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Leucine-rich Repeat (leucine-rich + repeat)
Terms modified by Leucine-rich Repeat Selected AbstractsLeucine-rich repeat proteins of synapsesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 13 2007Jaewon Ko Abstract Leucine-rich repeats (LRRs) are 20,29-aa motifs that mediate protein,protein interactions and are present in a variety of membrane and cytoplasmic proteins. Many LRR proteins with neuronal functions have been reported. Here, we summarize an emerging group of synaptic LRR proteins, which includes densin-180, Erbin, NGL, SALM, and LGI1. These proteins have been implicated in the formation, differentiation, maintenance, and plasticity of neuronal synapses. © 2007 Wiley-Liss, Inc. [source] Neuronal leucine-rich repeat 6 (XlNLRR-6) is required for late lens and retina development in Xenopus laevisDEVELOPMENTAL DYNAMICS, Issue 4 2006Adam D. Wolfe Abstract Leucine-rich repeat proteins expressed in the developing vertebrate nervous system comprise a complex, multifamily group, and little is known of their developmental function in vivo. We have identified a novel member of this group in Xenopus laevis, XlNLRR-6, and through sequence and phylogenetic analysis, have placed it within a defined family of vertebrate neuronal leucine-rich repeat proteins (NLRR). XlNLRR-6 is expressed in the developing nervous system and tissues of the eye beginning at the neural plate stage, and expression continues throughout embryonic and larval development. Using antisense morpholino oligonucleotide (MO) -mediated knockdown of XlNLRR-6, we demonstrate that this protein is critical for development of the lens, retina, and cornea. Reciprocal transplantation of presumptive lens ectoderm between MO-treated and untreated embryos demonstrate that XlNLRR-6 plays autonomous roles in the development of both the lens and retina. These findings represent the first in vivo functional analysis of an NLRR family protein and establish a role for this protein during late differentiation of tissues in the developing eye. Developmental Dynamics 235:1027,1041, 2006. © 2006 Wiley-Liss, Inc. [source] LRRN6A/LERN1 (leucine-rich repeat neuronal protein 1), a novel gene with enriched expression in limbic system and neocortexEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2003Laura Carim-Todd Abstract Human chromosome 15q24-q26 is a very complex genomic region containing several blocks of segmental duplications to which susceptibility to anxiety disorders has been mapped (Gratacos et al., 2001, Cell, 106, 367,379; Pujana et al., 2001, Genome Res., 11, 98,111). Through an in silico gene content analysis of the 15q24-q26 region we have identifie1d a novel gene, LRRN6A (leucine-rich repeat neuronal 6A), and confirmed its location to the centromeric end of this complex region. LRRN6A encodes a transmembrane leucine-rich repeat protein, LERN1 (leucine-rich repeat neuronal protein 1), with similarity to proteins involved in axonal guidance and migration, nervous system development and regeneration processes. The identification of homologous genes to LRRN6A on chromosomes 9 and 19 and the orthologous genes in the mouse genome and other organisms suggests that LERN proteins constitute a novel subfamily of LRR (leucine-rich repeat)-containing proteins. The LRRN6A expression pattern is specific to the central nervous system, highly and broadly expressed during early stages of development and gradually restricted to forebrain structures as development proceeds. Expression level in adulthood is lower in general but remains stable and significantly enriched in the limbic system and cerebral cortex. Taken together, the confirmation of LRRN6A's expression profile, its predicted protein structure and its similarity to nervous system-expressed LRR proteins with essential roles in nervous system development and maintenance suggest that LRRN6A is a novel gene of relevance in the molecular and cellular neurobiology of vertebrates. [source] An interaction between opticin and heparan sulfate may provide the molecular basis for vitreoretinal adhesionINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2004V. John Hindson Introduction Opticin is a member of the extracellular matrix small leucine-rich repeat (SLRP) proteoglycan/protein family, which was originally identified in the eye associated with the collagen fibrils of the vitreous humour. A putative heparin/heparan sulfate (HS) binding motif (RKERKRR) was identified at the N-terminus of human opticin, but this is absent in the bovine form. Furthermore, the strength of attachment between the vitreous