion of HEK-293 cells to GST-disintegrin protein. Immobilized GST-disintegrin domain perfused with renal cortical lysates specifically recovered the α3, α6, αv and β1 integrin chains by Western analysis. It is concluded that ADAM9 is a polyvalent ligand, through its disintegrin domain, for multiple renal integrins of the β1 class. PMID:15361064

  11. The disintegrin domain of ADAM9: a ligand for multiple beta1 renal integrins.

    PubMed

    Mahimkar, Rajeev M; Visaya, Orvin; Pollock, Allan S; Lovett, David H

    2005-01-15

    Renal tubular epithelial cells in all nephron segments express a distinct member of the metalloprotease-disintegrin family, ADAM9 (a disintegrin and metalloprotease 9), in a punctate basolateral distribution co-localized to the beta1 integrin chain [Mahimkar, Baricos, Visaya, Pollock and Lovett (2000) J. Am. Soc. Nephrol. 11, 595-603]. Discrete segments of the nephron express several defined beta1 integrins, suggesting that ADAM9 interacts with multiple renal integrins and thereby regulates epithelial cell-matrix interactions. Intact ADAM9 and a series of deletion constructs sequentially lacking the metalloprotease domain and the disintegrin domain were assembled as chimaeras with a C-terminal GFP (green fluorescent protein) tag. Stable expression of the ADAM9/GFP protein on the surface of HEK-293 cells (human embryonic kidney 293 cells) significantly decreased adhesion to types I and IV collagen, vitronectin and laminin, but had little effect on adhesion to fibronectin. Expression of the disintegrin/cysteine-rich/GFP construct yielded a similar, but more marked pattern of decreased adhesion. Expression of the cysteine-rich/GFP construct had no effect on adhesion, indicating that the disintegrin domain was responsible for the competitive inhibition of cell-matrix binding. To define the specific renal tubular beta1 integrins interacting with the ADAM9 disintegrin domain, a recombinant GST (glutathione S-transferase)-disintegrin protein was used as a substrate in adhesion assays in the presence or absence of specific integrin-blocking antibodies. Inclusion of antibodies to alpha1, alpha3, alpha6, alphav and beta1 blocked adhesion of HEK-293 cells to GST-disintegrin protein. Immobilized GST-disintegrin domain perfused with renal cortical lysates specifically recovered the alpha3, alpha6, alphav and beta1 integrin chains by Western analysis. It is concluded that ADAM9 is a polyvalent ligand, through its disintegrin domain, for multiple renal integrins of the beta1

  12. Tetramer formation in Arabidopsis MADS domain proteins: analysis of a protein-protein interaction network

    PubMed Central

    2014-01-01

    Background MADS domain proteins are transcription factors that coordinate several important developmental processes in plants. These proteins interact with other MADS domain proteins to form dimers, and it has been proposed that they are able to associate as tetrameric complexes that regulate transcription of target genes. Whether the formation of functional tetramers is a widespread property of plant MADS domain proteins, or it is specific to few of these transcriptional regulators remains unclear. Results We analyzed the structure of the network of physical interactions among MADS domain proteins in Arabidopsis thaliana. We determined the abundance of subgraphs that represent the connection pattern expected for a MADS domain protein heterotetramer. These subgraphs were significantly more abundant in the MADS domain protein interaction network than in randomized analogous networks. Importantly, these subgraphs are not significantly frequent in a protein interaction network of TCP plant transcription factors, when compared to expectation by chance. In addition, we found that MADS domain proteins in tetramer-like subgraphs are more likely to be expressed jointly than proteins in other subgraphs. This effect is mainly due to proteins in the monophyletic MIKC clade, as there is no association between tetramer-like subgraphs and co-expression for proteins outside this clade. Conclusions Our results support that the tendency to form functional tetramers is widespread in the MADS domain protein-protein interaction network. Our observations also suggest that this trend is prevalent, or perhaps exclusive, for proteins in the MIKC clade. Because it is possible to retrodict several experimental results from our analyses, our work can be an important aid to make new predictions and facilitates experimental research on plant MADS domain proteins. PMID:24468197

  13. A systematic analysis reveals an essential role for high-affinity iron uptake system, haemolysin and CFEM domain-containing protein in iron homoeostasis and virulence in Candida glabrata.

    PubMed

    Srivastava, Vivek Kumar; Suneetha, Korivi Jyothiraj; Kaur, Rupinder

    2014-10-01

    Iron is an essential nutrient for all living organisms and human pathogens employ a battery of factors to scavenge iron from the high-affinity iron-binding host proteins. In the present study, we have elucidated, via a candidate gene approach, major iron acquisition and homoeostatic mechanisms operational in an opportunistic human fungal pathogen Candida glabrata. Phenotypic, biochemical and molecular analysis of a set of 13 C. glabrata strains, deleted for proteins potentially implicated in iron metabolism, revealed that the high-affinity reductive iron uptake system is required for utilization of alternate carbon sources and for growth under both in vitro iron-limiting and in vivo conditions. Furthermore, we show for the first time that the cysteine-rich CFEM (common in fungal extracellular membranes) domain-containing cell wall structural protein, CgCcw14, and a putative haemolysin, CgMam3, are essential for maintenance of intracellular iron content, adherence to epithelial cells and virulence. Consistent with their roles in iron homoeostasis, mitochondrial aconitase activity was lower and higher in mutants disrupted for high-affinity iron transport, and haemolysin respectively. Additionally, we present evidence that the mitochondrial frataxin, CgYfh1, is pivotal to iron metabolism. Besides yielding insights into major in vitro and in vivo iron acquisition strategies, our findings establish high-affinity iron uptake mechanisms as critical virulence determinants in C. glabrata.

  14. 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

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

    PubMed

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

    2008-05-06

    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.

  16. Purification and Structural Analysis of LEM-Domain Proteins.

    PubMed

    Herrada, Isaline; Bourgeois, Benjamin; Samson, Camille; Buendia, Brigitte; Worman, Howard J; Zinn-Justin, Sophie

    2016-01-01

    LAP2-emerin-MAN1 (LEM)-domain proteins are modular proteins characterized by the presence of a conserved motif of about 50 residues. Most LEM-domain proteins localize at the inner nuclear membrane, but some are also found in the endoplasmic reticulum or nuclear interior. Their architecture has been analyzed by predicting the limits of their globular domains, determining the 3D structure of these domains and in a few cases calculating the 3D structure of specific domains bound to biological targets. The LEM domain adopts an α-helical fold also found in SAP and HeH domains of prokaryotes and unicellular eukaryotes. The LEM domain binds to BAF (barrier-to-autointegration factor; BANF1), which interacts with DNA and tethers chromatin to the nuclear envelope. LAP2 isoforms also share an N-terminal LEM-like domain, which binds DNA. The structure and function of other globular domains that distinguish LEM-domain proteins from each other have been characterized, including the C-terminal dimerization domain of LAP2α and C-terminal WH and UHM domains of MAN1. LEM-domain proteins also have large intrinsically disordered regions that are involved in intra- and intermolecular interactions and are highly regulated by posttranslational modifications in vivo.

  17. Selectivity filters and cysteine-rich extracellular loops in voltage-gated sodium, calcium, and NALCN channels

    PubMed Central

    Stephens, Robert F.; Guan, W.; Zhorov, Boris S.; Spafford, J. David

    2015-01-01

    How nature discriminates sodium from calcium ions in eukaryotic channels has been difficult to resolve because they contain four homologous, but markedly different repeat domains. We glean clues from analyzing the changing pore region in sodium, calcium and NALCN channels, from single-cell eukaryotes to mammals. Alternative splicing in invertebrate homologs provides insights into different structural features underlying calcium and sodium selectivity. NALCN generates alternative ion selectivity with splicing that changes the high field strength (HFS) site at the narrowest level of the hourglass shaped pore where the selectivity filter is located. Alternative splicing creates NALCN isoforms, in which the HFS site has a ring of glutamates contributed by all four repeat domains (EEEE), or three glutamates and a lysine residue in the third (EEKE) or second (EKEE) position. Alternative splicing provides sodium and/or calcium selectivity in T-type channels with extracellular loops between S5 and P-helices (S5P) of different lengths that contain three or five cysteines. All eukaryotic channels have a set of eight core cysteines in extracellular regions, but the T-type channels have an infusion of 4–12 extra cysteines in extracellular regions. The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in NALCN, Cav, and Nav channels, and pairing of shorter loops in Domains II and IV in T-type channel through disulfide bonds involving T-type specific cysteines. Extracellular turrets of increasing lengths in potassium channels (Kir2.2, hERG, and K2P1) contribute to a changing landscape above the pore selectivity filter that can limit drug access and serve as an ion pre-filter before ions reach the pore selectivity filter below. Pairing of extended loops likely contributes to the large extracellular appendage as seen in single particle electron cryo-microscopy images of the eel Nav1 channel. PMID

  18. AIDA: ab initio domain assembly for automated multi-domain protein structure prediction and domain–domain interaction prediction

    PubMed Central

    Xu, Dong; Jaroszewski, Lukasz; Li, Zhanwen; Godzik, Adam

    2015-01-01

    Motivation: Most proteins consist of multiple domains, independent structural and evolutionary units that are often reshuffled in genomic rearrangements to form new protein architectures. Template-based modeling methods can often detect homologous templates for individual domains, but templates that could be used to model the entire query protein are often not available. Results: We have developed a fast docking algorithm ab initio domain assembly (AIDA) for assembling multi-domain protein structures, guided by the ab initio folding potential. This approach can be extended to discontinuous domains (i.e. domains with ‘inserted’ domains). When tested on experimentally solved structures of multi-domain proteins, the relative domain positions were accurately found among top 5000 models in 86% of cases. AIDA server can use domain assignments provided by the user or predict them from the provided sequence. The latter approach is particularly useful for automated protein structure prediction servers. The blind test consisting of 95 CASP10 targets shows that domain boundaries could be successfully determined for 97% of targets. Availability and implementation: The AIDA package as well as the benchmark sets used here are available for download at http://ffas.burnham.org/AIDA/. Contact: adam@sanfordburnham.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25701568

  19. A kinetic study of domain swapping of Protein L.

    PubMed

    Moschen, Thomas; Tollinger, Martin

    2014-04-14

    Domain swapping of the B1 domain of Protein L isolated from Peptostreptococcus magnus can be induced by rational mutation. We show that the monomeric and the domain swapped dimeric forms of the G55A mutant of Protein L are directly observable by solution NMR spectroscopy under equilibrium conditions. The kinetics of the domain swapping process can be characterized by real-time NMR spectroscopic techniques, and the free energy landscape for domain swapping of Protein L can be probed by variation of denaturant concentration. Our data suggest that domain swapping of Protein L proceeds through a compact transition state, with an accessible surface area that is similar in size to the transition state for folding and unfolding. It is thus conceivable that domain swapping and folding of Protein L are mechanistically related at the level of their rate-limiting step(s), which might represent a branching point along the folding pathway.

  20. The architectural design of networks of protein domain architectures.

    PubMed

    Hsu, Chia-Hsin; Chen, Chien-Kuo; Hwang, Ming-Jing

    2013-08-23

    Protein domain architectures (PDAs), in which single domains are linked to form multiple-domain proteins, are a major molecular form used by evolution for the diversification of protein functions. However, the design principles of PDAs remain largely uninvestigated. In this study, we constructed networks to connect domain architectures that had grown out from the same single domain for every single domain in the Pfam-A database and found that there are three main distinctive types of these networks, which suggests that evolution can exploit PDAs in three different ways. Further analysis showed that these three different types of PDA networks are each adopted by different types of protein domains, although many networks exhibit the characteristics of more than one of the three types. Our results shed light on nature's blueprint for protein architecture and provide a framework for understanding architectural design from a network perspective.

  1. WW domain-containing proteins: retrospectives and the future.

    PubMed

    Salah, Zaidoun; Alian, Akram; Aqeilan, Rami I

    2012-01-01

    WW domains are protein modules that mediate protein-protein interactions through recognition of proline-rich peptide motifs (PRM) and phosphorylated serine/threonine-proline sites. WW domains are found in many different structural and signaling proteins that are involved in a variety of cellular processes, including RNA transcription and processing, protein trafficking and stability, receptor signaling, and control of the cytoskeleton. WW domain-containing proteins and complexes have been implicated in major human diseases including cancer as well as in major signaling cascades such as the Hippo tumor suppressor pathway, making them targets for new diagnostics and therapeutics. In this review, we discuss how WW domains provide versatile platforms that link individual proteins into physiologically important networks and the indispensible role of WW domain-containing proteins in biology and pathology, especially tumorogenesis.

  2. Structural and Biochemical Characterization of Chlamydia trachomatis DsbA Reveals a Cysteine-Rich and Weakly Oxidising Oxidoreductase

    PubMed Central

    Byriel, Karl; Huston, Wilhelmina M.; Furlong, Emily; Heras, Begoña; Martin, Jennifer L.

    2016-01-01

    The Gram negative bacteria Chlamydia trachomatis is an obligate intracellular human pathogen that can cause pelvic inflammatory disease, infertility and blinding trachoma. C. trachomatis encodes a homolog of the dithiol oxidoreductase DsbA. Bacterial DsbA proteins introduce disulfide bonds to folding proteins providing structural bracing for secreted virulence factors, consequently these proteins are potential targets for antimicrobial drugs. Despite sharing functional and structural characteristics, the DsbA enzymes studied to date vary widely in their redox character. In this study we show that the truncated soluble form of the predicted membrane anchored protein C. trachomatis DsbA (CtDsbA) has oxidase activity and redox properties broadly similar to other characterized DsbA proteins. However CtDsbA is distinguished from other DsbAs by having six cysteines, including a second disulfide bond, and an unusual dipeptide sequence in its catalytic motif (Cys-Ser-Ala-Cys). We report the 2.7 Å crystal structure of CtDsbA revealing a typical DsbA fold, which is most similar to that of DsbA-II type proteins. Consistent with this, the catalytic surface of CtDsbA is negatively charged and lacks the hydrophobic groove found in EcDsbA and DsbAs from other enterobacteriaceae. Biochemical characterization of CtDsbA reveals it to be weakly oxidizing compared to other DsbAs and with only a mildly destabilizing active site disulfide bond. Analysis of the crystal structure suggests that this redox character is consistent with a lack of contributing factors to stabilize the active site nucleophilic thiolate relative to more oxidizing DsbA proteins. PMID:28030602

  3. Fold of the conserved DTC domain in deltex proteins

    SciTech Connect

    Obiero, Josiah; Walker, John R.; Dhe-Paganon, Sirano

    2012-04-30

    Human Deltex 3-like (DTX3L) is a member of the Deltex family of proteins. Initially identified as a B-lymphoma and BAL-associated protein, DTX3L is an E3 ligase that regulates subcellular localization of its partner protein, BAL, by a dynamic nucleocytoplasmic trafficking mechanism. Unlike other members of the Deltex family of proteins, DTX3L lacks the highly basic N-terminal motif and the central proline-rich motif present in other Deltex proteins, and instead contains other unique N-terminal domains. The C-terminal domains are, however, homologous with other members of the Deltex family of proteins; these include a RING domain and a previously unidentified C-terminal domain. In this study, we report the high-resolution crystal structure of this previously uncharacterized C-terminal domain of human DTX3L, which we term the Deltex C-terminal domain.

  4. Fuzzy domains: new way of describing flexibility and interdependence of the protein domains.

    PubMed

    Yesylevskyy, Semen O; Kharkyanen, Valery N

    2009-03-01

    We proposed the innovative method of domain identification based on the concept of the fuzzy domains. In this method each residue of the protein can belong to several domains simultaneously with certain weights, which reflect to what extent this residue shares the motion pattern of the given domain. Our method allows describing the fuzzy boundaries between the domains and the gradual changes of the motion pattern from one domain to the other. It provides the reasonable compromise between the continuous change of the protein dynamics from one residue to the other and the discrete description of the structure in terms of small number of domains. We suggested quantitative criterion, which shows the overall degree of domain flexibility in the protein. The concept of the fuzzy domains provides an innovative way of visualization of domain flexibility, which makes the gradual transitions between the domains clearly visible and comparable to available experimental and structural data. In the future, the concept of the fuzzy domains can be used in the coarse-grained simulations of the domain dynamics in order to account for internal protein flexibility.

  5. 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.

  6. Comparative Analysis of SWIRM Domain-Containing Proteins in Plants

    PubMed Central

    Gao, Yan; Yang, Songguang; Yuan, Lianyu; Cui, Yuhai; Wu, Keqiang

    2012-01-01

    Chromatin-remodeling complexes affect gene expression by using the energy of ATP hydrolysis to locally disrupt or alter the association of histones with DNA. SWIRM (Swi3p, Rsc8p, and Moira) domain is an alpha-helical domain of about 85 residues in chromosomal proteins. SWIRM domain-containing proteins make up large multisubunit complexes by interacting with other chromatin modification factors and may have an important function in plants. However, little is known about SWIRM domain-containing proteins in plants. In this study, 67 SWIRM domain-containing proteins from 6 plant species were identified and analyzed. Plant SWIRM domain proteins can be divided into three distinct types: Swi-type, LSD1-type, and Ada2-type. Generally, the SWIRM domain forms a helix-turn-helix motif commonly found in DNA-binding proteins. The genes encoding SWIRM domain proteins in Oryza sativa are widely expressed, especially in pistils. In addition, OsCHB701 and OsHDMA701 were downregulated by cold stress, whereas OsHDMA701 and OsHDMA702 were significantly induced by heat stress. These observations indicate that SWIRM domain proteins may play an essential role in plant development and plant responses to environmental stress. PMID:22924025

  7. Identification of domains and domain interface residues in multidomain proteins from graph spectral method.

    PubMed

    Sistla, Ramesh K; K V, Brinda; Vishveshwara, Saraswathi

    2005-05-15

    We present a novel method for the identification of structural domains and domain interface residues in proteins by graph spectral method. This method converts the three-dimensional structure of the protein into a graph by using atomic coordinates from the PDB file. Domain definitions are obtained by constructing either a protein backbone graph or a protein side-chain graph. The graph is constructed based on the interactions between amino acid residues in the three-dimensional structure of the proteins. The spectral parameters of such a graph contain information regarding the domains and subdomains in the protein structure. This is based on the fact that the interactions among amino acids are higher within a domain than across domains. This is evident in the spectra of the protein backbone and the side-chain graphs, thus differentiating the structural domains from one another. Further, residues that occur at the interface of two domains can also be easily identified from the spectra. This method is simple, elegant, and robust. Moreover, a single numeric computation yields both the domain definitions and the interface residues. Copyright 2005 Wiley-Liss, Inc.

  8. 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.

  9. 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.

  10. Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant

    PubMed Central

    Horváth, Beatrix; Domonkos, Ágota; Szűcs, Attila; Ábrahám, Edit; Ayaydin, Ferhan; Bóka, Károly; Chen, Yuhui; Chen, Rujin; Murray, Jeremy D.; Udvardi, Michael K.; Kondorosi, Éva; Kaló, Péter

    2015-01-01

    Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula. PMID:26401023

  11. Convergent Evolution of Endosymbiont Differentiation in Dalbergioid and Inverted Repeat-Lacking Clade Legumes Mediated by Nodule-Specific Cysteine-Rich Peptides.

    PubMed

    Czernic, Pierre; Gully, Djamel; Cartieaux, Fabienne; Moulin, Lionel; Guefrachi, Ibtissem; Patrel, Delphine; Pierre, Olivier; Fardoux, Joël; Chaintreuil, Clémence; Nguyen, Phuong; Gressent, Frédéric; Da Silva, Corinne; Poulain, Julie; Wincker, Patrick; Rofidal, Valérie; Hem, Sonia; Barrière, Quentin; Arrighi, Jean-François; Mergaert, Peter; Giraud, Eric

    2015-10-01

    Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen-fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In Inverted Repeat-Lacking Clade (IRLC) legumes such as Medicago spp., the bacteroids are kept under control by an arsenal of nodule-specific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated, and polyploid state. Here, we show that in Aeschynomene spp. legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene spp. and that these bacteroids are terminally differentiated and polyploid, similar to bacteroids in IRLC legumes. Transcriptome, in situ hybridization, and proteome analyses demonstrated that the symbiotic cells in the Aeschynomene spp. nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNA interference-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes.

  12. Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant.

    PubMed

    Horváth, Beatrix; Domonkos, Ágota; Kereszt, Attila; Szűcs, Attila; Ábrahám, Edit; Ayaydin, Ferhan; Bóka, Károly; Chen, Yuhui; Chen, Rujin; Murray, Jeremy D; Udvardi, Michael K; Kondorosi, Éva; Kaló, Péter

    2015-12-08

    Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula.

  13. Bacteria-zinc co-localization implicates enhanced synthesis of cysteine-rich peptides in zinc detoxification when Brassica juncea is inoculated with Rhizobium leguminosarum.

    PubMed

    Adediran, Gbotemi A; Ngwenya, Bryne T; Mosselmans, J Frederick W; Heal, Kate V

    2016-01-01

    Some plant growth promoting bacteria (PGPB) are enigmatic in enhancing plant growth in the face of increased metal accumulation in plants. Since most PGPB colonize the plant root epidermis, we hypothesized that PGPB confer tolerance to metals through changes in speciation at the root epidermis. We employed a novel combination of fluorophore-based confocal laser scanning microscopic imaging and synchrotron based microscopic X-ray fluorescence mapping with X-ray absorption spectroscopy to characterize bacterial localization, zinc (Zn) distribution and speciation in the roots of Brassica juncea grown in Zn contaminated media (400 mg kg(-1) Zn) with the endophytic Pseudomonas brassicacearum and rhizospheric Rhizobium leguminosarum. PGPB enhanced epidermal Zn sequestration relative to PGBP-free controls while the extent of endophytic accumulation depended on the colonization mode of each PGBP. Increased root accumulation of Zn and increased tolerance to Zn was associated predominantly with R. leguminosarum and was likely due to the coordination of Zn with cysteine-rich peptides in the root endodermis, suggesting enhanced synthesis of phytochelatins or glutathione. Our mechanistic model of enhanced Zn accumulation and detoxification in plants inoculated with R. leguminosarum has particular relevance to PGPB enhanced phytoremediation of soils contaminated through mining and oxidation of sulphur-bearing Zn minerals or engineered nanomaterials such as ZnS. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  14. Convergent Evolution of Endosymbiont Differentiation in Dalbergioid and Inverted Repeat-Lacking Clade Legumes Mediated by Nodule-Specific Cysteine-Rich Peptides1

    PubMed Central

    Czernic, Pierre; Gully, Djamel; Cartieaux, Fabienne; Moulin, Lionel; Guefrachi, Ibtissem; Patrel, Delphine; Pierre, Olivier; Fardoux, Joël; Chaintreuil, Clémence; Nguyen, Phuong; Gressent, Frédéric; Da Silva, Corinne; Poulain, Julie; Wincker, Patrick; Rofidal, Valérie; Hem, Sonia; Barrière, Quentin; Arrighi, Jean-François; Mergaert, Peter; Giraud, Eric

    2015-01-01

    Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen-fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In Inverted Repeat-Lacking Clade (IRLC) legumes such as Medicago spp., the bacteroids are kept under control by an arsenal of nodule-specific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated, and polyploid state. Here, we show that in Aeschynomene spp. legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene spp. and that these bacteroids are terminally differentiated and polyploid, similar to bacteroids in IRLC legumes. Transcriptome, in situ hybridization, and proteome analyses demonstrated that the symbiotic cells in the Aeschynomene spp. nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNA interference-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes. PMID:26286718

  15. Bacteria–zinc co-localization implicates enhanced synthesis of cysteine-rich peptides in zinc detoxification when Brassica juncea is inoculated with Rhizobium leguminosarum

    PubMed Central

    Adediran, Gbotemi A; Ngwenya, Bryne T; Mosselmans, J Frederick W; Heal, Kate V

    2016-01-01

    Some plant growth promoting bacteria (PGPB) are enigmatic in enhancing plant growth in the face of increased metal accumulation in plants. Since most PGPB colonize the plant root epidermis, we hypothesized that PGPB confer tolerance to metals through changes in speciation at the root epidermis. We employed a novel combination of fluorophore-based confocal laser scanning microscopic imaging and synchrotron based microscopic X-ray fluorescence mapping with X-ray absorption spectroscopy to characterize bacterial localization, zinc (Zn) distribution and speciation in the roots of Brassica juncea grown in Zn contaminated media (400 mg kg−1 Zn) with the endophytic Pseudomonas brassicacearum and rhizospheric Rhizobium leguminosarum. PGPB enhanced epidermal Zn sequestration relative to PGBP-free controls while the extent of endophytic accumulation depended on the colonization mode of each PGBP. Increased root accumulation of Zn and increased tolerance to Zn was associated predominantly with R. leguminosarum and was likely due to the coordination of Zn with cysteine-rich peptides in the root endodermis, suggesting enhanced synthesis of phytochelatins or glutathione. Our mechanistic model of enhanced Zn accumulation and detoxification in plants inoculated with R. leguminosarum has particular relevance to PGPB enhanced phytoremediation of soils contaminated through mining and oxidation of sulphur-bearing Zn minerals or engineered nanomaterials such as ZnS. PMID:26263508

  16. Do proteins facilitate the formation of cholesterol-rich domains?

    PubMed

    Epand, Richard M

    2004-11-03

    Both biological and model membranes can exhibit the formation of domains. A brief review of some of the diverse methodologies used to identify the presence of domains in membranes is given. Some of these domains are enriched in cholesterol. The segregation of lipids into cholesterol-rich domains can occur in both pure lipid systems as well as membranes containing peptides and proteins. Peptides and proteins can promote the formation of cholesterol-rich domains not only by preferentially interacting with cholesterol and being sequestered into these regions of the membrane, but also indirectly as a consequence of being excluded from cholesterol-rich domains. The redistribution of components is dictated by the thermodynamics of the system. The formation of domains in a biological membrane is a consequence of all of the intermolecular interactions including those among lipid molecules as well as between lipids and proteins.

  17. Putative Domain-Domain Interactions in the Vesicular Stomatitis Virus L Polymerase Protein Appendage Region

    PubMed Central

    Ruedas, John B.

    2014-01-01

    ABSTRACT The multidomain polymerase protein (L) of nonsegmented negative-strand (NNS) RNA viruses catalyzes transcription and replication of the virus genome. The N-terminal half of the protein forms a ring-like polymerase structure, while the C-terminal half encoding viral mRNA transcript modifications consists of a flexible appendage with three distinct globular domains. To gain insight into putative transient interactions between L domains during viral RNA synthesis, we exchanged each of the four distinct regions encompassing the appendage region of vesicular stomatitis virus (VSV) Indiana serotype L protein with their counterparts from VSV New Jersey and analyzed effects on virus polymerase activity in a minigenome system. The methyltransferase domain exchange yielded a fully active polymerase protein, which functioned as well as wild-type L in the context of a recombinant virus. Exchange of the downstream C-terminal nonconserved region abolished activity, but coexchanging it with the methyltransferase domain generated a polymerase favoring replicase over transcriptase activity, providing strong evidence of interaction between these two regions. Exchange of the capping enzyme domain or the adjacent nonconserved region thought to function as an “unstructured” linker also abrogated polymerase activity even when either domain was coexchanged with other appendage domains. Further probing of the putative linker segment using in-frame enhanced green fluorescent protein (EGFP) insertions similarly abrogated activity. We discuss the implications of these findings with regard to L protein appendage domain structure and putative domain-domain interactions required for polymerase function. IMPORTANCE NNS viruses include many well-known human pathogens (e.g., rabies, measles, and Ebola viruses), as well as emerging viral threats (e.g., Nipah and Hendra viruses). These viruses all encode a large L polymerase protein similarly organized into multiple domains that work in

  18. 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

  19. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains.

    PubMed

    Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J; Eggeling, Christian; Hell, Stefan W; van den Bogaart, Geert; Jahn, Reinhard

    2015-01-30

    The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein-protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.

  20. Continuous and discontinuous domains: an algorithm for the automatic generation of reliable protein domain definitions.

    PubMed Central

    Siddiqui, A. S.; Barton, G. J.

    1995-01-01

    An algorithm is presented for the fast and accurate definition of protein structural domains from coordinate data without prior knowledge of the number or type of domains. The algorithm explicitly locates domains that comprise one or two continuous segments of protein chain. Domains that include more than two segments are also located. The algorithm was applied to a nonredundant database of 230 protein structures and the results compared to domain definitions obtained from the literature, or by inspection of the coordinates on molecular graphics. For 70% of the proteins, the derived domains agree with the reference definitions, 18% show minor differences and only 12% (28 proteins) show very different definitions. Three screens were applied to identify the derived domains least likely to agree with the subjective definition set. These screens revealed a set of 173 proteins, 97% of which agree well with the subjective definitions. The algorithm represents a practical domain identification tool that can be run routinely on the entire structural database. Adjustment of parameters also allows smaller compact units to be identified in proteins. PMID:7663343

  1. Domain structure and organisation in extracellular matrix proteins.

    PubMed

    Hohenester, Erhard; Engel, Jürgen

    2002-03-01

    Extracellular matrix (ECM) proteins are large modular molecules built up from a limited set of modules, or domains. The basic folds of many domains have now been determined by crystallography or NMR spectroscopy. Recent structures of domain pairs and larger tandem arrays, as well as of oligomerisation domains, have begun to reveal the principles underlying the higher order architecture of ECM proteins. Structural information, coupled with site-directed mutagenesis, has been instrumental in showing how adjacent domains can co-operate in ligand binding. Very recently, the first heterotypic ECM protein complexes have become available. Here, we review the advances of the last 5 years in understanding ECM protein structure, with special emphasis on those structures that have given insight into the biological functions of ECM proteins.

  2. The nature of protein domain evolution: shaping the interaction network.

    PubMed

    Bagowski, Christoph P; Bruins, Wouter; Te Velthuis, Aartjan J W

    2010-08-01

    The proteomes that make up the collection of proteins in contemporary organisms evolved through recombination and duplication of a limited set of domains. These protein domains are essentially the main components of globular proteins and are the most principal level at which protein function and protein interactions can be understood. An important aspect of domain evolution is their atomic structure and biochemical function, which are both specified by the information in the amino acid sequence. Changes in this information may bring about new folds, functions and protein architectures. With the present and still increasing wealth of sequences and annotation data brought about by genomics, new evolutionary relationships are constantly being revealed, unknown structures modeled and phylogenies inferred. Such investigations not only help predict the function of newly discovered proteins, but also assist in mapping unforeseen pathways of evolution and reveal crucial, co-evolving inter- and intra-molecular interactions. In turn this will help us describe how protein domains shaped cellular interaction networks and the dynamics with which they are regulated in the cell. Additionally, these studies can be used for the design of new and optimized protein domains for therapy. In this review, we aim to describe the basic concepts of protein domain evolution and illustrate recent developments in molecular evolution that have provided valuable new insights in the field of comparative genomics and protein interaction networks.

  3. The Nature of Protein Domain Evolution: Shaping the Interaction Network

    PubMed Central

    Bagowski, Christoph P; Bruins, Wouter; te Velthuis, Aartjan J.W

    2010-01-01

    The proteomes that make up the collection of proteins in contemporary organisms evolved through recombination and duplication of a limited set of domains. These protein domains are essentially the main components of globular proteins and are the most principal level at which protein function and protein interactions can be understood. An important aspect of domain evolution is their atomic structure and biochemical function, which are both specified by the information in the amino acid sequence. Changes in this information may bring about new folds, functions and protein architectures. With the present and still increasing wealth of sequences and annotation data brought about by genomics, new evolutionary relationships are constantly being revealed, unknown structures modeled and phylogenies inferred. Such investigations not only help predict the function of newly discovered proteins, but also assist in mapping unforeseen pathways of evolution and reveal crucial, co-evolving inter- and intra-molecular interactions. In turn this will help us describe how protein domains shaped cellular interaction networks and the dynamics with which they are regulated in the cell. Additionally, these studies can be used for the design of new and optimized protein domains for therapy. In this review, we aim to describe the basic concepts of protein domain evolution and illustrate recent developments in molecular evolution that have provided valuable new insights in the field of comparative genomics and protein interaction networks. PMID:21286315

  4. ELISA: a unified, multidimensional view of the protein domain universe.

    PubMed

    Shakhnovich, Boris E; Harvey, John Max; Delisi, Charles

    2004-01-01

    ELISA (http://romi.bu.edu/elisa/) is a database that was designed for flexibility in defining interesting queries about protein domain evolution. We have defined and included both the inherent characteristics of the domains such as structure and function and comparisons of these characteristics between domains. Thus, the database is useful in defining structural and functional links between related protein domains and by extension sequences that encode them. In this database we introduce and employ a novel method of functional annotation and comparison. For each protein domain we create a probabilistic functional annotation tree using GO. We have designed an algorithm that accurately compares these trees and thus provides a measure of "functional distance" between two protein domains. Along with functional annotation, we have also included structural comparison between protein domains and best sequence comparisons to all known genomes. The latter enables researchers to dynamically do searches for domains sharing similar phylogenetic profiles. This combination of data and tools enables the researcher to design complex queries to carry out research in the areas of protein domain evolution, structure prediction and functional annotation of novel sequences.

  5. Delineation of modular proteins: domain boundary prediction from sequence information.

    PubMed

    Kong, Lesheng; Ranganathan, Shoba

    2004-06-01

    The delineation of domain boundaries of a given sequence in the absence of known 3D structures or detectable sequence homology to known domains benefits many areas in protein science, such as protein engineering, protein 3D structure determination and protein structure prediction. With the exponential growth of newly determined sequences, our ability to predict domain boundaries rapidly and accurately from sequence information alone is both essential and critical from the viewpoint of gene function annotation. Anyone attempting to predict domain boundaries for a single protein sequence is invariably confronted with a plethora of databases that contain boundary information available from the internet and a variety of methods for domain boundary prediction. How are these derived and how well do they work? What definition of 'domain' do they use? We will first clarify the different definitions of protein domains, and then describe the available public databases with domain boundary information. Finally, we will review existing domain boundary prediction methods and discuss their strengths and weaknesses.

  6. Protein universe containing a PUA RNA-binding domain.

    PubMed

    Cerrudo, Carolina S; Ghiringhelli, Pablo D; Gomez, Daniel E

    2014-01-01

    Here, we review current knowledge about pseudouridine synthase and archaeosine transglycosylase (PUA)-domain-containing proteins to illustrate progress in this field. A methodological analysis of the literature about the topic was carried out, together with a 'qualitative comparative analysis' to give a more comprehensive review. Bioinformatics methods for whole-protein or protein-domain identification are commonly based on pairwise protein sequence comparisons; we added comparison of structures to detect the whole universe of proteins containing the PUA domain. We present an update of proteins having this domain, focusing on the specific proteins present in Homo sapiens (dyskerin, MCT1, Nip7, eIF2D and Nsun6), and explore the existence of these in other species. We also analyze the phylogenetic distribution of the PUA domain in different species and proteins. Finally, we performed a structural comparison of the PUA domain through data mining of structural databases, determining a conserved structural motif, despite the differences in the sequence, even among eukaryotes, archaea and bacteria. All data discussed in this review, both bibliographic and analytical, corroborate the functional importance of the PUA domain in RNA-binding proteins.

  7. Conserved central domains control the quaternary structure of Type I and Type II Hsp40 molecular chaperones

    PubMed Central

    Ramos, Carlos H.I.; Oliveira, Cristiano L.P.; Yang-Fan, Chung; Torriani, Iris L.; Cyr, Douglas M.

    2008-01-01

    Summary Hsp40s play an essential role in protein metabolism by regulating the polypeptide binding and release cycle of Hsp70. The Hsp40 family is large and specialized family members direct Hsp70 to perform highly specific tasks. Type I and Type II Hsp40s, such as yeast Ydj1 and Sis1, are homodimers that dictate functions of cytosolic Hsp70, but how they do so is unclear. Type I Hsp40s contain a conserved centrally located Cysteine-rich domain that is replaced by a Glycine and Methionine rich region in Type II Hsp40s, but the mechanism by which these unique domains influence Hsp40 structure and function is unknown. This is the case because high-resolution structures of full-length forms of these Hsp40s have not been solved. To fill this void we built low-resolution models of the quaternary structure of Ydj1 and Sis1 with information obtained from biophysical measurements of protein shape, small angle X-ray scattering and ab initio protein modeling. Low resolution models were also calculated for the chimeric Hsp40s YSY and SYS, in which the central domains of Ydj1 and Sis1 were exchanged. Similar to their human homologs, Ydj1 and Sis1 each has a unique shape with major structural differences apparently being the orientation of the J-domains relative to the long axis of the dimers. Central domain swapping in YSY and SYS correlates with the switched ability of YSY and SYS to perform unique functions of Sis1 and Ydj1, respectively. Models for the mechanism by which the conserved Cysteine-rich domain and Glycine and Methionine rich region confer structural and functional specificity to Type I and Type II Hsp40s are discussed. PMID:18723025

  8. Protein domain decomposition using a graph-theoretic approach.

    PubMed

    Xu, Y; Xu, D; Gabow, H N; Gabow, H

    2000-12-01

    Automatic decomposition of a multi-domain protein into individual domains represents a highly interesting and unsolved problem. As the number of protein structures in PDB is growing at an exponential rate, there is clearly a need for more reliable and efficient methods for protein domain decomposition simply to keep the domain databases up-to-date. We present a new algorithm for solving the domain decomposition problem, using a graph-theoretic approach. We have formulated the problem as a network flow problem, in which each residue of a protein is represented as a node of the network and each residue--residue contact is represented as an edge with a particular capacity, depending on the type of the contact. A two-domain decomposition problem is solved by finding a bottleneck (or a minimum cut) of the network, which minimizes the total cross-edge capacity, using the classical Ford--Fulkerson algorithm. A multi-domain decomposition problem is solved through repeatedly solving a series of two-domain problems. The algorithm has been implemented as a computer program, called DomainParser. We have tested the program on a commonly used test set consisting of 55 proteins. The decomposition results are 78.2% in agreement with the literature on both the number of decomposed domains and the assignments of residues to each domain, which compares favorably to existing programs. On the subset of two-domain proteins (20 in number), the program assigned 96.7% of the residues correctly when we require that the number of decomposed domains is two.

  9. Production of recombinant PvDBPII, receptor binding domain of Plasmodium vivax Duffy binding protein, and evaluation of immunogenicity to identify an adjuvant formulation for vaccine development.

    PubMed

    Bhardwaj, Rukmini; Shakri, Ahmad Rushdi; Hans, Dhiraj; Gupta, Pankaj; Fernandez-Becerra, Carmen; Del Portillo, Hernando A; Pandey, Gaurav; Chitnis, Chetan E

    2017-08-01

    Plasmodium vivax is dependent on interaction with the Duffy antigen receptor for chemokines (DARC) for invasion of human erythrocytes. The P. vivax Duffy binding protein (PvDBP) mediates interaction of P. vivax merozoites with DARC. The DARC receptor-binding domain lies in a conserved N-terminal cysteine-rich region of PvDBP referred to as region II (PvDBPII). PvDBPII is an attractive vaccine candidate since antibodies raised against PvDBPII block erythrocyte invasion by P. vivax. Here, we describe methods to produce recombinant PvDBPII in its correctly folded conformation. A synthetic gene optimized for expression of PvDBPII in Escherichia coli and fed batch fermentation process based on exponential feeding strategy was used to achieve high levels of expression of recombinant PvDBPII. Recombinant PvDBPII was isolated from inclusion bodies, refolded by rapid dilution and purified by ion exchange chromatography. Purified recombinant PvDBPII was characterized for identity, purity and functional activity using standardized release assays. Recombinant PvDBPII formulated with various human compatible adjuvants including glycosylpyranosyl lipid A-stable emulsion (GLA-SE) and alhydrogel was used for immunogenicity studies in small animals to downselect a suitable formulation for clinical development. Sera collected from immunized animals were tested for recognition of PvDBPII and inhibition of PvDBPII-DARC binding. GLA-SE formulations of PvDBPII yielded higher ELISA and binding inhibition titres compared to PvDBPII formulated with alhydrogel. These data support further development of a recombinant vaccine for P. vivax based on PvDBPII formulated with GLA-SE. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Unexpected diversity in Shisa-like proteins suggests the importance of their roles as transmembrane adaptors.

    PubMed

    Pei, Jimin; Grishin, Nick V

    2012-03-01

    The Shisa family of single-transmembrane proteins is characterized by an N-terminal cysteine-rich domain and a proline-rich C-terminal region. Its founding member, Xenopus Shisa, promotes head development by antagonizing Wnt and FGF signaling. Recently, a mouse brain-specific Shisa protein CKAMP44 (Shisa9) was shown to play an important role in AMPA receptor desensitization. We used sequence similarity searches against protein, genome and EST databases to study the evolutionary origin and phylogenetic distribution of Shisa homologs. In addition to nine Shisa subfamilies in vertebrates, we detected distantly related Shisa homologs that possess an N-terminal domain with six conserved cysteines. These Shisa-like proteins include FAM159 and KIAA1644 mainly from vertebrates, and members from various bilaterian invertebrates and Porifera, suggesting their presence in the last common ancestor of Metazoa. Shisa-like genes have undergone large expansions in Branchiostoma floridae and Saccoglossus kowalevskii, and appear to have been lost in certain insects. Pattern-based searches against eukaryotic proteomes also uncovered several other families of predicted single-transmembrane proteins with a similar cysteine-rich domain. We refer to these proteins (Shisa/Shisa-like, WBP1/VOPP1, CX, DUF2650, TMEM92, and CYYR1) as STMC6 proteins (single-transmembrane proteins with conserved 6 cysteines). STMC6 genes are widespread in Metazoa, with the human genome containing 17 members. Frequent occurrences of PY motifs in STMC6 proteins suggest that most of them could interact with WW-domain-containing proteins, such as the NEDD4 family E3 ubiquitin ligases, and could play critical roles in protein degradation and sorting. STMC6 proteins are likely transmembrane adaptors that regulate membrane proteins such as cell surface receptors.