and the retina was observed to be species-dependent and related to the presence or absence of this motif. We hypothesized that opticin cross-links the collagen fibrils of the vitreous to HS proteoglycans in the inner limiting lamina (a basement membrane on the inner surface of the retina), contributing towards vitreoretinal adhesion. Materials and methods Recombinant human and bovine opticin were expressed in 293-EBNA cells and purified to apparent homogeneity. Solid phase assays and surface plasmon resonance studies were used to characterize interactions between immobilized heparin/HS and opticin. Results Solid phase and BIAcore data revealed that human opticin binds heparin/HS and binds to heparin with a dissociation constant of approximately 20 nm. By contrast bovine opticin, which lacks the basic cluster, bound severalfold less tightly. Competition studies with heparin oligosaccharides indicated that the heparin/HS binding site is greater than 6 monosaccharides in length. Heparin, HS, chondroitin sulfate A (CS-A), dermatan sulfate and hyaluronan all competed with heparin for binding to human opticin but CS-C did not. Discussion Work to date suggests that the N-terminal sequence RKERKRR contributes significantly to the binding of opticin to heparin/HS. Vitreoretinal adhesion plays a key role in a number of eye diseases and inhibitors of the opticin,HS interaction could be of therapeutic value. [source] Biglycan binds to ,- and ,-sarcoglycan and regulates their expression during development,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2006Michael S. Rafii The dystrophin-associated protein complex (DAPC), which links the cytoskeleton to the extracellular matrix, is essential for muscle cell survival, and is defective in a wide range of muscular dystrophies. The DAPC contains two transmembrane subcomplexes,the dystroglycans and the sarcoglycans. Although several extracellular binding partners have been identified for the dystroglycans, none have been described for the sarcoglycan subcomplex. Here we show that the small leucine-rich repeat (LRR) proteoglycan biglycan binds to ,- and ,-sarcoglycan as judged by ligand blot overlay and co-immunoprecipitation assays. Our studies with biglycan-decorin chimeras show that ,- and ,-sarcoglycan bind to distinct sites on the polypeptide core of biglycan. Both biglycan proteoglycan as well as biglycan polypeptide lacking glycosaminoglycan (GAG) side chains are components of the dystrophin glycoprotein complex isolated from adult skeletal muscle membranes. Finally, our immunohistochemical and biochemical studies with biglycan null mice show that the expression of ,- and ,-sarcoglycan is selectively reduced in muscle from young (P14-P21) animals, while levels in adult muscle (,P35) are unchanged. We conclude that biglycan is a ligand for two members of the sarcoglycan complex and regulates their expression at discrete developmental ages. J. Cell. Physiol. 209: 439,447, 2006. © 2006 Wiley-Liss, Inc. [source] Research Article: The Cysteine Pairs in CLV2 are Not Necessary for Sensing the CLV3 Peptide in Shoot and Root MeristemsJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 9 2010Xiufen Song Receptor-like proteins (RLPs) are involved in both plant defense and developmental processes. Previous genetic and biochemical studies show that the leucine-rich repeat (LRR) receptor-like protein CLAVATA2 (CLV2) functions together with CLAVATA1 (CLV1) and CORYNE (CRN) in Arabidopsis to limit the stem cell number in shoot apical meristem, while in root it acts with CRN to trigger a premature differentiation of the stem cells after sensing the exogenously applied peptides of CLV3p, CLE19p or CLE40p. It has been proposed that disulfide bonds might be formed through two cysteine pairs in the extracellular LRR domains of CLV1 and CLV2 to stabilize the receptor complex. Here we tested the hypothesis by replacing these cysteines with alanines and showed that depletions of one or both of the cysteine pairs do not hamper the function of CLV2 in SAM maintenance. In vitro peptide assay also showed that removal of the cysteine pairs did not affect the perception of CLV3 peptides in roots. These observations allow us to conclude that the formation of disulfide bonds is not needed for the function of CLV2. [source] Cloning and Preliminary Characterization of Three Receptor-like Kinase Genes in SoybeanJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 11 2006Yuan-Yuan Ma Abstract Leaf senescence that occurs in the last stage of leaf development is a genetically programmed process. It is very significant to isolate the upstream