  11. Reconstituting Protein Interaction Networks Using Parameter-Dependent Domain-Domain Interactions

    DTIC Science & Technology

    2013-05-07

    for yeast ( Saccharomyces cerevisiae ) using sequences of 5,884 proteins,a downloaded from the Saccharomyces Genome Database (SGD) [31] and yeast...Riles L, Mortimer RK, et al: Genetic and physical maps of Saccharomyces cerevisiae . Nature 1997, 387(6632 Suppl):67–73. 32. Finn RD, Mistry J, Tate J...downloaded from the Saccharomyces Genome Database [31]. NP, proteins with at least one domain annotation. NS, protein-domain amino acid sequence

  12. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

    PubMed Central

    Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J.; Eggeling, Christian; Hell, Stefan W.; van den Bogaart, Geert; Jahn, Reinhard

    2015-01-01

    The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes. PMID:25635869

  13. Protein domain definition should allow for conditional disorder.

    PubMed

    Yegambaram, Kavestri; Bulloch, Esther M M; Kingston, Richard L

    2013-11-01

    Proteins are often classified in a binary fashion as either structured or disordered. However this approach has several deficits. Firstly, protein folding is always conditional on the physiochemical environment. A protein which is structured in some circumstances will be disordered in others. Secondly, it hides a fundamental asymmetry in behavior. While all structured proteins can be unfolded through a change in environment, not all disordered proteins have the capacity for folding. Failure to accommodate these complexities confuses the definition of both protein structural domains and intrinsically disordered regions. We illustrate these points with an experimental study of a family of small binding domains, drawn from the RNA polymerase of mumps virus and its closest relatives. Assessed at face value the domains fall on a structural continuum, with folded, partially folded, and near unstructured members. Yet the disorder present in the family is conditional, and these closely related polypeptides can access the same folded state under appropriate conditions. Any heuristic definition of the protein domain emphasizing conformational stability divides this domain family in two, in a way that makes no biological sense. Structural domains would be better defined by their ability to adopt a specific tertiary structure: a structure that may or may not be realized, dependent on the circumstances. This explicitly allows for the conditional nature of protein folding, and more clearly demarcates structural domains from intrinsically disordered regions that may function without folding.

  14. 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.

  15. Cholesterol and the interaction of proteins with membrane domains.

    PubMed

    Epand, Richard M

    2006-07-01

    Cholesterol is not uniformly distributed in biological membranes. One of the factors influencing the formation of cholesterol-rich domains in membranes is the unequal lateral distribution of proteins in membranes. Certain proteins are found in cholesterol-rich domains. In some of these cases, it is as a consequence of the proteins interacting directly with cholesterol. There are several structural features of a protein that result in the protein preferentially associating with cholesterol-rich domains. One of the best documented of these is certain types of lipidations. In addition, however, there are segments of a protein that can preferentially sequester cholesterol. We discuss two examples of these cholesterol-recognition elements: the cholesterol recognition/interaction amino acid consensus (CRAC) domain and the sterol-sensing domain (SSD). The requirements for a CRAC motif are quite flexible and predict that a large number of sequences could recognize cholesterol. There are, however, certain proteins that are known to interact with cholesterol-rich domains of cell membranes that have CRAC motifs, and synthetic peptides corresponding to these segments also promote the formation of cholesterol-rich domains. Modeling studies have provided a rationale for certain requirements of the CRAC motif. The SSD is a larger protein segment comprising five transmembrane domains. The amino acid sequence YIYF is found in several SSD and in certain other proteins for which there is evidence that they interact with cholesterol-rich domains. The CRAC sequences as well as YIYF are generally found adjacent to a transmembrane helical segment. These regions appear to have a strong influence of the localization of certain proteins into domains in biological membranes. In addition to the SSD, there is also a domain found in soluble proteins, the START domain, that binds lipids. Certain proteins with START domains specifically bind cholesterol and are believed to function in

  16. Discrete structure of van der Waals domains in globular proteins.

    PubMed

    Berezovsky, Igor N

    2003-03-01

    Most globular proteins are divisible by domains, distinct substructures of the globule. The notion of hierarchy of the domains was introduced earlier via van der Waals energy profiles that allow one to subdivide the proteins into domains (subdomains). The question remains open as to what is the possible structural connection of the energy profiles. The recent discovery of the loop-n-lock elements in the globular proteins suggests such a structural connection. A direct comparison of the segmentation by van der Waals energy criteria with the maps of the locked loops of nearly standard size reveals a striking correlation: domains in general appear to consist of one to several such loops. In addition, it was demonstrated that a variety of subdivisions of the same protein into domains is just a regrouping of the loop-n-lock elements.

  17. Allosteric properties of PH domains in Arf regulatory proteins.

    PubMed

    Roy, Neeladri Sekhar; Yohe, Marielle E; Randazzo, Paul A; Gruschus, James M

    2016-01-01

    Pleckstrin Homology (PH) domains bind phospholipids and proteins. They are critical regulatory elements of a number enzymes including guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) for Ras-superfamily guanine nucleotide binding proteins such as ADP-ribosylation factors (Arfs). Recent studies have indicated that many PH domains may bind more than one ligand cooperatively. Here we discuss the molecular basis of PH domain-dependent allosteric behavior of 2 ADP-ribosylation factor exchange factors, Grp1 and Brag2, cooperative binding of ligands to the PH domains of Grp1 and the Arf GTPase-activating protein, ASAP1, and the consequences for activity of the associated catalytic domains.

  18. The application of modular protein domains in proteomics

    PubMed Central

    Jadwin, Joshua A.; Ogiue-Ikeda, Mari; Machida, Kazuya

    2012-01-01

    The ability of modular protein domains to independently fold and bind short peptide ligands both in vivo and in vitro has allowed a significant number of protein-protein interaction studies to take advantage of them as affinity and detection reagents. Here, we refer to modular domain based proteomics as “domainomics” to draw attention to the potential of using domains and their motifs as tools in proteomics. In this review we describe core concepts of domainomics, established and emerging technologies, and recent studies by functional category. Accumulation of domain-motif binding data should ultimately provide the foundation for domain-specific interactomes, which will likely reveal the underlying substructure of protein networks as well as the selectivity and plasticity of signal transduction. PMID:22710164

  19. Inference of domain-disease associations from domain-protein, protein-disease and disease-disease relationships.

    PubMed

    Zhang, Wangshu; Coba, Marcelo P; Sun, Fengzhu

    2016-01-11

    Protein domains can be viewed as portable units of biological function that defines the functional properties of proteins. Therefore, if a protein is associated with a disease, protein domains might also be associated and define disease endophenotypes. However, knowledge about such domain-disease relationships is rarely available. Thus, identification of domains associated with human diseases would greatly improve our understanding of the mechanism of human complex diseases and further improve the prevention, diagnosis and treatment of these diseases. Based on phenotypic similarities among diseases, we first group diseases into overlapping modules. We then develop a framework to infer associations between domains and diseases through known relationships between diseases and modules, domains and proteins, as well as proteins and disease modules. Different methods including Association, Maximum likelihood estimation (MLE), Domain-disease pair exclusion analysis (DPEA), Bayesian, and Parsimonious explanation (PE) approaches are developed to predict domain-disease associations. We demonstrate the effectiveness of all the five approaches via a series of validation experiments, and show the robustness of the MLE, Bayesian and PE approaches to the involved parameters. We also study the effects of disease modularization in inferring novel domain-disease associations. Through validation, the AUC (Area Under the operating characteristic Curve) scores for Bayesian, MLE, DPEA, PE, and Association approaches are 0.86, 0.84, 0.83, 0.83 and 0.79, respectively, indicating the usefulness of these approaches for predicting domain-disease relationships. Finally, we choose the Bayesian approach to infer domains associated with two common diseases, Crohn's disease and type 2 diabetes. The Bayesian approach has the best performance for the inference of domain-disease relationships. The predicted landscape between domains and diseases provides a more detailed view about the disease

  20. Novel Strategy Utilizing Extracellular Cysteine-Rich Domain of Membrane Receptor for Constructing d-Peptide Mediated Targeted Drug Delivery Systems: A Case Study on Fn14.

    PubMed

    Li, Zhuoxuan; Xie, Jing; Peng, Shan; Liu, Sha; Wang, Ying; Lu, Weiyue; Shen, Jie; Li, Chong

    2017-08-16

    The development of proteolysis-resistant d-peptide ligands for targeted drug/gene delivery has been greatly limited, due to the challenge that lies in the chemical synthesis of membrane receptors without altering their structures. In the present research, a novel strategy utilizing self-stabilized extracellular CRD of the membrane receptor was developed to construct d-peptide ligands and their mediated targeted drug delivery systems. Fn14, a cell surface receptor overexpressed in many cancers including pancreatic and triple-negative breast cancers, was selected as the model receptor. Fn14 CRD was synthesized and folded, and used to screen Fn14 binding peptides using phage display (l-peptide) and mirror-image phage display (d-peptide) techniques, respectively. The d-peptide ligand successfully mediated targeted drug delivery to Fn14 positive tumor cells. In addition, the d-peptide possessed better target-binding affinity, stromal barrier permeability, and tumor targeting ability in vivo when conjugated with liposomes. More importantly, d-peptide mediated liposomal paclitaxel delivery significantly inhibited pancreatic tumor growth in a subcutaneous xenograft model and drastically prolonged survival in a lung metastasis of breast cancer mouse model. This study demonstrated that mirror-image phage display based on the CRD of membrane receptor can be a promising strategy to advance active targeted drug delivery via biostable d-peptides.

  1. Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains.

    PubMed

    Janganan, Thamarai K; Mullin, Nic; Tzokov, Svetomir B; Stringer, Sandra; Fagan, Robert P; Hobbs, Jamie K; Moir, Anne; Bullough, Per A

    2016-10-01

    Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75-100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Quantifying information transfer by protein domains: Analysis of the Fyn SH2 domain structure

    PubMed Central

    Lenaerts, Tom; Ferkinghoff-Borg, Jesper; Stricher, Francois; Serrano, Luis; Schymkowitz, Joost WH; Rousseau, Frederic

    2008-01-01

    Background Efficient communication between distant sites within a protein is essential for cooperative biological response. Although often associated with large allosteric movements, more subtle changes in protein dynamics can also induce long-range correlations. However, an appropriate formalism that directly relates protein structural dynamics to information exchange between functional sites is still lacking. Results Here we introduce a method to analyze protein dynamics within the framework of information theory and show that signal transduction within proteins can be considered as a particular instance of communication over a noisy channel. In particular, we analyze the conformational correlations between protein residues and apply the concept of mutual information to quantify information exchange. Mapping out changes of mutual information on the protein structure then allows visualizing how distal communication is achieved. We illustrate the approach by analyzing information transfer by the SH2 domain of Fyn tyrosine kinase, obtained from Monte Carlo dynamics simulations. Our analysis reveals that the Fyn SH2 domain forms a noisy communication channel that couples residues located in the phosphopeptide and specificity binding sites and a number of residues at the other side of the domain near the linkers that connect the SH2 domain to the SH3 and kinase domains. We find that for this particular domain, communication is affected by a series of contiguous residues that connect distal sites by crossing the core of the SH2 domain. Conclusion As a result, our method provides a means to directly map the exchange of biological information on the structure of protein domains, making it clear how binding triggers conformational changes in the protein structure. As such it provides a structural road, next to the existing attempts at sequence level, to predict long-range interactions within protein structures. PMID:18842137

  3. Identification of ADAM 31: a protein expressed in Leydig cells and specialized epithelia.

    PubMed

    Liu, L; Smith, J W

    2000-06-01

    A family of proteins containing a disintegrin and metalloproteinase domain (ADAMs) has been identified recently. Here, we report the identification of a novel member of the ADAM protein family from mouse. This protein is designated ADAM 31. The complementary DNA sequence of ADAM 31 predicts a transmembrane protein with metalloproteinase, disintegrin, cysteine-rich, and cytoplasmic domains. Messenger RNA encoding ADAM 31 was most abundant in testes, but was also detected in many other tissues. More significantly, the antibodies raised against ADAM 31 reveal that the protein has a unique and restricted expression pattern. ADAM 31 is expressed in Leydig cells of the testes, but unlike many other ADAMs, it is not found on developing sperm. Furthermore, ADAM 31 is highly expressed on four types of specialized epithelia: the cauda epididymidis, the vas deferens, the convoluted tubules of the kidney, and the parietal cells of the stomach.

  4. Automatic domain decomposition of proteins by a Gaussian Network Model.

    PubMed

    Kundu, Sibsankar; Sorensen, Dan C; Phillips, George N

    2004-12-01

    Proteins are often comprised of domains of apparently independent folding units. These domains can be defined in various ways, but one useful definition divides the protein into substructures that seem to move more or less independently. The same methods that allow fairly accurate calculation of motion can be used to help classify these substructures. We show how the Gaussian Network Model (GNM), commonly used for determining motion, can also be adapted to automatically classify domains in proteins. Parallels between this physical network model and graph theory implementation are apparent. The method is applied to a nonredundant set of 55 proteins, and the results are compared to the visual assignments by crystallographers. Apart from decomposing proteins into structural domains, the algorithm can generally be applied to any large macromolecular system to decompose it into motionally decoupled sub-systems. Copyright 2004 Wiley-Liss, Inc.

  5. Structure of synaptophysin: a hexameric MARVEL-domain channel protein.

    PubMed

    Arthur, Christopher P; Stowell, Michael H B

    2007-06-01

    Synaptophysin I (SypI) is an archetypal member of the MARVEL-domain family of integral membrane proteins and one of the first synaptic vesicle proteins to be identified and cloned. Most all MARVEL-domain proteins are involved in membrane apposition and vesicle-trafficking events, but their precise role in these processes is unclear. We have purified mammalian SypI and determined its three-dimensional (3D) structure by using electron microscopy and single-particle 3D reconstruction. The hexameric structure resembles an open basket with a large pore and tenuous interactions within the cytosolic domain. The structure suggests a model for Synaptophysin's role in fusion and recycling that is regulated by known interactions with the SNARE machinery. This 3D structure of a MARVEL-domain protein provides a structural foundation for understanding the role of these important proteins in a variety of biological processes.

  6. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    PubMed

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-10-23

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer.

  7. Domain tree-based analysis of protein architecture evolution.

    PubMed

    Forslund, Kristoffer; Henricson, Anna; Hollich, Volker; Sonnhammer, Erik L L

    2008-02-01

    Understanding the dynamics behind domain architecture evolution is of great importance to unravel the functions of proteins. Complex architectures have been created throughout evolution by rearrangement and duplication events. An interesting question is how many times a particular architecture has been created, a form of convergent evolution or domain architecture reinvention. Previous studies have approached this issue by comparing architectures found in different species. We wanted to achieve a finer-grained analysis by reconstructing protein architectures on complete domain trees. The prevalence of domain architecture reinvention in 96 genomes was investigated with a novel domain tree-based method that uses maximum parsimony for inferring ancestral protein architectures. Domain architectures were taken from Pfam. To ensure robustness, we applied the method to bootstrap trees and only considered results with strong statistical support. We detected multiple origins for 12.4% of the scored architectures. In a much smaller data set, the subset of completely domain-assigned proteins, the figure was 5.6%. These results indicate that domain architecture reinvention is a much more common phenomenon than previously thought. We also determined which domains are most frequent in multiply created architectures and assessed whether specific functions could be attributed to them. However, no strong functional bias was found in architectures with multiple origins.

  8. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

    PubMed Central

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

  9. The evolution of filamin-a protein domain repeat perspective.

    PubMed

    Light, Sara; Sagit, Rauan; Ithychanda, Sujay S; Qin, Jun; Elofsson, Arne

    2012-09-01

    Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. The evolution of filamin – A protein domain repeat perspective

    PubMed Central

    Light, Sara; Sagit, Rauan; Ithychanda, Sujay S.; Qin, Jun; Elofsson, Arne

    2013-01-01

    Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. PMID:22414427

  11. Classification of Domain Movements in Proteins Using Dynamic Contact Graphs

    PubMed Central

    Taylor, Daniel; Cawley, Gavin; Hayward, Steven

    2013-01-01

    A new method for the classification of domain movements in proteins is described and applied to 1822 pairs of structures from the Protein Data Bank that represent a domain movement in two-domain proteins. The method is based on changes in contacts between residues from the two domains in moving from one conformation to the other. We argue that there are five types of elemental contact changes and that these relate to five model domain movements called: “free”, “open-closed”, “anchored”, “sliding-twist”, and “see-saw.” A directed graph is introduced called the “Dynamic Contact Graph” which represents the contact changes in a domain movement. In many cases a graph, or part of a graph, provides a clear visual metaphor for the movement it represents and is a motif that can be easily recognised. The Dynamic Contact Graphs are often comprised of disconnected subgraphs indicating independent regions which may play different roles in the domain movement. The Dynamic Contact Graph for each domain movement is decomposed into elemental Dynamic Contact Graphs, those that represent elemental contact changes, allowing us to count the number of instances of each type of elemental contact change in the domain movement. This naturally leads to sixteen classes into which the 1822 domain movements are classified. PMID:24260562

  12. Classification of domain movements in proteins using dynamic contact graphs.

    PubMed

    Taylor, Daniel; Cawley, Gavin; Hayward, Steven

    2013-01-01

    A new method for the classification of domain movements in proteins is described and applied to 1822 pairs of structures from the Protein Data Bank that represent a domain movement in two-domain proteins. The method is based on changes in contacts between residues from the two domains in moving from one conformation to the other. We argue that there are five types of elemental contact changes and that these relate to five model domain movements called: "free", "open-closed", "anchored", "sliding-twist", and "see-saw." A directed graph is introduced called the "Dynamic Contact Graph" which represents the contact changes in a domain movement. In many cases a graph, or part of a graph, provides a clear visual metaphor for the movement it represents and is a motif that can be easily recognised. The Dynamic Contact Graphs are often comprised of disconnected subgraphs indicating independent regions which may play different roles in the domain movement. The Dynamic Contact Graph for each domain movement is decomposed into elemental Dynamic Contact Graphs, those that represent elemental contact changes, allowing us to count the number of instances of each type of elemental contact change in the domain movement. This naturally leads to sixteen classes into which the 1822 domain movements are classified.

  13. Selection of soluble protein expression constructs: the experimental determination of protein domain boundaries.

    PubMed

    Dyson, Michael R

    2010-08-01

    Proteins can contain multiple domains each of which is capable of possessing a separate independent function and three-dimensional structure. It is often useful to clone and express individual protein domains to study their biochemical properties and for structure determination. However, the annotated domain boundaries in databases such as Pfam or SMART are not always accurate. The present review summarizes various strategies for the experimental determination of protein domain boundaries.

  14. Exogenous agents that target transmembrane domains of proteins.

    PubMed

    Yin, Hang

    2008-01-01

    Although membrane proteins account for approximately one third of all proteins encoded in the human genome, the functions and structures of their transmembrane domains are much less understood than the water-soluble regions. A major hurdle in studying these transmembrane domains is the lack of appropriate exogenous agents that can be used as specific probes. Despite the daunting challenges, major strides have recently been made in targeting the transmembrane domains of a variety of membrane proteins. High affinity and selectivity have been achieved in model biophysical systems, membranes of bacteria, and mammalian cells.

  15. Cryptic protein-protein interaction motifs in the cytoplasmic domain of MHCI proteins.

    PubMed

    Frietze, Karla K; Pappy, Adlai L; Melson, Jack W; O'Driscoll, Emily E; Tyler, Carolyn M; Perlman, David H; Boulanger, Lisa M

    2016-07-19

    Major histocompatibility complex class I (MHCI) proteins present antigenic peptides for immune surveillance and play critical roles in nervous system development and plasticity. Most MHCI are transmembrane proteins. The extracellular domain of MHCI interacts with immunoreceptors, peptides, and co-receptors to mediate immune signaling. While the cytoplasmic domain also plays important roles in endocytic trafficking, cross-presentation of extracellularly derived antigens, and CTL priming, the molecular mediators of cytoplasmic signaling by MHCI remain largely unknown. Here we show that the cytoplasmic domain of MHCI contains putative protein-protein interaction domains known as PDZ (PSD95/disc large/zonula occludens-1) ligands. PDZ ligands are motifs that bind to PDZ domains to organize and mediate signaling at cell-cell contacts. PDZ ligands are short, degenerate motifs, and are therefore difficult to identify via sequence homology alone, but several lines of evidence suggest that putative PDZ ligand motifs in MHCI are under positive selective pressure. Putative PDZ ligands are found in all of the 99 MHCI proteins examined from diverse species, and are enriched in the cytoplasmic domain, where PDZ interactions occur. Both the position of the PDZ ligand and the class of ligand motif are conserved across species, as well as among genes within a species. Non-synonymous substitutions, when they occur, frequently preserve the motif. Of the many specific possible PDZ ligand motifs, a handful are strikingly and selectively overrepresented in MHCI's cytoplasmic domain, but not elsewhere in the same proteins. Putative PDZ ligands in MHCI encompass conserved serine and tyrosine residues that are targets of phosphorylation, a post-translational modification that can regulate PDZ interactions. Finally, proof-of-principle in vitro interaction assays demonstrate that the cytoplasmic domains of particular MHCI proteins can bind directly and specifically to PDZ1 and PDZ4&5 of MAGI

  16. An ambiguity principle for assigning protein structural domains.

    PubMed

    Postic, Guillaume; Ghouzam, Yassine; Chebrek, Romain; Gelly, Jean-Christophe

    2017-01-01

    Ambiguity is the quality of being open to several interpretations. For an image, it arises when the contained elements can be delimited in two or more distinct ways, which may cause confusion. We postulate that it also applies to the analysis of protein three-dimensional structure, which consists in dividing the molecule into subunits called domains. Because different definitions of what constitutes a domain can be used to partition a given structure, the same protein may have different but equally valid domain annotations. However, knowledge and experience generally displace our ability to accept more than one way to decompose the structure of an object-in this case, a protein. This human bias in structure analysis is particularly harmful because it leads to ignoring potential avenues of research. We present an automated method capable of producing multiple alternative decompositions of protein structure (web server and source code available at www.dsimb.inserm.fr/sword/). Our innovative algorithm assigns structural domains through the hierarchical merging of protein units, which are evolutionarily preserved substructures that describe protein architecture at an intermediate level, between domain and secondary structure. To validate the use of these protein units for decomposing protein structures into domains, we set up an extensive benchmark made of expert annotations of structural domains and including state-of-the-art domain parsing algorithms. The relevance of our "multipartitioning" approach is shown through numerous examples of applications covering protein function, evolution, folding, and structure prediction. Finally, we introduce a measure for the structural ambiguity of protein molecules.

  17. An ambiguity principle for assigning protein structural domains

    PubMed Central

    Postic, Guillaume; Ghouzam, Yassine; Chebrek, Romain; Gelly, Jean-Christophe

    2017-01-01

    Ambiguity is the quality of being open to several interpretations. For an image, it arises when the contained elements can be delimited in two or more distinct ways, which may cause confusion. We postulate that it also applies to the analysis of protein three-dimensional structure, which consists in dividing the molecule into subunits called domains. Because different definitions of what constitutes a domain can be used to partition a given structure, the same protein may have different but equally valid domain annotations. However, knowledge and experience generally displace our ability to accept more than one way to decompose the structure of an object—in this case, a protein. This human bias in structure analysis is particularly harmful because it leads to ignoring potential avenues of research. We present an automated method capable of producing multiple alternative decompositions of protein structure (web server and source code available at www.dsimb.inserm.fr/sword/). Our innovative algorithm assigns structural domains through the hierarchical merging of protein units, which are evolutionarily preserved substructures that describe protein architecture at an intermediate level, between domain and secondary structure. To validate the use of these protein units for decomposing protein structures into domains, we set up an extensive benchmark made of expert annotations of structural domains and including state-of-the-art domain parsing algorithms. The relevance of our “multipartitioning” approach is shown through numerous examples of applications covering protein function, evolution, folding, and structure prediction. Finally, we introduce a measure for the structural ambiguity of protein molecules. PMID:28097215

  18. Structural and biochemical characterization of an RNA/DNA binding motif in the N-terminal domain of RecQ4 helicases

    PubMed Central

    Marino, Francesca; Mojumdar, Aditya; Zucchelli, Chiara; Bhardwaj, Amit; Buratti, Emanuele; Vindigni, Alessandro; Musco, Giovanna; Onesti, Silvia

    2016-01-01

    The RecQ4 helicase belongs to the ubiquitous RecQ family but its exact role in the cell is not completely understood. In addition to the helicase domain, RecQ4 has a unique N-terminal part that is essential for viability and is constituted by a region homologous to the yeast Sld2 replication initiation factor, followed by a cysteine-rich region, predicted to fold as a Zn knuckle. We carried out a structural and biochemical analysis of both the human and Xenopus laevis RecQ4 cysteine-rich regions, and showed by NMR spectroscopy that the Xenopus fragment indeed assumes the canonical Zn knuckle fold, whereas the human sequence remains unstructured, consistent with the mutation of one of the Zn ligands. Both the human and Xenopus Zn knuckles bind to a variety of nucleic acid substrates, with a mild preference for RNA. We also investigated the effect of a segment located upstream the Zn knuckle that is highly conserved and rich in positively charged and aromatic residues, partially overlapping with the C-terminus of the Sld2-like domain. In both the human and Xenopus proteins, the presence of this region strongly enhances binding to nucleic acids. These results reveal novel possible roles of RecQ4 in DNA replication and genome stability. PMID:26888063

  19. Domain view: a web tool for protein domain visualization and analysis.

    PubMed

    Pan, Xiaokang; Bingman, Craig A; Wesenberg, Gary E; Sun, Zhaohui; Phillips, George N

    2010-12-01

    The identification of sequence-based protein domains and their boundaries is often a prelude to structure determination. An accurate prediction of disordered regions, secondary structures and low complexity segments of target protein sequences can improve the efficiency of selection in structural genomics and also aid in design of constructs for directed structural biology studies. At the Center for Eukaryotic Structural Genomics (CESG) we have developed DomainView, a web tool to visualize and analyze predicted protein domains, disordered regions, secondary structures and low complexity segments of target protein sequences for selection of experimental protein structure attempts. DomainView consists of a relational database and a web graphical-user interface. The database was developed based on MySQL, which stores data from target protein sequences and their domains, disordered regions, secondary structures and low complexity segments. The program of the web user interface is a Perl CGI script. When a user searches for a target protein sequence, the script displays the combinational information about the domains and other features of that target sequence graphically on a web page by querying the database. The graphical representation for each feature is linked to a web page showing more detailed annotation information or to a new window directly running the corresponding prediction program to show further information about that feature.

  20. 14-3-3 proteins, FHA domains and BRCT domains in the DNA damage response.

    PubMed

    Mohammad, Duaa H; Yaffe, Michael B

    2009-09-02

    The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of protein kinases, which includes ATM/ATR/DNA-PK, preferentially phosphorylate Ser-Gln sites, while their basophilic downstream effecter kinases, Chk1/Chk2/MK2 preferentially phosphorylate hydrophobic-X-Arg-X-X-Ser/Thr-hydrophobic sites. A subset of tandem BRCT domains act as phosphopeptide binding modules that bind to ATM/ATR/DNA-PK substrates after DNA damage. Conversely, 14-3-3 proteins interact with substrates of Chk1/Chk2/MK2. FHA domains have been shown to interact with substrates of ATM/ATR/DNA-PK and CK2. In this review we consider how substrate phosphorylation together with BRCT domains, FHA domains and 14-3-3 proteins function to regulate ionizing radiation-induced nuclear foci and help to establish the G(2)/M checkpoint. We discuss the role of MDC1 a molecular scaffold that recruits early proteins to foci, such as NBS1 and RNF8, through distinct phosphodependent interactions. In addition, we consider the role of 14-3-3 proteins and the Chk2 FHA domain in initiating and maintaining cell cycle arrest.

  1. Defining subdomains of the K domain important for protein-protein interactions of plant MADS proteins.

    PubMed

    Yang, Yingzhen; Jack, Thomas

    2004-05-01

    The MADS proteins APETALA3 (AP3), PISTILLATA (PI), SEPALLATAI (SEPI), SEP2, SEP3, AGAMOUS, and APETALA are required for proper floral organ identity in Arabidopsis flowers. All of these floral MADS proteins conserve two domains: the MADS domain that mediates DNA binding and dimerization, and the K domain that mediates protein protein interaction. The K domain is postulated to form a several amphipathic c-helices referred to as K1, K2, and K3. The K1 and K2 helicies are located entirely within the K domain while the K3 helix spans the K domain-C domain boundary. Here we report on our studies on the interactions of the B class MADS proteins AP3 and PI with the E class MADS proteins SEP1, SEP2, and SEP3. A comparative analysis of mutants in the K domain reveals that the subdomains mediating the PI/AP3 interaction are different from the subdomains mediating the PI/SEP3 (or PI/SEP1) interaction. The strong PI/SEP3 (or PI/SEP1) interaction requires K2, part of K3, and the interhelical region between K1 and K2. By contrast, K1, K2 and the region between K1 and K2 are important for strong AP3/PI interaction. Most of the K3 helix does not appear to be important for either the PI/AP3 or the PI/SEP3 (or PI/SEP1) interaction. Conserved hydrophobic positions are most important for the strength of both PI/AP3 and PI/SEP3 dimerization, though ionic and/or polar interactions appear to play a secondary role.

  2. Proteasomes and protein conjugation across domains of life.

    PubMed

    Maupin-Furlow, Julie

    2011-12-19

    Like other energy-dependent proteases, proteasomes, which are found across the three domains of life, are self-compartmentalized and important in the early steps of proteolysis. Proteasomes degrade improperly synthesized, damaged or misfolded proteins and hydrolyse regulatory proteins that must be specifically removed or cleaved for cell signalling. In eukaryotes, proteins are typically targeted for proteasome-mediated destruction through polyubiquitylation, although ubiquitin-independent pathways also exist. Interestingly, actinobacteria and archaea also covalently attach small proteins (prokaryotic ubiquitin-like protein (Pup) and small archaeal modifier proteins (Samps), respectively) to certain proteins, and this may serve to target the modified proteins for degradation by proteasomes.

  3. Identification of structural domains in proteins by a graph heuristic.

    PubMed

    Wernisch, L; Hunting, M; Wodak, S J

    1999-05-15

    A novel automatic procedure for identifying domains from protein atomic coordinates is presented. The procedure, termed STRUDL (STRUctural Domain Limits), does not take into account information on secondary structures and handles any number of domains made up of contiguous or non-contiguous chain segments. The core algorithm uses the Kernighan-Lin graph heuristic to partition the protein into residue sets which display minimum interactions between them. These interactions are deduced from the weighted Voronoi diagram. The generated partitions are accepted or rejected on the basis of optimized criteria, representing basic expected physical properties of structural domains. The graph heuristic approach is shown to be very effective, it approximates closely the exact solution provided by a branch and bound algorithm for a number of test proteins. In addition, the overall performance of STRUDL is assessed on a set of 787 representative proteins from the Protein Data Bank by comparison to domain definitions in the CATH protein classification. The domains assigned by STRUDL agree with the CATH assignments in at least 81% of the tested proteins. This result is comparable to that obtained previously using PUU (Holm and Sander, Proteins 1994;9:256-268), the only other available algorithm designed to identify domains with any number of non-contiguous chain segments. A detailed discussion of the structures for which our assignments differ from those in CATH brings to light some clear inconsistencies between the concept of structural domains based on minimizing inter-domain interactions and that of delimiting structural motifs that represent acceptable folding topologies or architectures. Considering both concepts as complementary and combining them in a layered approach might be the way forward.

  4. Evidence for the presence of collagenous domains in Candida albicans cell surface proteins.

    PubMed Central

    Sepúlveda, P; Murgui, A; López-Ribot, J L; Casanova, M; Timoneda, J; Martínez, J P

    1995-01-01

    Rabbit polyclonal antibodies (PAbs) directed towards the amino-terminal cysteine-rich 7S domain (PAb anti-7S), the major internal collagenous domain (PAb anti-type IV), and the C-terminal noncollagenous region (PAb anti-NC1) of the type IV collagen molecule were probed by indirect immunofluorescence against Candida albicans blastoconidia and germinated blastoconidia. Most nongerminating cells and mother blastoconidia from which germ tubes originated showed strong fluorescence when PAb anti-7S was used, whereas with PAb anti-type IV, fluorescence was found almost exclusively on the surface of filamentous forms. A patched fluorescent pattern rather than a homogenous confluent fluorescence was observed in all cases. No fluorescent cells were observed with PAb anti-NC1. By Western immunoblotting, PAb anti-type IV cross-reacted primarily with a polypeptide of 37 kDa present in wall extracts obtained from intact cells of both growth forms by treatment with beta-mercaptoethanol, whereas PAb anti-7S recognized a major 58-kDa antigen also present in both extracts, along with some other high-molecular-mass (> 106-kDa) polydisperse species present only in the material released from blastoconidia with beta-mercaptoethanol. No reactive bands were observed when PAb anti-NC1 was used as a probe in Western immunoblotting experiments. The sensitivities or resistances to collagenase digestion of the different polypeptides that cross-reacted with PAbs anti-type IV and anti-7S suggest the existence of cell wall components in C. albicans that contain epitopes that mimic the collagenous domains of the type IV collagen molecule. PMID:7768595

  5. Domain fusion analysis by applying relational algebra to protein sequence and domain databases

    PubMed Central

    Truong, Kevin; Ikura, Mitsuhiko

    2003-01-01

    Background Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. Results This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at . Conclusion As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time. PMID:12734020

  6. Domain fusion analysis by applying relational algebra to protein sequence and domain databases.

    PubMed

    Truong, Kevin; Ikura, Mitsuhiko

    2003-05-06

    Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at http://calcium.uhnres.utoronto.ca/pi. As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time.

  7. RNA polymerase II conserved protein domains as platforms for protein-protein interactions

    PubMed Central

    García-López, M Carmen

    2011-01-01

    RNA polymerase II establishes many protein-protein interactions with transcriptional regulators to coordinate gene expression, but little is known about protein domains involved in the contact with them. We use a new approach to look for conserved regions of the RNA pol II of S. cerevisiae located at the surface of the structure of the complex, hypothesizing that they might be involved in the interaction with transcriptional regulators. We defined five different conserved domains and demonstrate that all of them make contact with transcriptional regulators. PMID:21922063

  8. Neuregulin 1 Expression and Electrophysiological Abnormalities in the Neuregulin 1 Transmembrane Domain Heterozygous Mutant Mouse

    PubMed Central

    Frank, Elisabeth; Shaw, Alex; Liu, Shijie; Huang, Xu-Feng; Pinault, Didier; Karl, Tim; O’Brien, Terence J.; Shannon Weickert, Cynthia; Jones, Nigel C.

    2015-01-01

    Background The Neuregulin 1 transmembrane domain heterozygous mutant (Nrg1 TM HET) mouse is used to investigate the role of Nrg1 in brain function and schizophrenia-like behavioural phenotypes. However, the molecular alterations in brain Nrg1 expression that underpin the behavioural observations have been assumed, but not directly determined. Here we comprehensively characterise mRNA Nrg1 transcripts throughout development of the Nrg1 TM HET mouse. In addition, we investigate the regulation of high-frequency (gamma) electrophysiological oscillations in this mutant mouse to associate molecular changes in Nrg1 with a schizophrenia-relevant neurophysiological profile. Methods Using exonic probes spanning the cysteine-rich, epidermal growth factor (EGF)-like, transmembrane and intracellular domain encoding regions of Nrg1, mRNA levels were measured using qPCR in hippocampus and frontal cortex from male and female Nrg1 TM HET and wild type-like (WT) mice throughout development. We also performed electrophysiological recordings in adult mice and analysed gamma oscillatory at baseline, in responses to auditory stimuli and to ketamine. Results In both hippocampus and cortex, Nrg1 TM HET mice show significantly reduced expression of the exon encoding the transmembrane domain of Nrg1 compared with WT, but unaltered mRNA expression encoding the extracellular bioactive EGF-like and the cysteine-rich (type III) domains, and development-specific and region-specific reductions in the mRNA encoding the intracellular domain. Hippocampal Nrg1 protein expression was not altered, but NMDA receptor NR2B subunit phosphorylation was lower in Nrg1 TM HET mice. We identified elevated ongoing and reduced sensory-evoked gamma power in Nrg1 TM HET mice. Interpretation We found no evidence to support the claim that the Nrg1 TM HET mouse represents a simple haploinsufficient model. Further research is required to explore the possibility that mutation results in a gain of Nrg1 function. PMID

  9. Predicting PDZ domain mediated protein interactions from structure

    PubMed Central

    2013-01-01

    Background PDZ domains are structural protein domains that recognize simple linear amino acid motifs, often at protein C-termini, and mediate protein-protein interactions (PPIs) in important biological processes, such as ion channel regulation, cell polarity and neural development. PDZ domain-peptide interaction predictors have been developed based on domain and peptide sequence information. Since domain structure is known to influence binding specificity, we hypothesized that structural information could be used to predict new interactions compared to sequence-based predictors. Results We developed a novel computational predictor of PDZ domain and C-terminal peptide interactions using a support vector machine trained with PDZ domain structure and peptide sequence information. Performance was estimated using extensive cross validation testing. We used the structure-based predictor to scan the human proteome for ligands of 218 PDZ domains and show that the predictions correspond to known PDZ domain-peptide interactions and PPIs in curated databases. The structure-based predictor is complementary to the sequence-based predictor, finding unique known and novel PPIs, and is less dependent on training–testing domain sequence similarity. We used a functional enrichment analysis of our hits to create a predicted map of PDZ domain biology. This map highlights PDZ domain involvement in diverse biological processes, some only found by the structure-based predictor. Based on this analysis, we predict novel PDZ domain involvement in xenobiotic metabolism and suggest new interactions for other processes including wound healing and Wnt signalling. Conclusions We built a structure-based predictor of PDZ domain-peptide interactions, which can be used to scan C-terminal proteomes for PDZ interactions. We also show that the structure-based predictor finds many known PDZ mediated PPIs in human that were not found by our previous sequence-based predictor and is less dependent on

  10. EVEREST: a collection of evolutionary conserved protein domains

    PubMed Central

    Portugaly, Elon; Linial, Nathan; Linial, Michal

    2007-01-01

    Protein domains are subunits of proteins that recur throughout the protein world. There are many definitions attempting to capture the essence of a protein domain, and several systems that identify protein domains and classify them into families. EVEREST, recently described in Portugaly et al. (2006) BMC Bioinformatics, 7, 277, is one such system that performs the task automatically, using protein sequence alone. Herein we describe EVEREST release 2.0, consisting of 20 029 families, each defined by one or more HMMs. The current EVEREST database was constructed by scanning UniProt 8.1 and all PDB sequences (total over 3 000 000 sequences) with each of the EVEREST families. EVEREST annotates 64% of all sequences, and covers 59% of all residues. EVEREST is available at . The website provides annotations given by SCOP, CATH, Pfam A and EVEREST. It allows for browsing through the families of each of those sources, graphically visualizing the domain organization of the proteins in the family. The website also provides access to analyzes of relationships between domain families, within and across domain definition systems. Users can upload sequences for analysis by the set of EVEREST families. Finally an advanced search form allows querying for families matching criteria regarding novelty, phylogenetic composition and more. PMID:17099230

  11. EVEREST: a collection of evolutionary conserved protein domains.

    PubMed

    Portugaly, Elon; Linial, Nathan; Linial, Michal

    2007-01-01

    Protein domains are subunits of proteins that recur throughout the protein world. There are many definitions attempting to capture the essence of a protein domain, and several systems that identify protein domains and classify them into families. EVEREST, recently described in Portugaly et al. (2006) BMC Bioinformatics, 7, 277, is one such system that performs the task automatically, using protein sequence alone. Herein we describe EVEREST release 2.0, consisting of 20,029 families, each defined by one or more HMMs. The current EVEREST database was constructed by scanning UniProt 8.1 and all PDB sequences (total over 3,000,000 sequences) with each of the EVEREST families. EVEREST annotates 64% of all sequences, and covers 59% of all residues. EVEREST is available at http://www.everest.cs.huji.ac.il/. The website provides annotations given by SCOP, CATH, Pfam A and EVEREST. It allows for browsing through the families of each of those sources, graphically visualizing the domain organization of the proteins in the family. The website also provides access to analyzes of relationships between domain families, within and across domain definition systems. Users can upload sequences for analysis by the set of EVEREST families. Finally an advanced search form allows querying for families matching criteria regarding novelty, phylogenetic composition and more.