components in the senescence signaling pathway and to elucidate the molecular mechanisms that control the initiation and progression of leaf senescence. In this study, full-length cDNAs of three receptor-like protein kinase genes, designated rlpk1, rlpk2 and rlpk3, were cloned from artificially-induced senescent soybean (Glycine max L.) primary leaves (GenBank accession AY687390, AY687391, AF338813). The deduced amino acid sequences indicated that they belonged to a receptor-like kinase family. Each of rlpk1 and rlpk2 encodes a leucine-rich repeat (LRR) receptor-like protein kinase. They both comprise a typical signal peptide, several LRR motifs, a single-pass transmem-brane domain, and a cytoplasmic protein kinase domain. No typical extracellular domain of RLPK3 was predicted. Organ-specific expression pattern analysis by reverse-transcription polymerase chain reaction (RT-PCR) revealed higher expression levels of the three genes in cotyledons, roots and flowers. Phylogenetic analysis indicated that RLPK1 and RLPK2 belonged to an independent branch, whereas RLPK3 shared common nodes with several known RLKs responding to abiotic and biotic stresses. The evident alternations of expression profiles of rlpk1 and rlpk2 induced by the artificial senescence-inducing treatment implied involvements of these two RLKs in regulating soybean leaf senescence. (Managing editor: Li-Hui Zhao) [source] Identification and molecular analysis of candidate genes homologous to HcrVf genes for scab resistance in applePLANT BREEDING, Issue 1 2009A. Boudichevskaia Abstract The genetic locus for resistance to apple scab most frequently used in apple breeding is Vf, derived from Malus floribunda 821. For the Vf locus a cluster of four resistance gene paralogs (called as HcrVf genes) encoding receptor-like proteins (RLP) with similarity to the tomato Cf resistance genes is known. Based on published sequences for HcrVf1 and HcrVf2 PCR primers were designed from the domain B and the variable leucine-rich repeat (LRR) C1 subdomain. PCR products with high amino acid identity (85,100%) to HcrVf1 and HcrVf2 were obtained not only from M. floribunda 821 and Vf cultivars but also from other apple scab resistance sources, such as ,Russian Seedling' R12740-7A (Vr resistance) or ,Antonovka polutorafuntovaya' (VA resistance). A series of 13 HcrVf candidate genes have been partly cloned from the PCR fragments spanning N-terminal LRRs 20,30. A considerable number of amino acid exchanges within the solvent-exposed xxLxLxx structural motives were detected among the homologous sequences. Expression analyses and mapping focused on a selected Vf- homologous candidate gene (called Vf2ARD) identified in resistant Malus genotypes known for carrying other scab resistance genes than Vf. RT-PCR experiments showed that Vf2ARD is expressed under pathogen-free conditions. The results of a quantitative PCR-based transcription profiling suggest that this gene is scab-inducible in some resistant cultivars. Vf2ARD has been mapped on linkage group LG 1. It is separated from the Vf gene cluster with a genetic distance of about 2 cM and might be a member of a second Vf - like locus on apple linkage group LG 1. [source] The hydroxyproline-rich glycoprotein domain of the Arabidopsis LRX1 requires Tyr for function but not for insolubilization in the cell wallTHE PLANT JOURNAL, Issue 4 2010Christoph Ringli Summary Extensins, hydroxyproline-rich repetitive glycoproteins with Ser,Hyp4 motifs, are structural proteins in plant cell walls. The leucine-rich repeat extensin 1 (LRX1) of Arabidopsis thaliana is an extracellular protein with both a leucine-rich repeat and an extensin domain, and has been demonstrated to be important for cell-wall formation in root hairs. lrx1 mutants develop defective cell walls, resulting in a strong root hair phenotype. The extensin domain is essential for protein function and is thought to confer insolubilization of LRX1 in the cell wall. Here, in vivo characterization of the LRX1 extensin domain is described. First, a series of LRX1 extensin deletion constructs was produced that led to identification of a much shorter, functional extensin domain. Tyr residues can induce intra- and inter-molecular cross-links in extensins, and substitution of Tyr in the extensin domain by Phe led to reduced activity of the corresponding LRX1 protein. An additional function of Tyr (or Phe) is provided by the aromatic nature of the side chain. This suggests that these residues might be involved in hydrophobic stacking, possibly as a mechanism of protein assembly. Finally, modified LRX1 proteins lacking Tyr in the extensin domain are still insolubilized in the cell wall, indicating strong interactions of extensins within the cell wall in addition to the well-described Tyr cross-links. [source] Signaling requirements and role of salicylic acid in HRT - and rrt -mediated resistance to turnip crinkle virus in ArabidopsisTHE PLANT JOURNAL, Issue 5 2004A.C. Chandra-Shekara Summary Inoculation of turnip crinkle virus (TCV) on the resistant Arabidopsis ecotype Di-17 elicits a hypersensitive response (HR), which is accompanied by increased expression of pathogenesis-related (PR) genes. Previous genetic analyses revealed that the HR to TCV is conferred by HRT, which encodes a coiled-coil (CC), nucleotide-binding site (NBS) and leucine-rich repeat (LRR) class resistance (R) protein. In contrast to the HR, resistance to TCV requires both HRT and a recessive allele at a second locus designated rrt. Here, we demonstrate that unlike most CC-NBS-LRR R genes, HRT/rrt -mediated resistance is dependent on EDS1 and independent of NDR1. Resistance is also independent of RAR1 and SGT1. HRT/rrt -mediated resistance is compromised in plants with reduced salicylic acid (SA) content as a consequence of mutations eds5, pad4, or sid2. By contrast, HR is not affected by mutations in eds1, eds5, pad4, sid2, ndr1, rar1, or sgt1b. Resistance to TCV is restored in both SA-deficient Di-17 plants expressing the nahG transgene and mutants containing the eds1, eds5, or sid2 mutations by exogenous application of SA or the SA analog benzo(1,2,3)thiadiazole-7-carbothioic acid (BTH). In contrast, SA/BTH treatment failed to enhance resistance in HRT pad4, Col-0, or hrt homozygous progeny of a cross between Di-17 and Col-0. Thus, HRT and PAD4 are required for SA-induced resistance. Exogenously supplied SA or high endogenous levels of SA, due to the ssi2 mutation, overcame the suppressive effects of RRT and enhanced resistance to TCV, provided the HRT allele was present. High levels of SA upregulate HRT expression via a PAD4 -dependent pathway. As Col-0 transgenic lines expressing high levels of HRT were resistant to TCV, but lines expressing moderate to low levels of HRT were not, we conclude that SA enhances resistance in the RRT background by upregulating HRT expression. These data suggest that the HRT-TCV interaction is unable to generate sufficient amounts of SA required for a stable resistance phenotype, and the presence of rrt possibly corrects this deficiency. [source] A single-amino acid substitution in the sixth leucine-rich repeat of barley MLA6 and MLA13 alleviates dependence on RAR1 for disease resistance signalingTHE PLANT JOURNAL, Issue 2 2004Dennis A. Halterman Summary Interactions between barley and the powdery mildew pathogen, Blumeria graminis f. sp. hordei, (Bgh) are determined by unique combinations of host resistance genes, designated Mildew-resistance locus (Ml), and cognate pathogen avirulence genes. These interactions occur both dependent and independent of Rar1 (required for Mla12 resistance) and Sgt1 (Suppressor of G-two allele of skp1), which are differentially required for diverse plant disease-resistance pathways. We have isolated two new functional Mla alleles, Rar1 -independent Mla7 and Rar1 -dependent Mla10, as well as the Mla paralogs, Mla6-2 and Mla13-2. Utilizing the inherent diversity amongst Mla -encoded proteins, we identified the only two amino acids exclusively conserved in RAR1-dependent MLA6, MLA10, MLA12, and MLA13 that differ at the corresponding position in RAR1-independent MLA1 and MLA7. Two- and three-dimensional modeling places these residues on a predicted surface of the sixth leucine-rich repeat (LRR) domain at positions distinct from those within the ,-sheets hypothesized to determine resistance specificity. Site-directed mutagenesis of these residues indicates that RAR1 independence requires the presence of an aspartate at position 721, as mutation of this residue to a structurally similar, but uncharged, asparagine did not alter RAR1 dependence. These results demonstrate that a single-amino acid substitution in the sixth MLA LRR can alter host signaling but not resistance specificity to B. graminis. [source] Role of the leucine-rich repeat domain of cryopyrin/NALP3 in monosodium urate crystal,induced inflammation in miceARTHRITIS & RHEUMATISM, Issue 7 2010Hal M. Hoffman Objective The mechanism by which monosodium urate monohydrate (MSU) crystals intracellularly activate the cryopyrin inflammasome is unknown. The aim of this study was to use a mouse molecular genetics,based approach to test whether the leucine-rich repeat (LRR) domain of cryopyrin