  12. CDD: a Conserved Domain Database for protein classification.

    PubMed

    Marchler-Bauer, Aron; Anderson, John B; Cherukuri, Praveen F; DeWeese-Scott, Carol; Geer, Lewis Y; Gwadz, Marc; He, Siqian; Hurwitz, David I; Jackson, John D; Ke, Zhaoxi; Lanczycki, Christopher J; Liebert, Cynthia A; Liu, Chunlei; Lu, Fu; Marchler, Gabriele H; Mullokandov, Mikhail; Shoemaker, Benjamin A; Simonyan, Vahan; Song, James S; Thiessen, Paul A; Yamashita, Roxanne A; Yin, Jodie J; Zhang, Dachuan; Bryant, Stephen H

    2005-01-01

    The Conserved Domain Database (CDD) is the protein classification component of NCBI's Entrez query and retrieval system. CDD is linked to other Entrez databases such as Proteins, Taxonomy and PubMed, and can be accessed at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. CD-Search, which is available at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, is a fast, interactive tool to identify conserved domains in new protein sequences. CD-Search results for protein sequences in Entrez are pre-computed to provide links between proteins and domain models, and computational annotation visible upon request. Protein-protein queries submitted to NCBI's BLAST search service at http://www.ncbi.nlm.nih.gov/BLAST are scanned for the presence of conserved domains by default. While CDD started out as essentially a mirror of publicly available domain alignment collections, such as SMART, Pfam and COG, we have continued an effort to update, and in some cases replace these models with domain hierarchies curated at the NCBI. Here, we report on the progress of the curation effort and associated improvements in the functionality of the CDD information retrieval system.

  13. Targeting of passenger protein domains to multiple intracellular membranes.

    PubMed Central

    Janiak, F; Glover, J R; Leber, B; Rachubinski, R A; Andrews, D W

    1994-01-01

    The role of passenger domains in protein targeting was examined by fusing previously characterized targeting motifs to different protein sequences. To compare the targeting requirements for a variety of subcellular compartments, targeting of the fusion proteins was examined for endoplasmic reticulum, mitochondria and peroxisomes in vitro and in yeast. Although most passenger domains were only partially passive to translocation, motif-dependent targeting via motifs positioned at either end of one passenger domain (gPA) was demonstrated for all of the subcellular compartments tested. The data presented extend earlier suggestions that translocation competence is an intrinsic property of the passenger protein. However, the properties that determine protein targeting are not mutually exclusive for the compartments tested. Therefore, although the primary determinant of specificity is the targeting motif, our results suggest that translocation competence of the targeted protein augments the fidelity of transport. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8198533

  14. Assembly of cell regulatory systems through protein interaction domains.

    PubMed

    Pawson, Tony; Nash, Piers

    2003-04-18

    The sequencing of complete genomes provides a list that includes the proteins responsible for cellular regulation. However, this does not immediately reveal what these proteins do, nor how they are assembled into the molecular machines and functional networks that control cellular behavior. The regulation of many different cellular processes requires the use of protein interaction domains to direct the association of polypeptides with one another and with phospholipids, small molecules, or nucleic acids. The modular nature of these domains, and the flexibility of their binding properties, have likely facilitated the evolution of cellular pathways. Conversely, aberrant interactions can induce abnormal cellular behavior and disease. The fundamental properties of protein interaction domains are discussed in this review and in detailed reviews on individual domains at Science's STKE at http://www.sciencemag.org/cgi/content/full/300/5618/445/DC1.

  15. Evolution of a protein domain interaction network

    NASA Astrophysics Data System (ADS)

    Gao, Li-Feng; Shi, Jian-Jun; Guan, Shan

    2010-01-01

    In this paper, we attempt to understand complex network evolution from the underlying evolutionary relationship between biological organisms. Firstly, we construct a Pfam domain interaction network for each of the 470 completely sequenced organisms, and therefore each organism is correlated with a specific Pfam domain interaction network; secondly, we infer the evolutionary relationship of these organisms with the nearest neighbour joining method; thirdly, we use the evolutionary relationship between organisms constructed in the second step as the evolutionary course of the Pfam domain interaction network constructed in the first step. This analysis of the evolutionary course shows: (i) there is a conserved sub-network structure in network evolution; in this sub-network, nodes with lower degree prefer to maintain their connectivity invariant, and hubs tend to maintain their role as a hub is attached preferentially to new added nodes; (ii) few nodes are conserved as hubs; most of the other nodes are conserved as one with very low degree; (iii) in the course of network evolution, new nodes are added to the network either individually in most cases or as clusters with relative high clustering coefficients in a very few cases.

  16. Tandem-repeat protein domains across the tree of life

    PubMed Central

    Jernigan, Kristin K.

    2015-01-01

    Tandem-repeat protein domains, composed of repeated units of conserved stretches of 20–40 amino acids, are required for a wide array of biological functions. Despite their diverse and fundamental functions, there has been no comprehensive assessment of their taxonomic distribution, incidence, and associations with organismal lifestyle and phylogeny. In this study, we assess for the first time the abundance of armadillo (ARM) and tetratricopeptide (TPR) repeat domains across all three domains in the tree of life and compare the results to our previous analysis on ankyrin (ANK) repeat domains in this journal. All eukaryotes and a majority of the bacterial and archaeal genomes analyzed have a minimum of one TPR and ARM repeat. In eukaryotes, the fraction of ARM-containing proteins is approximately double that of TPR and ANK-containing proteins, whereas bacteria and archaea are enriched in TPR-containing proteins relative to ARM- and ANK-containing proteins. We show in bacteria that phylogenetic history, rather than lifestyle or pathogenicity, is a predictor of TPR repeat domain abundance, while neither phylogenetic history nor lifestyle predicts ARM repeat domain abundance. Surprisingly, pathogenic bacteria were not enriched in TPR-containing proteins, which have been associated within virulence factors in certain species. Taken together, this comparative analysis provides a newly appreciated view of the prevalence and diversity of multiple types of tandem-repeat protein domains across the tree of life. A central finding of this analysis is that tandem repeat domain-containing proteins are prevalent not just in eukaryotes, but also in bacterial and archaeal species. PMID:25653910

  17. de Gennes Narrowing Describes the Relative Motion of Protein Domains

    NASA Astrophysics Data System (ADS)

    Hong, Liang; Smolin, Nikolai; Smith, Jeremy C.

    2014-04-01

    The relative motion of structural domains is essential for the biological function of many proteins. Here, by analyzing neutron scattering data and performing molecular dynamics simulations, we find that interdomain motion in several proteins obeys the principle of de Gennes narrowing, in which the wave vector dependence of the interdomain diffusion coefficient is inversely proportional to the interdomain structure factor. Thus, the rate of interdomain motion is inversely proportional to the probability distribution of the spatial configurations of domains.

  18. Stochastic lattice model of synaptic membrane protein domains

    NASA Astrophysics Data System (ADS)

    Li, Yiwei; Kahraman, Osman; Haselwandter, Christoph A.

    2017-05-01

    Neurotransmitter receptor molecules, concentrated in synaptic membrane domains along with scaffolds and other kinds of proteins, are crucial for signal transmission across chemical synapses. In common with other membrane protein domains, synaptic domains are characterized by low protein copy numbers and protein crowding, with rapid stochastic turnover of individual molecules. We study here in detail a stochastic lattice model of the receptor-scaffold reaction-diffusion dynamics at synaptic domains that was found previously to capture, at the mean-field level, the self-assembly, stability, and characteristic size of synaptic domains observed in experiments. We show that our stochastic lattice model yields quantitative agreement with mean-field models of nonlinear diffusion in crowded membranes. Through a combination of analytic and numerical solutions of the master equation governing the reaction dynamics at synaptic domains, together with kinetic Monte Carlo simulations, we find substantial discrepancies between mean-field and stochastic models for the reaction dynamics at synaptic domains. Based on the reaction and diffusion properties of synaptic receptors and scaffolds suggested by previous experiments and mean-field calculations, we show that the stochastic reaction-diffusion dynamics of synaptic receptors and scaffolds provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the observed single-molecule trajectories, and spatial heterogeneity in the effective rates at which receptors and scaffolds are recycled at the cell membrane. Our work sheds light on the physical mechanisms and principles linking the collective properties of membrane protein domains to the stochastic dynamics that rule their molecular components.

  19. Singlet CH domain containing human multidomain proteins: an inventory.

    PubMed

    Friedberg, Felix

    2010-03-01

    The actin cytoskeleton presents the basic force in processes such as cytokinesis, endocytosis, vesicular trafficking and cell migration. Here, we list 30 human singlet CH (calpononin homology/actin binding) containing multidomain molecules, each encoded by one gene. We show the domain distributions as given by the SMART program. These mosaic proteins organize geographically the placement of selected proteins in proximity within the cell. In most instances, their precise location, their actin binding capacity by way of the singlet CH (or by other domains?) and their physiological functions need further elucidation. A dendrogram based solely on the relationship for the human singlet CH domains (in terms of AA sequences) for the various molecules that possess the domain, implies that the singlet descended from a common ancestor which in turn sprouted three main branches of protein products. Each branch bifurcated multiple times thus accounting for a cornucopia of products. Wherever, additional (unassigned), highly homologous regions exist in related proteins (e.g., in LIM and LMO7 or in Tangerin and EH/BP1), these unrecognized domain regions await assignment as specific functional domains. Frequently genes coding multidomain proteins duplicated. The varying modular nature within multidomain proteins should have accelerated evolutionary changes to a degree not feasible to achieve by means of mere post-duplication mutational changes.

  20. Positive selection within sperm-egg adhesion domains of fertilin: an ADAM gene with a potential role in fertilization.

    PubMed

    Civetta, Alberto

    2003-01-01

    Genes with a role in fertilization show a common pattern of rapid evolution. The role played by positive selection versus lack of selective constraints has been more difficult to establish. One problem arises from attempts to detect selection in an overall gene sequence analysis. I have analyzed the pattern of molecular evolution of fertilin, a gene coding for a heterodimeric sperm protein belonging to the ADAM (A disintegrin and A metalloprotease) gene family. A nonsynonymous to synonymous rate ratio (d(N)/d(S)) analysis for different protein domains of fertilin alpha and fertilin beta showed d(N)/d(S) < 1, suggesting that purifying selection has shaped fertilin's evolution. However, an analysis of the distribution of single positively selected codon sites using phylogentic analysis by maximum likelihood (PAML) showed sites within adhesion domains (disintegrin and cysteine-rich) of fertilin beta evolving under positive selection. The region 3' to the EGF-like domain of fertilin alpha, where the transmembrane and cytoplasmic tail regions are supposed to be localized, showed higher d(N) and d(S) than any other fertilin alpha region. However, it was not possible to identify positively selected codon sites due to ambiguous alignments of the carboxy-end region (ClustalX vs. DiAlign2). When this region was excluded from the PAML analysis, most single positively selected codon sites were concentrated within adhesion domains (cysteine-rich and EGF-like). The use of an ancestral sequence prior to a recent duplication event of fertilin alpha among non-Hominidae primates (Macaca, Papio, and Saguinus) revealed that the duplication is partially responsible for masking the detection of positively selected sites within the disintegrin domain. Finally, most ADAM genes with a potential role in sperm maturation and/or fertilization showed significantly higher d(N) estimates than other ADAM genes.

  1. Conditional random field approach to prediction of protein-protein interactions using domain information.

    PubMed

    Hayashida, Morihiro; Kamada, Mayumi; Song, Jiangning; Akutsu, Tatsuya

    2011-06-20

    For understanding cellular systems and biological networks, it is important to analyze functions and interactions of proteins and domains. Many methods for predicting protein-protein interactions have been developed. It is known that mutual information between residues at interacting sites can be higher than that at non-interacting sites. It is based on the thought that amino acid residues at interacting sites have coevolved with those at the corresponding residues in the partner proteins. Several studies have shown that such mutual information is useful for identifying contact residues in interacting proteins. We propose novel methods using conditional random fields for predicting protein-protein interactions. We focus on the mutual information between residues, and combine it with conditional random fields. In the methods, protein-protein interactions are modeled using domain-domain interactions. We perform computational experiments using protein-protein interaction datasets for several organisms, and calculate AUC (Area Under ROC Curve) score. The results suggest that our proposed methods with and without mutual information outperform EM (Expectation Maximization) method proposed by Deng et al., which is one of the best predictors based on domain-domain interactions. We propose novel methods using conditional random fields with and without mutual information between domains. Our methods based on domain-domain interactions are useful for predicting protein-protein interactions.

  2. Anchors aweigh: protein localization and transport mediated by transmembrane domains.

    PubMed

    Cosson, Pierre; Perrin, Jackie; Bonifacino, Juan S

    2013-10-01

    The transmembrane domains (TMDs) of integral membrane proteins have emerged as major determinants of intracellular localization and transport in the secretory and endocytic pathways. Unlike sorting signals in cytosolic domains, TMD sorting determinants are not conserved amino acid sequences but physical properties such as the length and hydrophilicity of the transmembrane span. The underlying sorting machinery is still poorly characterized, but several mechanisms have been proposed, including TMD recognition by transmembrane sorting receptors and partitioning into membrane lipid domains. Here we review the nature of TMD sorting determinants and how they may dictate transmembrane protein localization and transport.

  3. Anchors Aweigh: Protein Traffic Mediated by Transmembrane Domains

    PubMed Central

    Cosson, Pierre; Perrin, Jackie; Bonifacino, Juan S.

    2013-01-01

    The transmembrane domains (TMDs) of integral membrane proteins have emerged as major determinants of intracellular localization and transport in the secretory and endocytic pathways. Unlike sorting signals in the cytosolic domains, TMD sorting determinants are not conserved amino-acid sequences but physical properties such as length and hydrophilicity of the transmembrane span. The underlying sorting machinery is still poorly characterized but several mechanisms have been proposed, including TMD recognition by transmembrane sorting receptors and partitioning into membrane lipid domains. Here we review the nature of TMD sorting determinants and how they may dictate transmembrane protein localization and transport. PMID:23806646

  4. 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.

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

    PubMed Central

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

    2011-01-01

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

  6. Cloning, expression and purification of the SRCR domains of glycoprotein 340.

    PubMed

    Purushotham, Sangeetha; Deivanayagam, Champion

    2013-08-01

    Glycoprotein 340 (gp340), an innate immunity molecule is secreted luminally by monolayered epithelia and associated glands within the human oral cavity. Gp340 contains 14 scavenger receptor cysteine rich (SRCR) domains, two CUB (C1r/C1s Uegf Bmp1) domains and one zona pellucida (ZP) domain. Oral streptococci are known to adhere to the tooth immobilized gp340 via its surface protein Antigen I/II (AgI/II), which is considered to be the critical first step in pathogenesis that eventually results in colonization and infection. In order to decipher the interactions between gp340's domains and oral streptococcal AgI/II domains, we undertook to express human gp340's first SRCR domain (SRCR1) and the first three tandem SRCR domains (SRCR123) in Drosophila S2 cells. While our initial attempts with human codons did not produce optimal results, codon-optimization for expression in Drosophila S2 cells and usage of inducible/secretory Drosophila expression system (DES) pMT/BiP/V5-HisA vector greatly enhanced the expression of the SRCR domains. Here we report the successful cloning, expression, and purification of the SRCR domains of gp340. Recognition of expressed SRCRs by the conformational dependent gp340 antibody indicate that these domains are appropriately folded and furthermore, surface plasmon resonance studies confirmed functional adherence of the SRCR domains to AgI/II.

  7. Domain organizations of modular extracellular matrix proteins and their evolution.

    PubMed

    Engel, J

    1996-11-01

    Multidomain proteins which are composed of modular units are a rather recent invention of evolution. Domains are defined as autonomously folding regions of a protein, and many of them are similar in sequence and structure, indicating common ancestry. Their modular nature is emphasized by frequent repetitions in identical or in different proteins and by a large number of different combinations with other domains. The extracellular matrix is perhaps the largest biological system composed of modular mosaic proteins, and its astonishing complexity and diversity are based on them. A cluster of minireviews on modular proteins is being published in Matrix Biology. These deal with the evolution of modular proteins, the three-dimensional structure of domains and the ways in which these interact in a multidomain protein. They discuss structure-function relationships in calcium binding domains, collagen helices, alpha-helical coiled-coil domains and C-lectins. The present minireview is focused on some general aspects and serves as an introduction to the cluster.

  8. Structural basis of Smoothened regulation by its extracellular domains

    NASA Astrophysics Data System (ADS)

    Byrne, Eamon F. X.; Sircar, Ria; Miller, Paul S.; Hedger, George; Luchetti, Giovanni; Nachtergaele, Sigrid; Tully, Mark D.; Mydock-McGrane, Laurel; Covey, Douglas F.; Rambo, Robert P.; Sansom, Mark S. P.; Newstead, Simon; Rohatgi, Rajat; Siebold, Christian

    2016-07-01

    Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.

  9. 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

  10. CD-Search: protein domain annotations on the fly.

    PubMed

    Marchler-Bauer, Aron; Bryant, Stephen H

    2004-07-01

    We describe the Conserved Domain Search service (CD-Search), a web-based tool for the detection of structural and functional domains in protein sequences. CD-Search uses BLAST(R) heuristics to provide a fast, interactive service, and searches a comprehensive collection of domain models. Search results are displayed as domain architecture cartoons and pairwise alignments between the query and domain-model consensus sequences. Search results may be visualized in further detail by embedding the query sequence into multiple alignment displays and by mapping onto three-dimensional molecular graphic displays of known structures within the domain family. CD-Search can be accessed at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi.

  11. Domain conservation in several volvocalean cell wall proteins.

    PubMed

    Woessner, J P; Molendijk, A J; van Egmond, P; Klis, F M; Goodenough, U W; Haring, M A

    1994-11-01

    Based on our previous work demonstrating that (SerPro)x epitopes are common to extensin-like cell wall proteins in Chlamydomonas' reinhardtii, we looked for similar proteins in the distantly related species C. eugametos. Using a polyclonal antiserum against a (SerPro)10 oligopeptide, we found distinct sets of stage-specific polypeptides immunoprecipitated from in vitro translations of C. eugametos RNA. Screening of a C. eugametos cDNA expression library with the antiserum led to the isolation of a cDNA (WP6) encoding a (SerPro)x-rich multidomain wall protein. Analysis of a similarly selected cDNA (VSP-3) from a C. reinhardtii cDNA expression library revealed that it also coded for a (SerPro)x-rich multidomain wall protein. The C-terminal rod domains of VSP-3 and WP6 are highly homologous, while the N-terminal domains are dissimilar; however, the N-terminal domain of VSP-3 is homologous to the globular domain of a cell wall protein from Volvox carteri. Exon shuffling might be responsible for this example of domain conservation over 350 million years of volvocalean cell wall protein evolution.

  12. Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

    SciTech Connect

    Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong

    2012-06-05

    A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

  13. The PUB domain: a putative protein-protein interaction domain implicated in the ubiquitin-proteasome pathway.

    PubMed

    Suzuki, T; Park, H; Till, E A; Lennarz, W J

    2001-10-12

    Cytoplasmic peptide:N-glycanase (PNGase) is a de-N-glycosylating enzyme which may be involved in the proteasome-dependent pathway for degradation of misfolded glycoproteins formed in the endoplasmic reticulum (ER) that are exported into the cytoplasm. A cytoplasmic PNGase found in Saccharomyces cerevisiae, Png1p, is widely distributed in higher eukaryotes as well as in yeast (Suzuki, T., et al. J. Cell Biol. 149, 1039-1051, 2000). The recently uncovered complete genome sequence of Arabidopsis thaliana prompted us to search for the protein homologue of Png1p in this organism. Interestingly, when the mouse Png1p homologue sequence was used as a query, not only a Png1p homologue containing a transglutaminase-like domain that is believed to contain a catalytic triad for PNGase activity, but also four proteins which had a domain of 46 amino acids in length that exhibited significant similarity to the N-terminus of mouse Png1p were identified. Moreover, three of these homologous proteins were also found to possess a UBA or UBX domain, which are found in various proteins involved in the ubiquitin-related pathway. We name this newly found homologous region the PUB (Peptide:N-glycanase/UBA or UBX-containing proteins) domain and propose that this domain may mediate protein-protein interactions.

  14. The history of the CATH structural classification of protein domains.

    PubMed

    Sillitoe, Ian; Dawson, Natalie; Thornton, Janet; Orengo, Christine

    2015-12-01

    This article presents a historical review of the protein structure classification database CATH. Together with the SCOP database, CATH remains comprehensive and reasonably up-to-date with the now more than 100,000 protein structures in the PDB. We review the expansion of the CATH and SCOP resources to capture predicted domain structures in the genome sequence data and to provide information on the likely functions of proteins mediated by their constituent domains. The establishment of comprehensive function annotation resources has also meant that domain families can be functionally annotated allowing insights into functional divergence and evolution within protein families. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Viral Macro Domains Reverse Protein ADP-Ribosylation.

    PubMed

    Li, Changqing; Debing, Yannick; Jankevicius, Gytis; Neyts, Johan; Ahel, Ivan; Coutard, Bruno; Canard, Bruno

    2016-10-01

    ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD(+) to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant. Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular integrity are demodified

  16. Viral Macro Domains Reverse Protein ADP-Ribosylation

    PubMed Central

    Li, Changqing; Debing, Yannick; Jankevicius, Gytis; Neyts, Johan; Ahel, Ivan

    2016-01-01

    ABSTRACT ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD+ to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant. IMPORTANCE Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular

  17. On the detection of functionally coherent groups of protein domains with an extension to protein annotation

    PubMed Central

    McLaughlin, William A; Chen, Ken; Hou, Tingjun; Wang, Wei

    2007-01-01

    Background Protein domains coordinate to perform multifaceted cellular functions, and domain combinations serve as the functional building blocks of the cell. The available methods to identify functional domain combinations are limited in their scope, e.g. to the identification of combinations falling within individual proteins or within specific regions in a translated genome. Further effort is needed to identify groups of domains that span across two or more proteins and are linked by a cooperative function. Such functional domain combinations can be useful for protein annotation. Results Using a new computational method, we have identified 114 groups of domains, referred to as domain assembly units (DASSEM units), in the proteome of budding yeast Saccharomyces cerevisiae. The units participate in many important cellular processes such as transcription regulation, translation initiation, and mRNA splicing. Within the units the domains were found to function in a cooperative manner; and each domain contributed to a different aspect of the unit's overall function. The member domains of DASSEM units were found to be significantly enriched among proteins contained in transcription modules, defined as genes sharing similar expression profiles and presumably similar functions. The observation further confirmed the functional coherence of DASSEM units. The functional linkages of units were found in both functionally characterized and uncharacterized proteins, which enabled the assessment of protein function based on domain composition. Conclusion A new computational method was developed to identify groups of domains that are linked by a common function in the proteome of Saccharomyces cerevisiae. These groups can either lie within individual proteins or span across different proteins. We propose that the functional linkages among the domains within the DASSEM units can be used as a non-homology based tool to annotate uncharacterized proteins. PMID:17937820

  18. Protein domain repetition is enriched in Streptococcal cell-surface proteins.

    PubMed

    Lin, I-Hsuan; Hsu, Ming-Ta; Chang, Chuan-Hsiung

    2012-12-01

    Tandem repetition of domain in protein sequence occurs in all three domains of life. It creates protein diversity and adds functional complexity in organisms. In this work, we analyzed 52 streptococcal genomes and found 3748 proteins contained domain repeats. Proteins not harboring domain repeats are significantly enriched in cytoplasm, whereas proteins with domain repeats are significantly enriched in cytoplasmic membrane, cell wall and extracellular locations. Domain repetition occurs most frequently in S. pneumoniae and least in S. thermophilus and S. pyogenes. DUF1542 is the highest repeated domain in a single protein, followed by Rib, CW_binding_1, G5 and HemolysinCabind. 3D structures of 24 repeat-containing proteins were predicted to investigate the structural and functional effect of domain repetition. Several repeat-containing streptococcal cell surface proteins are known to be virulence-associated. Surface-associated tandem domain-containing proteins without experimental functional characterization may be potentially involved in the pathogenesis of streptococci and deserve further investigation. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Crystal structure of Bacillus subtilis anti-TRAP protein, an antagonist of TRAP/RNA interaction

    PubMed Central

    Shevtsov, Mikhail B.; Chen, Yanling; Gollnick, Paul; Antson, Alfred A.

    2005-01-01

    In Bacillus subtilis the anti-TRAP protein (AT) is produced in response to the accumulation of uncharged tRNATrp. AT regulates expression of genes involved in tryptophan biosynthesis and transport by binding to the tryptophan-activated trp RNA-binding attenuation protein (TRAP) and preventing its interaction with several mRNAs. Here, we report the x-ray structure of AT at 2.8 Å resolution, showing that the protein subunits assemble into tight trimers. Four such trimers are further associated into a 12-subunit particle in which individual trimers are related by twofold and threefold symmetry axes. Twelve DnaJ-like, cysteine-rich zinc-binding domains form spikes on the surface of the dodecamer. Available data suggest several possible ways for AT to interact with the 11-subunit TRAP. Interaction between the two symmetry-mismatching molecules could be assisted by the flexible nature of AT zinc-binding domains. PMID:16306262

  20. Quantifying protein–protein interactions in high throughput using protein domain microarrays

    PubMed Central

    Kaushansky, Alexis; Allen, John E; Gordus, Andrew; Stiffler, Michael A; Karp, Ethan S; Chang, Bryan H; MacBeath, Gavin

    2011-01-01

    Protein microarrays provide an efficient way to identify and quantify protein–protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain–peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (KDs) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein–ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein–protein interaction networks. PMID:20360771

  1. Understanding protein domain-swapping using structure-based models of protein folding.

    PubMed

    Mascarenhas, Nahren Manuel; Gosavi, Shachi

    2017-09-01

    In domain-swapping, two or more identical protein monomers exchange structural elements and fold into dimers or multimers whose units are structurally similar to the original monomer. Domain-swapping is of biotechnological interest because inhibiting domain-swapping can reduce disease-causing fibrillar protein aggregation. To achieve such inhibition, it is important to understand both the energetics that stabilize the domain-swapped structure and the protein dynamics that enable the swapping. Structure-based models (SBMs) encode the folded structure of the protein in their potential energy functions. SBMs have been successfully used to understand diverse aspects of monomer folding. Symmetrized SBMs model interactions between two identical protein chains using only intra-monomer interactions. Molecular dynamics simulations of such symmetrized SBMs have been used to correctly predict the domain-swapped structure and to understand the mechanism of domain-swapping. Here, we review such models and illustrate that monomer topology determines key aspects of domain-swapping. However, in some proteins, specifics of local energetic interactions modulate domain-swapping and these need to be added to the symmetrized SBMs. We then summarize some general principles of the mechanism of domain-swapping that emerge from the symmetrized SBM simulations. Finally, using our own results, we explore how symmetrized SBMs could be used to design domain-swapping in proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Partitioning protein structures into domains: why is it so difficult?

    PubMed

    Holland, Timothy A; Veretnik, Stella; Shindyalov, Ilya N; Bourne, Philip E

    2006-08-18

    This analysis takes an in-depth look into the difficulties encountered by automatic methods for domain decomposition from three-dimensional structure. The analysis involves a multi-faceted set of criteria including the integrity of secondary structure elements, the tendency toward fragmentation of domains, domain boundary consistency and topology. The strength of the analysis comes from the use of a new comprehensive benchmark dataset, which is based on consensus among experts (CATH, SCOP and AUTHORS of the 3D structures) and covers 30 distinct architectures and 211 distinct topologies as defined by CATH. Furthermore, over 66% of the structures are multi-domain proteins; each domain combination occurring once per dataset. The performance of four automatic domain assignment methods, DomainParser, NCBI, PDP and PUU, is carefully analyzed using this broad spectrum of topology combinations and knowledge of rules and assumptions built into each algorithm. We conclude that it is practically impossible for an automatic method to achieve the level of performance of human experts. However, we propose specific improvements to automatic methods as well as broadening the concept of a structural domain. Such work is prerequisite for establishing improved approaches to domain recognition. (The benchmark dataset is available from http://pdomains.sdsc.edu).

  3. Characterization of Two Dinoflagellate Cold Shock Domain Proteins

    PubMed Central

    Beauchemin, Mathieu; Roy, Sougata; Pelletier, Sarah; Averback, Alexandra; Lanthier, Frederic

    2016-01-01

    ABSTRACT Roughly two-thirds of the proteins annotated as transcription factors in dinoflagellate transcriptomes are cold shock domain-containing proteins (CSPs), an uncommon condition in eukaryotic organisms. However, no functional analysis has ever been reported for a dinoflagellate CSP, and so it is not known if they do in fact act as transcription factors. We describe here some of the properties of two CSPs from the dinoflagellate Lingulodinium polyedrum, LpCSP1 and LpCSP2, which contain a glycine-rich C-terminal domain and an N-terminal cold shock domain phylogenetically related to those in bacteria. However, neither of the two LpCSPs act like the bacterial CSP, since they do not functionally complement the Escherichia coli quadruple cold shock domain protein mutant BX04, and cold shock does not induce LpCSP1 and LpCSP2 to detectable levels, based on two-dimensional gel electrophoresis. Both CSPs bind to RNA and single-stranded DNA in a nonspecific manner in electrophoretic mobility shift assays, and both proteins also bind double-stranded DNA nonspecifically, albeit more weakly. These CSPs are thus unlikely to act alone as sequence-specific transcription factors. IMPORTANCE Dinoflagellate transcriptomes contain cold shock domain proteins as the major component of the proteins annotated as transcription factors. We show here that the major family of cold shock domain proteins in the dinoflagellate Lingulodinium do not bind specific sequences, suggesting that transcriptional control is not a predominant mechanism for regulating gene expression in this group of protists. PMID:27303711

  4. Rapid similarity search of proteins using alignments of domain arrangements.

    PubMed

    Terrapon, Nicolas; Weiner, January; Grath, Sonja; Moore, Andrew D; Bornberg-Bauer, Erich

    2014-01-15

    Homology search methods are dominated by the central paradigm that sequence similarity is a proxy for common ancestry and, by extension, functional similarity. For determining sequence similarity in proteins, most widely used methods use models of sequence evolution and compare amino-acid strings in search for conserved linear stretches. Probabilistic models or sequence profiles capture the position-specific variation in an alignment of homologous sequences and can identify conserved motifs or domains. While profile-based search methods are generally more accurate than simple sequence comparison methods, they tend to be computationally more demanding. In recent years, several methods have emerged that perform protein similarity searches based on domain composition. However, few methods have considered the linear arrangements of domains when conducting similarity searches, despite strong evidence that domain order can harbour considerable functional and evolutionary signal. Here, we introduce an alignment scheme that uses a classical dynamic programming approach to the global alignment of domains. We illustrate that representing proteins as strings of domains (domain arrangements) and comparing these strings globally allows for a both fast and sensitive homology search. Further, we demonstrate that the presented methods complement existing methods by finding similar proteins missed by popular amino-acid-based comparison methods. An implementation of the presented algorithms, a web-based interface as well as a command-line program for batch searching against the UniProt database can be found at http://rads.uni-muenster.de. Furthermore, we provide a JAVA API for programmatic access to domain-string–based search methods.

  5. Segmental, Domain-Selective Perdeuteration and Small-Angle Neutron Scattering for Structural Analysis of Multi-Domain Proteins.

    PubMed

    Sonntag, Miriam; Jagtap, Pravin Kumar Ankush; Simon, Bernd; Appavou, Marie-Sousai; Geerlof, Arie; Stehle, Ralf; Gabel, Frank; Hennig, Janosch; Sattler, Michael

    2017-08-01

    Multi-domain proteins play critical roles in fine-tuning essential processes in cellular signaling and gene regulation. Typically, multiple globular domains that are connected by flexible linkers undergo dynamic rearrangements upon binding to protein, DNA or RNA ligands. RNA binding proteins (RBPs) represent an important class of multi-domain proteins, which regulate gene expression by recognizing linear or structured RNA sequence motifs. Here, we employ segmental perdeuteration of the three RNA recognition motif (RRM) domains in the RBP TIA-1 using Sortase A mediated protein ligation. We show that domain-selective perdeuteration combined with contrast-matched small-angle neutron scattering (SANS), SAXS and computational modeling provides valuable information to precisely define relative domain arrangements. The approach is generally applicable to study conformational arrangements of individual domains in multi-domain proteins and changes induced by ligand binding. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Dynamics of domain coverage of the protein sequence universe.

    PubMed

    Rekapalli, Bhanu; Wuichet, Kristin; Peterson, Gregory D; Zhulin, Igor B

    2012-11-16

    The currently known protein sequence space consists of millions of sequences in public databases and is rapidly expanding. Assigning sequences to families leads to a better understanding of protein function and the nature of the protein universe. However, a large portion of the current protein space remains unassigned and is referred to as its "dark matter". Here we suggest that true size of "dark matter" is much larger than stated by current definitions. We propose an approach to reducing the size of "dark matter" by identifying and subtracting regions in protein sequences that are not likely to contain any domain. Recent improvements in computational domain modeling result in a decrease, albeit slowly, in the relative size of "dark matter"; however, its absolute size increases substantially with the growth of sequence data.

  7. Dynamics of domain coverage of the protein sequence universe

    PubMed Central

    2012-01-01

    Background The currently known protein sequence space consists of millions of sequences in public databases and is rapidly expanding. Assigning sequences to families leads to a better understanding of protein function and the nature of the protein universe. However, a large portion of the current protein space remains unassigned and is referred to as its “dark matter”. Results Here we suggest that true size of “dark matter” is much larger than stated by current definitions. We propose an approach to reducing the size of “dark matter” by identifying and subtracting regions in protein sequences that are not likely to contain any domain. Conclusions Recent improvements in computational domain modeling result in a decrease, albeit slowly, in the relative size of “dark matter”; however, its absolute size increases substantially with the growth of sequence data. PMID:23157439

  8. Repeat proteins challenge the concept of structural domains.

    PubMed

    Espada, Rocío; Parra, R Gonzalo; Sippl, Manfred J; Mora, Thierry; Walczak, Aleksandra M; Ferreiro, Diego U

    2015-10-01

    Structural domains are believed to be modules within proteins that can fold and function independently. Some proteins show tandem repetitions of apparent modular structure that do not fold independently, but rather co-operate in stabilizing structural forms that comprise several repeat-units. For many natural repeat-proteins, it has been shown that weak energetic links between repeats lead to the breakdown of co-operativity and the appearance of folding sub-domains within an apparently regular repeat array. The quasi-1D architecture of repeat-proteins is crucial in detailing how the local energetic balances can modulate the folding dynamics of these proteins, which can be related to the physiological behaviour of these ubiquitous biological systems.

  9. How to hide zinc in a small protein.

    PubMed

    Blindauer, Claudia A; Sadler, Peter J

    2005-01-01

    Small cysteine-rich proteins (metallothioneins) and related domains of some large proteins (e.g., lysine methyltransferases) bind tri- and tetranuclear zinc clusters with topologies resembling fragments of Zn(II) sulfide minerals. These clusters are ubiquitous in animals, plants, and bacteria. Bacterial metallothioneins can also contain histidines as cluster ligands and embed Zn(II) with a "treble-clef"-like finger fold. This unusual embedded Zn(II) is "hidden" and surprisingly inert toward Zn or Cd exchange. Clearly, proteins can exert fine control over both the thermodynamics and kinetics of zinc binding in thiolate clusters. Genome sequences suggest that related zinc-finger sites are common in a variety of bacteria.

  10. Facilitation of Endosomal Recycling by an IRG Protein Homolog Maintains Apical Tubule Structure in Caenorhabditis elegans

    PubMed Central

    Grussendorf, Kelly A.; Trezza, Christopher J.; Salem, Alexander T.; Al-Hashimi, Hikmat; Mattingly, Brendan C.; Kampmeyer, Drew E.; Khan, Liakot A.; Hall, David H.; Göbel, Verena; Ackley, Brian D.; Buechner, Matthew

    2016-01-01

    Determination of luminal diameter is critical to the function of small single-celled tubes. A series of EXC proteins, including EXC-1, prevent swelling of the tubular excretory canals in Caenorhabditis elegans. In this study, cloning of exc-1 reveals it to encode a homolog of mammalian IRG proteins, which play roles in immune response and autophagy and are associated with Crohn’s disease. Mutants in exc-1 accumulate early endosomes, lack recycling endosomes, and exhibit abnormal apical cytoskeletal structure in regions of enlarged tubules. EXC-1 interacts genetically with two other EXC proteins that also affect endosomal trafficking. In yeast two-hybrid assays, wild-type and putative constitutively active EXC-1 binds to the LIM-domain protein EXC-9, whose homolog, cysteine-rich intestinal protein, is enriched in mammalian intestine. These results suggest a model for IRG function in forming and maintaining apical tubule structure via regulation of endosomal recycling. PMID:27334269

  11. An Algebro-Topological Description of Protein Domain Structure

    PubMed Central

    Penner, Robert Clark; Knudsen, Michael; Wiuf, Carsten; Andersen, Jørgen Ellegaard

    2011-01-01

    The space of possible protein structures appears vast and continuous, and the relationship between primary, secondary and tertiary structure levels is complex. Protein structure comparison and classification is therefore a difficult but important task since structure is a determinant for molecular interaction and function. We introduce a novel mathematical abstraction based on geometric topology to describe protein domain structure. Using the locations of the backbone atoms and the hydrogen bonds, we build a combinatorial object – a so-called fatgraph. The description is discrete yet gives rise to a 2-dimensional mathematical surface. Thus, each protein domain corresponds to a particular mathematical surface with characteristic topological invariants, such as the genus (number of holes) and the number of boundary components. Both invariants are global fatgraph features reflecting the interconnectivity of the domain by hydrogen bonds. We introduce the notion of robust variables, that is variables that are robust towards minor changes in the structure/fatgraph, and show that the genus and the number of boundary components are robust. Further, we invesigate the distribution of different fatgraph variables and show how only four variables are capable of distinguishing different folds. We use local (secondary) and global (tertiary) fatgraph features to describe domain structures and illustrate that they are useful for classification of domains in CATH. In addition, we combine our method with two other methods thereby using primary, secondary, and tertiary structure information, and show that we can identify a large percentage of new and unclassified structures in CATH. PMID:21629687

  12. Bpur, the Lyme Disease Spirochete's PUR Domain Protein

    PubMed Central

    Jutras, Brandon L.; Chenail, Alicia M.; Carroll, Dustin W.; Miller, M. Clarke; Zhu, Haining; Bowman, Amy; Stevenson, Brian

    2013-01-01

    The PUR domain is a nucleic acid-binding motif found in critical regulatory proteins of higher eukaryotes and in certain species of bacteria. During investigations into mechanisms by which the Lyme disease spirochete controls synthesis of its Erp surface proteins, it was discovered that the borrelial PUR domain protein, Bpur, binds with high affinity to double-stranded DNA adjacent to the erp transcriptional promoter. Bpur was found to enhance the effects of the erp repressor protein, BpaB. Bpur also bound single-stranded DNA and RNA, with relative affinities RNA > double-stranded DNA > single-stranded DNA. Rational site-directed mutagenesis of Bpur identified amino acid residues and domains critical for interactions with nucleic acids, and it revealed that the PUR domain has a distinct mechanism of interaction with each type of nucleic acid ligand. These data shed light on both gene regulation in the Lyme spirochete and functional mechanisms of the widely distributed PUR domain. PMID:23846702

  13. Formation and organization of protein domains in the immunological synapse

    NASA Astrophysics Data System (ADS)

    Carlson, Andreas; Mahadevan, L.

    2014-11-01

    The cellular basis for the adaptive immune response during antigen recognition relies on a specialized protein interface known as the immunological synapse. Here, we propose a minimal mathematical model for the dynamics of the IS that encompass membrane mechanics, hydrodynamics and protein kinetics. Simple scaling laws describe the dynamics of protein clusters as a function of membrane stiffness, rigidity of the adhesive proteins, and fluid flow in the synaptic cleft. Numerical simulations complement the scaling laws by quantifying the nucleation, growth and stabilization of proteins domains on the size of the cell. Direct comparison with experiment suggests that passive dynamics suffices to describe the short-time formation and organization of protein clusters, while the stabilization and long time dynamics of the synapse is likely determined by active cytoskeleton processes triggered by receptor binding. Our study reveals that the fluid flow generated by the interplay between membrane deformation and protein binding kinetics can assist immune cells in regulating protein sorting.

  14. Effective Moment Feature Vectors for Protein Domain Structures

    PubMed Central

    Shi, Jian-Yu; Yiu, Siu-Ming; Zhang, Yan-Ning; Chin, Francis Yuk-Lun

    2013-01-01

    Imaging processing techniques have been shown to be useful in studying protein domain structures. The idea is to represent the pairwise distances of any two residues of the structure in a 2D distance matrix (DM). Features and/or submatrices are extracted from this DM to represent a domain. Existing approaches, however, may involve a large number of features (100–400) or complicated mathematical operations. Finding fewer but more effective features is always desirable. In this paper, based on some key observations on DMs, we are able to decompose a DM image into four basic binary images, each representing the structural characteristics of a fundamental secondary structure element (SSE) or a motif in the domain. Using the concept of moments in image processing, we further derive 45 structural features based on the four binary images. Together with 4 features extracted from the basic images, we represent the structure of a domain using 49 features. We show that our feature vectors can represent domain structures effectively in terms of the following. (1) We show a higher accuracy for domain classification. (2) We show a clear and consistent distribution of domains using our proposed structural vector space. (3) We are able to cluster the domains according to our moment features and demonstrate a relationship between structural variation and functional diversity. PMID:24391828

  15. 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

  16. Protein domain recurrence and order can enhance prediction of protein functions.

    PubMed

    Messih, Mario Abdel; Chitale, Meghana; Bajic, Vladimir B; Kihara, Daisuke; Gao, Xin

    2012-09-15

    Burgeoning sequencing technologies have generated massive amounts of genomic and proteomic data. Annotating the functions of proteins identified in this data has become a big and crucial problem. Various computational methods have been developed to infer the protein functions based on either the sequences or domains of proteins. The existing methods, however, ignore the recurrence and the order of the protein domains in this function inference. We developed two new methods to infer protein functions based on protein domain recurrence and domain order. Our first method, DRDO, calculates the posterior probability of the Gene Ontology terms based on domain recurrence and domain order information, whereas our second method, DRDO-NB, relies on the naïve Bayes methodology using the same domain architecture information. Our large-scale benchmark comparisons show strong improvements in the accuracy of the protein function inference achieved by our new methods, demonstrating that domain recurrence and order can provide important information for inference of protein functions. The new models are provided as open source programs at http://sfb.kaust.edu.sa/Pages/Software.aspx. dkihara@cs.purdue.edu, xin.gao@kaust.edu.sa Supplementary data are available at Bioinformatics Online.