is required for MSU crystal,induced inflammation. Methods Cryopyrin-knockout lacZ (Cryo,Z/,Z) mice and mice with the cryopyrin LRR domain deleted and fused to the lacZ reporter (Cryo,LRR Z/,LRR Z) were generated using bacterial artificial chromosome,based targeting vectors, which allow for large genomic deletions. Bone marrow,derived macrophages from Cryo,LRR Z/,LRR Z mice, Cryo,Z/,Z mice, and congenic wild-type (WT) mice were challenged with endotoxin-free MSU crystals under serum-free conditions. Phagocytosis and cytokine expression were assessed by flow cytometry and enzyme-linked immunosorbent assay. MSU crystals also were injected into mouse synovial-like subcutaneous air pouches. The in vivo inflammatory responses were examined. Results Release of interleukin-1, (IL-1,), but not CXCL1 and tumor necrosis factor ,, was impaired in Cryo,LRR Z/,LRR Z and Cryo,Z/,Z mouse bone marrow,derived macrophages compared with WT mouse bone marrow,derived macrophages in response to not only MSU crystals but also other known stimuli that activate the cryopyrin inflammasome. In addition, a comparable percentage of MSU crystals taken up by each type of bone marrow,derived macrophage was observed. Moreover, total leukocyte infiltration in the air pouch and IL-1, production were attenuated in Cryo,Z/,Z and Cryo,LRR Z/,LRR Z mice at 6 hours postinjection of MSU crystals compared with WT mice. Conclusion MSU crystal,induced inflammatory responses were comparably attenuated both in vitro and in vivo in Cryo,LRR Z/,LRR Z and Cryo,Z/,Z mice. Hence, the LRR domain of cryopyrin plays a role in mediating MSU crystal,induced inflammation in this model. [source] Functional consequences of a germline mutation in the leucine-rich repeat domain of NLRP3 identified in an atypical autoinflammatory disorderARTHRITIS & RHEUMATISM, Issue 4 2010Isabelle Jéru Objective To gain insight into the pathophysiology of an atypical familial form of an autoinflammatory disorder, characterized by autosomal-dominant sensorineural hearing loss, systemic inflammation, increased secretion of interleukin-1, (IL-1,), and the absence of any cutaneous manifestations, and to assess the functional consequences of a missense mutation identified in the leucine-rich repeat (LRR) domain of NLRP3. Methods Microsatellite markers were used to test the familial segregation of the NLRP3 locus with the disease phenotype. All NLRP3 exons were screened for mutations by sequencing. Functional assays were performed in HEK 293T cells to determine the effects of mutated (versus normal) NLRP3 proteins on NF-,B activation, caspase 1 signaling, and speck formation. Results A heterozygous NLRP3 missense mutation (p.Tyr859Cys) was identified in exon 6, which encodes the LRR domain of the protein. This mutation was found to segregate with the disease phenotype within the family, and had a moderate activating effect on speck formation and procaspase 1 processing and did not alter the inhibitory properties of NLRP3 on NF-,B signaling. Conclusion This report is the first to describe a familial form of a cryopyrinopathy associated with a mutation outside of exon 3 of NLRP3. This finding, together with the known efficacy of anti,IL-1 treatments in these disorders, underlines the importance of screening all exons of NLRP3 in patients who present with atypical manifestations. In addition, the gain of function associated with this mutation in terms of activation of caspase 1 signaling was consistent with the observed inflammatory phenotype. Therefore, this study of the functional consequences of an LRR mutation sheds new light on the clinical relevance of in vitro assays. [source] The NOD2 defect in Blau syndrome does not result in excess interleukin-1 activityARTHRITIS & RHEUMATISM, Issue 2 2009Tammy M. Martin Objective Blau syndrome is a rare, autosomal-dominant, autoinflammatory disorder characterized by granulomatous arthritis, uveitis, and dermatitis. Genetics studies have shown that the disease is caused by single nonsynonymous substitutions in NOD-2, a member of the NOD-like receptor or NACHT,leucine-rich repeat (NLR) family of intracellular proteins. Several NLRs function in the innate immune system as sensors of pathogen components and participate in immune-mediated cellular responses via the caspase 1 inflammasome. Mutations in a gene related to NOD-2, NLRP3, are responsible for excess caspase 1,dependent interleukin-1, (IL-1,) in cryopyrinopathies such as Muckle-Wells syndrome. Furthermore, functional studies demonstrate that caspase 1,mediated release of IL-1, also involves NOD-2. The aim of this study was to test the