  17. Analysis of familial hemophagocytic lymphohistiocytosis type 4 (FHL-4) mutant proteins reveals that S-acylation is required for the function of syntaxin 11 in natural killer cells.

    PubMed

    Hellewell, Andrew L; Foresti, Ombretta; Gover, Nicola; Porter, Morwenna Y; Hewitt, Eric W

    2014-01-01

    Natural killer (NK) cell secretory lysosome exocytosis and cytotoxicity are impaired in familial hemophagocytic lymphohistiocytosis type 4 (FHL-4), a disorder caused by mutations in the gene encoding the SNARE protein syntaxin 11. We show that syntaxin 11 binds to SNAP23 in NK cells and that this interaction is reduced by FHL-4 truncation and frameshift mutation proteins that delete all or part of the SNARE domain of syntaxin 11. In contrast the FHL-4 mutant proteins bound to the Sec-1/Munc18-like (SM) protein Munc18-2. We demonstrate that the C-terminal cysteine rich region of syntaxin 11, which is deleted in the FHL-4 mutants, is S-acylated. This posttranslational modification is required for the membrane association of syntaxin 11 and for its polarization to the immunological synapse in NK cells conjugated to target cells. Moreover, we show that Munc18-2 is recruited by syntaxin 11 to intracellular membranes in resting NK cells and to the immunological synapse in activated NK cells. This recruitment of Munc18-2 is abolished by deletion of the C-terminal cysteine rich region of syntaxin 11. These results suggest a pivotal role for S-acylation in the function of syntaxin 11 in NK cells.

  18. Analysis of Familial Hemophagocytic Lymphohistiocytosis Type 4 (FHL-4) Mutant Proteins Reveals that S-Acylation Is Required for the Function of Syntaxin 11 in Natural Killer Cells

    PubMed Central

    Hellewell, Andrew L.; Foresti, Ombretta; Gover, Nicola; Porter, Morwenna Y.; Hewitt, Eric W.

    2014-01-01

    Natural killer (NK) cell secretory lysosome exocytosis and cytotoxicity are impaired in familial hemophagocytic lymphohistiocytosis type 4 (FHL-4), a disorder caused by mutations in the gene encoding the SNARE protein syntaxin 11. We show that syntaxin 11 binds to SNAP23 in NK cells and that this interaction is reduced by FHL-4 truncation and frameshift mutation proteins that delete all or part of the SNARE domain of syntaxin 11. In contrast the FHL-4 mutant proteins bound to the Sec-1/Munc18-like (SM) protein Munc18-2. We demonstrate that the C-terminal cysteine rich region of syntaxin 11, which is deleted in the FHL-4 mutants, is S-acylated. This posttranslational modification is required for the membrane association of syntaxin 11 and for its polarization to the immunological synapse in NK cells conjugated to target cells. Moreover, we show that Munc18-2 is recruited by syntaxin 11 to intracellular membranes in resting NK cells and to the immunological synapse in activated NK cells. This recruitment of Munc18-2 is abolished by deletion of the C-terminal cysteine rich region of syntaxin 11. These results suggest a pivotal role for S-acylation in the function of syntaxin 11 in NK cells. PMID:24910990

  19. Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins

    PubMed Central

    Smith, Everett Clinton; Smith, Stacy E.; Carter, James R.; Webb, Stacy R.; Gibson, Kathleen M.; Hellman, Lance M.; Fried, Michael G.; Dutch, Rebecca Ellis

    2013-01-01

    Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion. PMID:24178297

  20. SNP@Domain: a web resource of single nucleotide polymorphisms (SNPs) within protein domain structures and sequences

    PubMed Central

    Han, Areum; Kang, Hyo Jin; Cho, Yoobok; Lee, Sunghoon; Kim, Young Joo; Gong, Sungsam

    2006-01-01

    The single nucleotide polymorphisms (SNPs) in conserved protein regions have been thought to be strong candidates that alter protein functions. Thus, we have developed SNP@Domain, a web resource, to identify SNPs within human protein domains. We annotated SNPs from dbSNP with protein structure-based as well as sequence-based domains: (i) structure-based using SCOP and (ii) sequence-based using Pfam to avoid conflicts from two domain assignment methodologies. Users can investigate SNPs within protein domains with 2D and 3D maps. We expect this visual annotation of SNPs within protein domains will help scientists select and interpret SNPs associated with diseases. A web interface for the SNP@Domain is freely available at and from . PMID:16845090

  1. Pleiotropic roles of cold shock domain proteins in plants.

    PubMed

    Sasaki, Kentaro; Imai, Ryozo

    2011-01-01

    The cold shock domain (CSD) is a nucleic acid binding domain that is widely conserved from bacteria to higher plants and animals. In Escherichia coli, cold shock proteins (CSPs) are composed solely of a CSD and function as RNA chaperones that destabilize RNA secondary structures. Cellular RNAs tend to be folded into unfavorable structures under low temperature conditions, and RNA chaperones resolve these structures, recovering functionality of the RNAs. CSP functions are associated mainly with cold adaptation, but they are also involved in other biological processes under normal growth conditions. Eukaryotic CSD proteins contain auxiliary domains in addition to the CSD and regulate many biological processes such as development and stress tolerance. In plants, it has been demonstrated that CSD proteins play essential roles in acquiring freezing tolerance. In addition, it has been suggested that some plant CSD proteins regulate embryo development, flowering time, and fruit development. In this review, we summarize the pleiotropic biological functions of CSP proteins in plants and discuss possible mechanisms by which plant CSD proteins regulate the functions of RNA molecules.

  2. Structural basis for Smoothened regulation by its extracellular domains

    PubMed Central

    Miller, Paul S.; Hedger, George; Luchetti, Giovanni; Nachtergaele, Sigrid; Tully, Mark D.; Mydock-McGrane, Laurel; Covey, Douglas F.; Rambo, Robert P.; Sansom, Mark S. P.; Rohatgi, Rajat; Siebold, Christian

    2016-01-01

    Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class G-protein coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How such large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened (SMO), which contains two distinct ligand-binding sites in its TMD and CRD. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain (LD). Structure-guided mutations show that the interface between the CRD, LD and TMD stabilises the inactive state of SMO. Unexpectedly, we find a cholesterol molecule bound to SMO in the CRD-binding site. Mutations predicted to prevent cholesterol binding impair the ability of SMO to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-LD-TMD interface. Our work elucidates the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains. PMID:27437577

  3. BRCT-domain protein BRIT1 influences class switch recombination.

    PubMed

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

    2017-07-19

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

  4. Methods of use of cellulose binding domain proteins

    SciTech Connect

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1997-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  5. Methods of use of cellulose binding domain proteins

    SciTech Connect

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1997-09-23

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  6. Methods of use of cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1997-09-23

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  7. Inferring Selection on Amino Acid Preference in Protein Domains

    PubMed Central

    Durbin, Richard

    2009-01-01

    Models that explicitly account for the effect of selection on new mutations have been proposed to account for “codon bias” or the excess of “preferred” codons that results from selection for translational efficiency and/or accuracy. In principle, such models can be applied to any mutation that results in a preferred allele, but in most cases, the fitness effect of a specific mutation cannot be predicted. Here we show that it is possible to assign preferred and unpreferred states to amino acid changing mutations that occur in protein domains. We propose that mutations that lead to more common amino acids (at a given position in a domain) can be considered “preferred alleles” just as are synonymous mutations leading to codons for more abundant tRNAs. We use genome-scale polymorphism data to show that alleles for preferred amino acids in protein domains occur at higher frequencies in the population, as has been shown for preferred codons. We show that this effect is quantitative, such that there is a correlation between the shift in frequency of preferred alleles and the predicted fitness effect. As expected, we also observe a reduction in the numbers of polymorphisms and substitutions at more important positions in domains, consistent with stronger selection at those positions. We examine the derived allele frequency distribution and polymorphism to divergence ratios of preferred and unpreferred differences and find evidence for both negative and positive selections acting to maintain protein domains in the human population. Finally, we analyze a model for selection on amino acid preferences in protein domains and find that it is consistent with the quantitative effects that we observe. PMID:19095755

  8. Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly.

    PubMed

    Sawatsky, Bevan; Bente, Dennis A; Czub, Markus; von Messling, Veronika

    2016-05-01

    The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle.

  9. The modular organization of domain structures: insights into protein-protein binding.

    PubMed

    del Sol, Antonio; Carbonell, Pablo

    2007-12-01

    Domains are the building blocks of proteins and play a crucial role in protein-protein interactions. Here, we propose a new approach for the analysis and prediction of domain-domain interfaces. Our method, which relies on the representation of domains as residue-interacting networks, finds an optimal decomposition of domain structures into modules. The resulting modules comprise highly cooperative residues, which exhibit few connections with other modules. We found that non-overlapping binding sites in a domain, involved in different domain-domain interactions, are generally contained in different modules. This observation indicates that our modular decomposition is able to separate protein domains into regions with specialized functions. Our results show that modules with high modularity values identify binding site regions, demonstrating the predictive character of modularity. Furthermore, the combination of modularity with other characteristics, such as sequence conservation or surface patches, was found to improve our predictions. In an attempt to give a physical interpretation to the modular architecture of domains, we analyzed in detail six examples of protein domains with available experimental binding data. The modular configuration of the TEM1-beta-lactamase binding site illustrates the energetic independence of hotspots located in different modules and the cooperativity of those sited within the same modules. The energetic and structural cooperativity between intramodular residues is also clearly shown in the example of the chymotrypsin inhibitor, where non-binding site residues have a synergistic effect on binding. Interestingly, the binding site of the T cell receptor beta chain variable domain 2.1 is contained in one module, which includes structurally distant hot regions displaying positive cooperativity. These findings support the idea that modules possess certain functional and energetic independence. A modular organization of binding sites confers

  10. A conserved region in the EBL proteins is implicated in microneme targeting of the malaria parasite Plasmodium falciparum.

    PubMed

    Treeck, Moritz; Struck, Nicole S; Haase, Silvia; Langer, Christine; Herrmann, Susann; Healer, Julie; Cowman, Alan F; Gilberger, Tim W

    2006-10-20

    The proliferation of the malaria parasite Plasmodium falciparum within the human host is dependent upon invasion of erythrocytes. This process is accomplished by the merozoite, a highly specialized form of the parasite. Secretory organelles including micronemes and rhoptries play a pivotal role in the invasion process by storing and releasing parasite proteins. The mechanism of protein sorting to these compartments is unclear. Using a transgenic approach we show that trafficking of the most abundant micronemal proteins (members of the EBL-family: EBA-175, EBA-140/BAEBL, and EBA-181/JSEBL) is independent of their cytoplasmic and transmembrane domains, respectively. To identify the minimal sequence requirements for microneme trafficking, we generated parasites expressing EBA-GFP chimeric proteins and analyzed their distribution within the infected erythrocyte. This revealed that: (i) a conserved cysteine-rich region in the ectodomain is necessary for protein trafficking to the micronemes and (ii) correct sorting is dependent on accurate timing of expression.

  11. Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins

    PubMed Central

    Xu, Zhixiong; Meng, Xianzhang; Cai, Ying; Liang, Hong; Nagarajan, Lalitha; Brandt, Stephen J.

    2007-01-01

    The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and β-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins. PMID:17437998

  12. Inter-Domain Dynamics in a Two-Domain Protein Studied by NMR

    NASA Astrophysics Data System (ADS)

    Ryabov, Yaroslav; Fushman, David

    2006-03-01

    Domain orientation and dynamics often play an important role in regulation of multidomain proteins function. Here we consider a two-domain system, Lys48-linked di-ubiquitin (Ub2), which is the simplest model of the polyubiquitin chain involved in the ubiquitin-proteasome pathway. Under physiological conditions Ub2 adopts a compact conformation, in which the functionally important hydrophobic residues are sequestered at the interface between the two Ub2 domains. Here we present a dynamic model that combines the anisotropic overall rotational diffusion with intra- and interdomain dynamics. This model describes the interdomain motion as a transition between two distinct conformational states. The model is applied to experimental ^15N relaxation data for Lys48-linked Ub2 acquired at neutral (pH 6.8) and acidic (pH 4.5) conditions. The model provides complete picture of Ub2 domain mobility including domain orientations, time scales of domain motions, and occupation probabilities for both states of Ub2. The obtained results are consistent with independent data on chemical shift perturbation mapping and spin labeling.

  13. TOPDOM: database of conservatively located domains and motifs in proteins.

    PubMed

    Varga, Julia; Dobson, László; Tusnády, Gábor E

    2016-09-01

    The TOPDOM database-originally created as a collection of domains and motifs located consistently on the same side of the membranes in α-helical transmembrane proteins-has been updated and extended by taking into consideration consistently localized domains and motifs in globular proteins, too. By taking advantage of the recently developed CCTOP algorithm to determine the type of a protein and predict topology in case of transmembrane proteins, and by applying a thorough search for domains and motifs as well as utilizing the most up-to-date version of all source databases, we managed to reach a 6-fold increase in the size of the whole database and a 2-fold increase in the number of transmembrane proteins. TOPDOM database is available at http://topdom.enzim.hu The webpage utilizes the common Apache, PHP5 and MySQL software to provide the user interface for accessing and searching the database. The database itself is generated on a high performance computer. tusnady.gabor@ttk.mta.hu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

  14. Targeting proteins to liquid-ordered domains in lipid membranes.

    PubMed

    Stachowiak, Jeanne C; Hayden, Carl C; Sanchez, Mari Angelica A; Wang, Julia; Bunker, Bruce C; Voigt, James A; Sasaki, Darryl Y

    2011-02-15

    We demonstrate the construction of novel protein-lipid assemblies through the design of a lipid-like molecule, DPIDA, endowed with tail-driven affinity for specific lipid membrane phases and head-driven affinity for specific proteins. In studies performed on giant unilamellar vesicles (GUVs) with varying mole fractions of dipalymitoylphosphatidylcholine (DPPC), cholesterol, and diphytanoylphosphatidyl choline (DPhPC), DPIDA selectively partitioned into the more ordered phases, either solid or liquid-ordered (L(o)) depending on membrane composition. Fluorescence imaging established the phase behavior of the resulting quaternary lipid system. Fluorescence correlation spectroscopy confirmed the fluidity of the L(o) phase containing DPIDA. In the presence of CuCl(2), the iminodiacetic acid (IDA) headgroup of DPIDA forms the Cu(II)-IDA complex that exhibits a high affinity for histidine residues. His-tagged proteins were bound specifically to domains enriched in DPIDA, demonstrating the capacity to target protein binding selectively to both solid and L(o) phases. Steric pressure from the crowding of surface-bound proteins transformed the domains into tubules with persistence lengths that depended on the phase state of the lipid domains.

  15. 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.

  16. Definition of structural elements in Plasmodium vivax and P. knowlesi Duffy-binding domains necessary for erythrocyte invasion.

    PubMed

    Singh, Saurabh K; Singh, Agam P; Pandey, Sunita; Yazdani, Syed S; Chitnis, Chetan E; Sharma, Amit

    2003-08-15

    Plasmodium vivax and P. knowlesi use the Duffy antigen as a receptor to invade human erythrocytes. Duffy-binding ligands belong to a family of erythrocyte-binding proteins that bind erythrocyte receptors to mediate invasion. Receptor-binding domains in erythrocyte-binding proteins lie in conserved cysteine-rich regions called Duffy-binding-like domains. In the present study, we report an analysis of the overall three-dimensional architecture of P. vivax and P. knowlesi Duffy-binding domains based on mild proteolysis and supportive-functional assays. Our proteolysis experiments indicate that these domains are built of two distinct subdomains. The N-terminal region from Cys-1-4 (C1-C4) forms a stable non-functional subdomain. The region spanning C5-C12 forms another subdomain, which is capable of binding Duffy antigen. These subdomains are joined by a protease-sensitive linker. Results from deletion constructs, designed for expression of truncated proteins on COS cell surface, show that regions containing C5-C8 of the Duffy-binding domains are sufficient for the binding receptor. Therefore the central region of Duffy-binding domains, which is flanked by two non-functional regions, is responsible for receptor recognition. Moreover, the minimal Duffy-binding region identified here is capable of folding into a functionally competent module. These studies pave the way for understanding the architecture of Duffy-binding domains and their interactions with host receptors.

  17. A Bayesian Sampler for Optimization of Protein Domain Hierarchies

    PubMed Central

    2014-01-01

    Abstract The process of identifying and modeling functionally divergent subgroups for a specific protein domain class and arranging these subgroups hierarchically has, thus far, largely been done via manual curation. How to accomplish this automatically and optimally is an unsolved statistical and algorithmic problem that is addressed here via Markov chain Monte Carlo sampling. Taking as input a (typically very large) multiple-sequence alignment, the sampler creates and optimizes a hierarchy by adding and deleting leaf nodes, by moving nodes and subtrees up and down the hierarchy, by inserting or deleting internal nodes, and by redefining the sequences and conserved patterns associated with each node. All such operations are based on a probability distribution that models the conserved and divergent patterns defining each subgroup. When we view these patterns as sequence determinants of protein function, each node or subtree in such a hierarchy corresponds to a subgroup of sequences with similar biological properties. The sampler can be applied either de novo or to an existing hierarchy. When applied to 60 protein domains from multiple starting points in this way, it converged on similar solutions with nearly identical log-likelihood ratio scores, suggesting that it typically finds the optimal peak in the posterior probability distribution. Similarities and differences between independently generated, nearly optimal hierarchies for a given domain help distinguish robust from statistically uncertain features. Thus, a future application of the sampler is to provide confidence measures for various features of a domain hierarchy. PMID:24494927

  18. Membrane shape instabilities induced by BAR domain proteins

    NASA Astrophysics Data System (ADS)

    Baumgart, Tobias

    2014-03-01

    Membrane curvature has developed into a forefront of membrane biophysics. Numerous proteins involved in membrane curvature sensing and membrane curvature generation have recently been discovered, including proteins containing the crescent-shaped BAR domain as membrane binding and shaping module. Accordingly, the structure determination of these proteins and their multimeric complexes is increasingly well-understood. Substantially less understood, however, are thermodynamic and kinetic aspects and the detailed mechanisms of how these proteins interact with membranes in a curvature-dependent manner. New experimental approaches need to be combined with established techniques to be able to fill in these missing details. Here we use model membrane systems in combination with a variety of biophysical techniques to characterize mechanistic aspects of BAR domain protein function. This includes a characterization of membrane curvature sensing and membrane generation. We also establish kinetic and thermodynamic aspects of BAR protein dimerization in solution, and investigate kinetic aspects of membrane binding. We present two new approaches to investigate membrane shape instabilities and demonstrate that membrane shape instabilities can be controlled by protein binding and lateral membrane tension. This work is supported through NIH grant GM-097552 and NSF grant CBET-1053857.