hypothesis that IL-1, may mediate the inflammation seen in patients with Blau syndrome. Methods IL-1, release was measured in peripheral blood mononuclear cells cultured in vitro, obtained from 5 Blau syndrome individuals with a NOD2 (CARD15) mutation. Results We observed no evidence for increased IL-1, production in cells obtained from subjects with Blau syndrome compared with healthy control subjects. Furthermore, we presented 2 cases of Blau syndrome in which recombinant human IL-1 receptor antagonist (anakinra) was ineffective treatment. Conclusion Taken together, these data suggest that in contrast to related IL-1,,dependent autoinflammatory cryopyrinopathies, Blau syndrome is not mediated by excess IL-1, or other IL-1 activity. [source] Structure of internalin C from Listeria monocytogenesACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2006Amy Ooi The crystal structure of internalin C (InlC) from Listeria monocytogenes has been determined at 2.0,Ĺ resolution. Several observations implicate InlC in infection: inlC has the same transcriptional activator as other virulence genes, it is only present in pathogenic Listeria strains and an inlC deletion mutant is significantly less virulent. While the extended concave receptor-binding surfaces of the leucine-rich repeat (LRR) domains of internalins A and B have aromatic clusters involved in receptor binding, the corresponding surface of InlC is smaller, flatter and more hydrophilic, suggesting that InlC may be involved in weak or transient associations with receptors; this may help explain why no receptor has yet been discovered for InlC. In contrast, the Ig-like domain, to which the LRR domain is fused, has surface aromatics that may be of functional importance, possibly being involved in binding to the surface of the bacteria or in receptor binding. [source] The leucine-rich repeat domain in plant innate immunity: a wealth of possibilitiesCELLULAR MICROBIOLOGY, Issue 2 2009Meenu Padmanabhan Summary The innate immune system of both plants and animals uses immune receptors to detect pathogens and trigger defence responses. Despite having distinct evolutionary origin, most plant and animal immune receptors have a leucine-rich repeat (LRR) domain. The LRR domain adopts a slender conformation that maximizes surface area and has been shown to be ideal for mediating protein,protein interactions. Although the LRR domain was expected to be a platform for pathogen recognition, the NB-LRR class of plant innate immune receptors uses its LRR domain to carry out many other roles. This review discusses the domain architecture of plant LRRs and the various roles ascribed to this motif. [source] Novel genes involved in canonical Wnt/, -catenin signaling pathway in early Ciona intestinalis embryosDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 4 2008Shuichi Wada We report here characterization of five genes for novel components of the canonical Wnt/, -catenin signaling pathway. These genes were identified in the ascidian Ciona intestinalis through a loss-of-function screening for genes required for embryogenesis with morpholinos, and four of them have counterparts in vertebrates. The five genes we studied are as follows: Ci-PGAP1, a Ciona orthologue of human PGAP1, which encodes GPI (glycosylphosphatidylinositol) inositol-deacylase, Ci-ZF278, a gene encoding a C2H2 zinc-finger protein, Ci-C10orf11, a Ciona orthologue of human C10orf11 that encodes a protein with leucine-rich repeats, Ci-Spatial/C4orf17, a single counterpart for two human genes Spatial and C4orf17, and Ci-FLJ10634, a Ciona orthologue of human FLJ10634 that encodes a member of the J-protein family. Knockdown of each of the genes mimicked , -catenin knockdown and resulted in suppression of the expression of , -catenin downstream genes (Ci-FoxD, Ci-Lhx3, Ci-Otx and Ci-Fgf9/16/20) and subsequent endoderm formation. For every gene, defects in knockdown embryos were rescued by overexpression of a constitutively active form, but not wild-type, of Ci- , -catenin. Dosage-sensitive interactions were found between Ci-,-catenin and each of the genes. These results suggest that these five genes act upstream of or parallel to Ci- , -catenin in the Wnt/, -catenin signaling pathway in early Ciona embryos. [source] Characterization of two novel proteins, NgRH1 and NgRH2, structurally and biochemically homologous to the Nogo-66 receptorJOURNAL OF NEUROCHEMISTRY, Issue 3 2003V. Pignot Abstract Nogo-66 receptor (NgR) has recently been identified as the neuronal receptor of the myelin-associated proteins Nogo-A, oligodendrocyte protein (OMgp) and myelin-associated glycoprotein (MAG), and mediates inhibition of axonal regeneration both in vitro and in vivo. Through database searches, we have identified two novel proteins (NgRH1 and NgRH2) that turned out to be homologous in their primary structures, biochemical properties and expression patterns to NgR. Like NgR, the homologues contain eight leucine-rich repeats (LRR) flanked by a leucine-rich repeat C-terminus (LRRCT) and a leucine-rich repeat N-terminus (LRRNT), and also have a C-terminal GPI signal sequence. Northern blot analysis showed predominant expression of NgRH1 and NgRH2 mRNA in the brain. In situ hybridization and immunohistochemistry on rat brain slices revealed neuronal expression of the genes. NgRH1 and NgRH2 were detected on the cell surface of recombinant cell lines as N-glycosylated GPI anchored proteins and, consistent with other GPI anchored proteins, were localized within the lipid rafts of cellular membranes. In addition, an N-terminal proteolytic fragment of NgR comprising the majority of the ectodomain was found to be constitutively secreted from cells. Our data indicate that NgR, NgRH1 and NgRH2 constitute a novel receptor protein family, which may play related roles within the CNS. [source] Binding of platelet glycoprotein Ib, through the convex surface of leucine-rich repeats domain of glycoprotein IXJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 9 2009X. MO Summary.,Background: The mechanism of assembly of the platelet glycoprotein (GP) Ib-IX complex from GPIb,, GPIb, and GPIX subunits is not entirely clear. In this complex, ectodomains of both GPIb, and GPIX subunits contain two leucine-rich repeats (LRR) and share high sequence similarity. However, they differ noticeably in stability, hampering further analysis of their interaction. Objectives and methods: Guided by analysis of the LRR structure, we report a well-folded Ib,/IX chimera and its usage in dissecting GPIX function. Results: In this chimera, three non-contiguous sequences that may constitute the putative convex surface of the GPIb, ectodomain are replaced by their GPIX counterparts. Like GPIb, but unlike GPIX ectodomain, it can secrete from transfected Chinese hamster ovary cells and fold into a stable conformation. Furthermore, replacing the ectodomain in GPIX with the Ib,/IX chimera, but not the GPIb, ectodomain, preserved its interaction with GPIb, as demonstrated by its native-like GPIb,-induced increase in surface expression and coimmunoprecipitation. Conclusions: The putative convex surface of the LRR domain in GPIX is sufficient, in the context of full-length subunit, to mediate its association with GPIb,. [source] Ice recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic Antarctic hair grass Deschampsia antarctica E. Desv.PLANT CELL & ENVIRONMENT, Issue 4 2009ULRIK P. JOHN ABSTRACT Antarctic hair grass (Deschampsia antarctica E. Desv.), the only grass indigenous to Antarctica, has well-developed freezing tolerance, strongly induced by cold acclimation. Here, we show that in response to low temperatures, D. antarctica expresses potent recrystallization inhibition (RI) activity that, inhibits the growth of small ice crystals into potentially damaging large ones, is proteinaceous and localized to the apoplasm. A gene family from D. antarctica encoding putative homologs of an ice recrystallization inhibition protein (IRIP) has been isolated and characterized. IRIPs are apoplastically targeted proteins with two potential ice-binding motifs: 1,9 leucine-rich repeats (LRRs) and c. 16 ,IRIP' repeats. IRIP genes appear to be confined to the grass subfamily Pooideae and their products, exhibit sequence similarity to phytosulphokine receptors and are predicted to adopt conformations with two ice-binding surfaces. D. antarctica IRIP (DaIRIP) transcript levels are greatly enhanced in leaf tissue following cold acclimation. Transgenic Arabidopsis thaliana expressing a DaIRIP has novel RI activity, and purified DaIRIP, when added back to extracts of leaves from non-acclimated D. antarctica, can reconstitute the activity found in acclimated plants. We propose that IRIP-mediated RI activity may contribute to the cryotolerance of D. antarctica, and thus to its unique ability to have colonized Antarctica. [source] The N-terminal region of Pseudomonas type III effector AvrPtoB elicits Pto-dependent immunity and has two distinct virulence determinantsTHE PLANT JOURNAL, Issue 4 2007Fangming Xiao Summary Resistance to bacterial speck disease in tomato is activated by the physical interaction of the host Pto kinase with either of the sequence-dissimilar type III effector