  19. EVEREST: automatic identification and classification of protein domains in all protein sequences

    PubMed Central

    Portugaly, Elon; Harel, Amir; Linial, Nathan; Linial, Michal

    2006-01-01

    Background Proteins are comprised of one or several building blocks, known as domains. Such domains can be classified into families according to their evolutionary origin. Whereas sequencing technologies have advanced immensely in recent years, there are no matching computational methodologies for large-scale determination of protein domains and their boundaries. We provide and rigorously evaluate a novel set of domain families that is automatically generated from sequence data. Our domain family identification process, called EVEREST (EVolutionary Ensembles of REcurrent SegmenTs), begins by constructing a library of protein segments that emerge in an all vs. all pairwise sequence comparison. It then proceeds to cluster these segments into putative domain families. The selection of the best putative families is done using machine learning techniques. A statistical model is then created for each of the chosen families. This procedure is then iterated: the aforementioned statistical models are used to scan all protein sequences, to recreate a library of segments and to cluster them again. Results Processing the Swiss-Prot section of the UniProt Knoledgebase, release 7.2, EVEREST defines 20,230 domains, covering 85% of the amino acids of the Swiss-Prot database. EVEREST annotates 11,852 proteins (6% of the database) that are not annotated by Pfam A. In addition, in 43,086 proteins (20% of the database), EVEREST annotates a part of the protein that is not annotated by Pfam A. Performance tests show that EVEREST recovers 56% of Pfam A families and 63% of SCOP families with high accuracy, and suggests previously unknown domain families with at least 51% fidelity. EVEREST domains are often a combination of domains as defined by Pfam or SCOP and are frequently sub-domains of such domains. Conclusion The EVEREST process and its output domain families provide an exhaustive and validated view of the protein domain world that is automatically generated from sequence data. The

  20. EVEREST: automatic identification and classification of protein domains in all protein sequences.

    PubMed

    Portugaly, Elon; Harel, Amir; Linial, Nathan; Linial, Michal

    2006-06-02

    Proteins are comprised of one or several building blocks, known as domains. Such domains can be classified into families according to their evolutionary origin. Whereas sequencing technologies have advanced immensely in recent years, there are no matching computational methodologies for large-scale determination of protein domains and their boundaries. We provide and rigorously evaluate a novel set of domain families that is automatically generated from sequence data. Our domain family identification process, called EVEREST (EVolutionary Ensembles of REcurrent SegmenTs), begins by constructing a library of protein segments that emerge in an all vs. all pairwise sequence comparison. It then proceeds to cluster these segments into putative domain families. The selection of the best putative families is done using machine learning techniques. A statistical model is then created for each of the chosen families. This procedure is then iterated: the aforementioned statistical models are used to scan all protein sequences, to recreate a library of segments and to cluster them again. Processing the Swiss-Prot section of the UniProt Knoledgebase, release 7.2, EVEREST defines 20,230 domains, covering 85% of the amino acids of the Swiss-Prot database. EVEREST annotates 11,852 proteins (6% of the database) that are not annotated by Pfam A. In addition, in 43,086 proteins (20% of the database), EVEREST annotates a part of the protein that is not annotated by Pfam A. Performance tests show that EVEREST recovers 56% of Pfam A families and 63% of SCOP families with high accuracy, and suggests previously unknown domain families with at least 51% fidelity. EVEREST domains are often a combination of domains as defined by Pfam or SCOP and are frequently sub-domains of such domains. The EVEREST process and its output domain families provide an exhaustive and validated view of the protein domain world that is automatically generated from sequence data. The EVEREST library of domain

  1. BC-box protein domain-related mechanism for VHL protein degradation

    PubMed Central

    Pozzebon, Maria Elena; Varadaraj, Archana; Mattoscio, Domenico; Jaffray, Ellis G.; Miccolo, Claudia; Galimberti, Viviana; Tommasino, Massimo; Hay, Ronald T.; Chiocca, Susanna

    2013-01-01

    The tumor suppressor VHL (von Hippel–Lindau) protein is a substrate receptor for Ubiquitin Cullin Ring Ligase complexes (CRLs), containing a BC-box domain that associates to the adaptor Elongin B/C. VHL targets hypoxia-inducible factor 1α to proteasome-dependent degradation. Gam1 is an adenoviral protein, which also possesses a BC-box domain that interacts with the host Elongin B/C, thereby acting as a viral substrate receptor. Gam1 associates with both Cullin2 and Cullin5 to form CRL complexes targeting the host protein SUMO enzyme SAE1 for proteasomal degradation. We show that Gam1 protein expression induces VHL protein degradation leading to hypoxia-inducible factor 1α stabilization and induction of its downstream targets. We also characterize the CRL-dependent mechanism that drives VHL protein degradation via proteasome. Interestingly, expression of Suppressor of Cytokine Signaling (SOCS) domain-containing viral proteins and cellular BC-box proteins leads to VHL protein degradation, in a SOCS domain-containing manner. Our work underscores the exquisite ability of viral domains to uncover new regulatory mechanisms by hijacking key cellular proteins. PMID:24145437

  2. Domain formation in membranes caused by lipid wetting of protein.

    PubMed

    Akimov, Sergey A; Frolov, Vladimir A J; Kuzmin, Peter I; Zimmerberg, Joshua; Chizmadzhev, Yuri A; Cohen, Fredric S

    2008-05-01

    Formation of rafts and other domains in cell membranes is considered as wetting of proteins by lipids. The membrane is modeled as a continuous elastic medium. Thermodynamic functions of the lipid films that wet proteins are calculated using a mean-field theory of liquid crystals as adapted to biomembranes. This approach yields the conditions necessary for a macroscopic wetting film to form; its thickness could also be determined. It is shown that films of macroscopic thicknesses form around large (tens nanometers in diameter) lipid-protein aggregates; only thin adsorption films form around single proteins or small complexes. The means by which wetting films can facilitate the merger of these aggregates is considered. It is shown that a wetting film prevents a protein from leaving an aggregate. Using experimentally derived values of elastic moduli and spontaneous curvatures as well as height mismatch between aggregates and bulk membrane, we obtained numerical results, which can be compared with the experimental data.

  3. Prediction of Cancer Proteins by Integrating Protein Interaction, Domain Frequency, and Domain Interaction Data Using Machine Learning Algorithms

    PubMed Central

    2015-01-01

    Many proteins are known to be associated with cancer diseases. It is quite often that their precise functional role in disease pathogenesis remains unclear. A strategy to gain a better understanding of the function of these proteins is to make use of a combination of different aspects of proteomics data types. In this study, we extended Aragues's method by employing the protein-protein interaction (PPI) data, domain-domain interaction (DDI) data, weighted domain frequency score (DFS), and cancer linker degree (CLD) data to predict cancer proteins. Performances were benchmarked based on three kinds of experiments as follows: (I) using individual algorithm, (II) combining algorithms, and (III) combining the same classification types of algorithms. When compared with Aragues's method, our proposed methods, that is, machine learning algorithm and voting with the majority, are significantly superior in all seven performance measures. We demonstrated the accuracy of the proposed method on two independent datasets. The best algorithm can achieve a hit ratio of 89.4% and 72.8% for lung cancer dataset and lung cancer microarray study, respectively. It is anticipated that the current research could help understand disease mechanisms and diagnosis. PMID:25866773

  4. Domain Hierarchy and closed Loops (DHcL): a server for exploring hierarchy of protein domain structure

    PubMed Central

    Koczyk, Grzegorz; Berezovsky, Igor N.

    2008-01-01

    Domain hierarchy and closed loops (DHcL) (http://sitron.bccs.uib.no/dhcl/) is a web server that delineates energy hierarchy of protein domain structure and detects domains at different levels of this hierarchy. The server also identifies closed loops and van der Waals locks, which constitute a structural basis for the protein domain hierarchy. The DHcL can be a useful tool for an express analysis of protein structures and their alternative domain decompositions. The user submits a PDB identifier(s) or uploads a 3D protein structure in a PDB format. The results of the analysis are the location of domains at different levels of hierarchy, closed loops, van der Waals locks and their interactive visualization. The server maintains a regularly updated database of domains, closed loop and van der Waals locks for all X-ray structures in PDB. DHcL server is available at: http://sitron.bccs.uib.no/dhcl. PMID:18502776

  5. ADDA: a domain database with global coverage of the protein universe.

    PubMed

    Heger, Andreas; Wilton, Christopher Andrew; Sivakumar, Ashwin; Holm, Liisa

    2005-01-01

    We used the Automatic Domain Decomposition Algorithm (ADDA) to generate a database of protein domain families with complete coverage of all protein sequences. Sequences are split into domains and domains are grouped into protein domain families in a completely automated process. The current database contains domains for more than 1.5 million sequences in more than 40,000 domain families. In particular, there are 3828 novel domain families that do not overlap with the curated domain databases Pfam, SCOP and InterPro. The data are freely available for downloading and querying via a web interface (http://ekhidna.biocenter.helsinki.fi:9801/sqgraph/pairsdb).

  6. Evolutionary history and genome organization of DUF1220 protein domains.

    PubMed

    O'Bleness, Majesta S; Dickens, C Michael; Dumas, Laura J; Kehrer-Sawatzki, Hildegard; Wyckoff, Gerald J; Sikela, James M

    2012-09-01

    DUF1220 protein domains exhibit the most extreme human lineage-specific (HLS) copy number increase of any protein coding region in the human genome and have recently been linked to evolutionary and pathological changes in brain size (e.g., 1q21-associated microcephaly). These findings lend support to the view that DUF1220 domain dosage is a key factor in the determination of primate (and human) brain size. Here we analyze 41 animal genomes and present the most complete account to date of the evolutionary history and genome organization of DUF1220 domains and the gene family that encodes them (NBPF). Included among the novel features identified by this analysis is a DUF1220 domain precursor in nonmammalian vertebrates, a unique predicted promoter common to all mammalian NBPF genes, six distinct clades into which DUF1220 sequences can be subdivided, and a previously unknown member of the NBPF gene family (NBPF25). Most importantly, we show that the exceptional HLS increase in DUF1220 copy number (from 102 in our last common ancestor with chimp to 272 in human; an average HLS increase of ~28 copies every million years since the Homo/Pan split) was driven by intragenic domain hyperamplification. This increase primarily involved a 4.7 kb, tandemly repeated three DUF1220 domain unit we have named the HLS DUF1220 triplet, a motif that is a likely candidate to underlie key properties unique to the Homo sapiens brain. Interestingly, all copies of the HLS DUF1220 triplet lie within a human-specific pericentric inversion that also includes the 1q12 C-band, a polymorphic heterochromatin expansion that is unique to the human genome. Both cytogenetic features likely played key roles in the rapid HLS DUF1220 triplet hyperamplification, which is among the most striking genomic changes specific to the human lineage.

  7. Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides

    PubMed Central

    Safavi-Hemami, Helena; Li, Qing; Jackson, Ronneshia L.; Song, Albert S.; Boomsma, Wouter; Bandyopadhyay, Pradip K.; Gruber, Christian W.; Purcell, Anthony W.; Yandell, Mark; Olivera, Baldomero M.

    2016-01-01

    Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed conotoxin-specific PDIs, significantly and differentially accelerate the kinetics of disulfide-bond formation of several conotoxins. Our results are consistent with a unique biological scenario associated with protein folding: The diversification of a family of foldases can be correlated with the rapid evolution of an unprecedented diversity of disulfide-rich structural domains expressed by venomous marine snails in the superfamily Conoidea. PMID:26957604

  8. Control of domain swapping in bovine odorant-binding protein.

    PubMed Central

    Ramoni, Roberto; Vincent, Florence; Ashcroft, Alison E; Accornero, Paolo; Grolli, Stefano; Valencia, Christel; Tegoni, Mariella; Cambillau, Christian

    2002-01-01

    As revealed by the X-ray structure, bovine odorant-binding protein (OBPb) is a domain swapped dimer [Tegoni, Ramoni, Bignetti, Spinelli and Cambillau (1996) Nat. Struct. Biol. 3, 863-867; Bianchet, Bains, Petosi, Pevsner, Snyder, Monaco and Amzel (1996) Nat. Struct. Biol. 3, 934-939]. This contrasts with all known mammalian OBPs, which are monomers, and in particular with porcine OBP (OBPp), sharing 42.3% identity with OBPb. By the mechanism of domain swapping, monomers are proposed to evolve into dimers and oligomers, as observed in human prion. Comparison of bovine and porcine OBP sequences pointed at OBPp glycine 121, in the hinge linking the beta-barrel to the alpha-helix. The absence of this residue in OBPb might explain why the normal lipocalin beta-turn is not formed. In order to decipher the domain swapping determinants we have produced a mutant of OBPb in which a glycine residue was inserted after position 121, and a mutant of OBPp in which glycine 121 was deleted. The latter mutation did not result in dimerization, while OBPb-121Gly+ became monomeric, suggesting that domain swapping was reversed. Careful structural analysis revealed that besides the presence of a glycine in the hinge, the dimer interface formed by the C-termini and by the presence of the lipocalins conserved disulphide bridge may also control domain swapping. PMID:11931632

  9. The functional domain grouping of microtubule associated proteins

    PubMed Central

    Deane, Charlotte M; Wakefield, James G

    2008-01-01

    Microtubules (MTs), which play crucial roles in normal cell function, are regulated by MT associated proteins (MAPs). Using a combinatorial approach that includes biochemistry, proteomics and bioinformatics, we have recently identified 270 putative MAPs from Drosophila embryos and characterized some of those required for correct progression through mitosis. Here we identify functional groups of these MAPs using a reciprocal hits sequence alignment technique and assign InterPro functional domains to 28 previously uncharacterized proteins. This approach gives insight into the potential functions of MAPs and how their roles may affect MTs. PMID:19704789

  10. Roles for the coat protein telokin-like domain and the scaffolding protein amino-terminus

    PubMed Central

    Suhanovsky, Margaret M.; Teschke, Carolyn M.

    2011-01-01

    Assembly of icosahedral capsids of proper size and symmetry is not understood. Residue F170 in bacteriophage P22 coat protein is critical for conformational switching during assembly. Substitutions at this site cause assembly of tubes of hexamerically arranged coat protein. Intragenic suppressors of the ts phenotype of F170A and F170K coat protein mutants were isolated. Suppressors were repeatedly found in the coat protein telokin-like domain at position 285, which caused coat protein to assemble into petite procapsids and capsids. Petite capsid assembly strongly correlated to the side chain volume of the substituted amino acid. We hypothesize that larger side chains at position 285 torque the telokin-like domain, changing flexibility of the subunit and intercapsomer contacts. Thus, a single amino acid substitution in coat protein is sufficient to change capsid size. In addition, the products of assembly of the variant coat proteins were affected by the size of the internal scaffolding protein. PMID:21784500

  11. Single-domain protein folding: a multi-faceted problem

    NASA Astrophysics Data System (ADS)

    Junier, Ivan; Ritort, Felix

    2006-08-01

    We review theoretical approaches, experiments and numerical simulations that have been recently proposed to investigate the folding problem in single-domain proteins. From a theoretical point of view, we emphasize the energy landscape approach. As far as experiments are concerned, we focus on the recent development of single-molecule techniques. In particular, we compare the results obtained with two main techniques: single protein force measurements with optical tweezers and single-molecule fluorescence in studies on the same protein (RNase H). This allows us to point out some controversial issues such as the nature of the denatured and intermediate states and possible folding pathways. After reviewing the various numerical simulation techniques, we show that on-lattice protein-like models can help to understand many controversial issues.

  12. The KP4 killer protein gene family

    USDA-ARS?s Scientific Manuscript database

    Killer protein 4 (KP4) is a well studied toxin secreted by the maize smut fungus Ustilago maydis that kills sensitive Ustilago strains as well as inhibits Fusarium and plant root growth. This small, cysteine rich protein is encoded by a virus that depends on host survival for replication. KP4 functi...

  13. Identification and phylogenetic analyses of VASt, an uncharacterized protein domain associated with lipid-binding domains in Eukaryotes.

    PubMed

    Khafif, Mehdi; Cottret, Ludovic; Balagué, Claudine; Raffaele, Sylvain

    2014-06-26

    Several regulators of programmed cell death (PCD) in plants encode proteins with putative lipid-binding domains. Among them, VAD1 is a regulator of PCD propagation harboring a GRAM putative lipid-binding domain. However the function of VAD1 at the subcellular level is unknown and the domain architecture of VAD1 has not been analyzed in details. We analyzed sequence conservation across the plant kingdom in the VAD1 protein and identified an uncharacterized VASt (VAD1 Analog of StAR-related lipid transfer) domain. Using profile hidden Markov models (profile HMMs) and phylogenetic analysis we found that this domain is conserved among eukaryotes and generally associates with various lipid-binding domains. Proteins containing both a GRAM and a VASt domain include notably the yeast Ysp2 cell death regulator and numerous uncharacterized proteins. Using structure-based phylogeny, we found that the VASt domain is structurally related to Bet v1-like domains. We identified a novel protein domain ubiquitous in Eukaryotic genomes and belonging to the Bet v1-like superfamily. Our findings open perspectives for the functional analysis of VASt-containing proteins and the characterization of novel mechanisms regulating PCD.

  14. Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins.

    PubMed

    Martin-Serrano, Juan; Yarovoy, Anton; Perez-Caballero, David; Bieniasz, Paul D; Yaravoy, Anton

    2003-10-14

    The release of enveloped viruses from infected cells often requires a virally encoded activity, termed a late-budding domain (L domain), encoded by essential PTAP, PPXY, or YPDL sequence motifs. PTAP-type L domains recruit one of three endosomal sorting complexes required for transport (ESCRT-I). However, subsequent events in viral budding are poorly defined, and neither YPDL nor PPXY-type L domains require ESCRT-I. Here, we show that ESCRT-I and other class E vacuolar protein sorting (VPS) factors are linked by a complex series of protein-protein interactions. In particular, interactions between ESCRT-I and ESCRT-III are bridged by AIP-1/ALIX, a mammalian orthologue of the yeast class E VPS factor, Bro1. Expression of certain ESCRT-III components as fusion proteins induces a late budding defect that afflicts all three L-domain types, suggesting that ESCRT-III integrity is required in a general manner. Notably, the prototype YPDL-type L domain encoded by equine infectious anemia virus (EIAV) acts by recruiting AIP-1/ALIX and expression of a truncated form of AIP-1/ALIX or small interfering RNA-induced AIP-1/ALIX depletion specifically inhibits EIAV YPDL-type L-domain function. Overall, these findings indicate that L domains subvert a subset of class E VPS factors to mediate viral budding, some of which are required for each of the L-domain types, whereas others apparently act as adaptors to physically link specific L-domain types to the class E VPS machinery.

  15. Proteins on the catwalk: modelling the structural domains of the CCN family of proteins.

    PubMed

    Holbourn, Kenneth P; Perbal, Bernard; Ravi Acharya, K

    2009-03-01

    The CCN family of proteins (CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6) are multifunctional mosaic proteins that play keys roles in crucial areas of physiology such as angiogenesis, skeletal development tumourigenesis, cell proliferation, adhesion and survival. This expansive repertoire of functions comes through a modular structure of 4 discrete domains that act both independently and in concert. How these interactions with ligands and with neighbouring domains lead to the biological effects is still to be explored but the molecular structure of the domains is likely to play an important role in this. In this review we have highlighted some of the key features of the individual domains of CCN family of proteins based on their biological effects using a homology modelling approach.

  16. PDZ domain protein GIPC interacts with the cytoplasmic tail of melanosomal membrane protein gp75 (tyrosinase-related protein-1).

    PubMed

    Liu, T F; Kandala, G; Setaluri, V

    2001-09-21

    Tyrosinase and tyrosinase-related proteins (TRPs) are a family of melanosomal membrane proteins involved in mammalian pigmentation. Whereas the melanogenic functions of TRPs are localized in their amino-terminal domains that reside within the lumen of melanosomes, the sorting and targeting of these proteins to melanosomes is mediated by signals in their cytoplasmic domains. To identify proteins that interact with the cytoplasmic tail of gp75 (TRP-1), the most abundant melanosomal membrane protein, we performed yeast two-hybrid screening of a melanocyte cDNA library. Here, we show that the cytoplasmic domain of gp75 interacts with a PDZ domain-containing protein. The gp75-interacting protein is identical to GIPC, an RGS (regulator of G protein signaling)/GAIP-interacting protein, and to SEMCAP-1, a transmembrane semaphorin-binding protein. Carboxyl-terminal amino acid residues, Ser-Val-Val, of gp75 are necessary and sufficient for interaction of gp75 with the single PDZ domain in GIPC. Although endogenous and transfected GIPCs bind efficiently to transiently expressed gp75, only a small amount of GIPC is found associated with gp75 at steady state. Using a strategy to selectively synchronize the biosynthesis of endogenous gp75, we demonstrate that only newly synthesized gp75 associates with GIPC, primarily in the juxtanuclear Golgi region. Our data suggest that GIPC/SEMCAP-1 plays a role in biosynthetic sorting of proteins, specifically gp75, to melanosomes.

  17. The β1 domain of protein G can replace the chorismate mutase domain of the T-protein.

    PubMed

    Osuna, Joel; Flores, Humberto; Saab-Rincón, Gloria

    2012-02-17

    T-protein is composed of chorismate mutase (AroQ(T)) fused to the N-terminus of prephenate dehydrogenase (TyrA). Here, we report the replacement of AroQ(T) with the β1-domain of protein G (Gβ1). The TyrA domain shows a strong dehydrogenase activity within the context of this fusion, and our data indicate that Gβ1-TyrA folds into a dimeric conformation. Amino acid substitutions in the Gβ1 domain of Gβ1-TyrA identified residues involved in stabilizing the TyrA dimeric conformation. Gβ1 substitutions in the N-terminal β-hairpin eliminated Gβ1-TyrA expression, whereas Gβ1-TyrA tolerated Gβ1 substitutions in the C-terminal β-hairpin and in the α-helix. All of the characterized variants folded into a dimeric conformation. The importance of the