proteins AvrPto or AvrPtoB (HopAB2) from Pseudomonas syringae pv. tomato. Pto-mediated immunity requires Prf, a protein with a nucleotide-binding site and leucine-rich repeats. The N-terminal 307 amino acids of AvrPtoB were previously reported to interact with the Pto kinase, and we show here that this region (AvrPtoB1-307) is sufficient for eliciting Pto/Prf-dependent immunity against P. s. pv. tomato. AvrPtoB1-307 was also found to be sufficient for a virulence activity that enhances ethylene production and increases growth of P. s. pv. tomato and severity of speck disease on susceptible tomato lines lacking either Pto or Prf. Moreover, we found that residues 308,387 of AvrPtoB are required for the previously reported ability of AvrPtoB to suppress pathogen-associated molecular patterns-induced basal defenses in Arabidopsis. Thus, the N-terminal region of AvrPtoB has two structurally distinct domains involved in different virulence-promoting mechanisms. Random and targeted mutagenesis identified five tightly clustered residues in AvrPtoB1-307 that are required for interaction with Pto and for elicitation of immunity to P. s. pv. tomato. Mutation of one of the five clustered residues abolished the ethylene-associated virulence activity of AvrPtoB1-307. However, individual mutations of the other four residues, despite abolishing interaction with Pto and avirulence activity, had no effect on AvrPtoB1-307 virulence activity. None of these mutations affected the basal defense-suppressing activity of AvrPtoB1-387. Based on sequence alignments, estimates of helical propensity, and the previously reported structure of AvrPto, we hypothesize that the Pto-interacting domains of AvrPto and AvrPtoB1-307 have structural similarity. Together, these data support a model in which AvrPtoB1-307 promotes ethylene-associated virulence by interaction not with Pto but with another unknown host protein. [source] A leucine-rich repeat protein is required for growth promotion and enhanced seed production mediated by the endophytic fungus Piriformospora indica in Arabidopsis thalianaTHE PLANT JOURNAL, Issue 1 2007Bationa Shahollari Summary Piriformospora indica, a basidiomycete of the Sebacinaceae family, promotes the growth, development and seed production of a variety of plant species. Arabidopsis plants colonized with the fungus produce 22% more seeds than uncolonized plants. Deactivating the Arabidopsis single-copy gene DMI-1, which encodes an ion carrier required for mycorrihiza formation in legumes, does not affect the beneficial interaction between the two symbiotic partners. We used cellular and molecular responses initiated during the establishment of the interaction between P. indica and Arabidopsis roots to isolate mutants that fail to respond to the fungus. An ethylmethane sulfonate mutant (Piriformospora indica - insensitive-2; pii-2), and a corresponding insertion line, are impaired in a leucine-rich repeat protein (At1g13230). The protein pii-2, which contains a putative endoplasmic reticulum retention signal, is also found in Triton X-100-insoluble plasma membrane microdomains, suggesting that it is present in the endoplasmic reticulum/plasma membrane continuum in Arabidopsis roots. The microdomains also contain an atypical receptor protein (At5g16590) containing leucine-rich repeats, the message of which is transiently upregulated in Arabidopsis roots in response to P. indica. This response is not detectable in At1g13230 mutants, and the protein is not detectable in the At1g13230 mutant microdomains. Partial deactivation of a gene for a sphingosine kinase, which is required for the biosynthesis of sphingolipid found in plasma membrane microdomains, also affects the Arabidopsis/P. indica interaction. Thus, pii-2, and presumably also At5g16590, two proteins present in plasma membrane microdomains, appear to be involved in P. indica -induced growth promotion and enhanced seed production in Arabidopsis thaliana. [source] PYPAF1 nonsense mutation in a patient with an unusual autoinflammatory syndrome: Role of PYPAF1 in inflammationARTHRITIS & RHEUMATISM, Issue 2 2006I. Jéru Objective To gain insight into the pathophysiology of an unusual autoinflammatory syndrome, in a patient of Armenian origin, that mimicked familial Mediterranean fever (FMF) but with episodes triggered by generalized exposure to cold, and to further elucidate the controversial function of the protein encoded by PYPAF1, whose mutations (exclusively missense to date) have been identified in 3 hereditary recurrent fever syndromes. Methods The patient's DNA was screened for mutations in both MEFV, the gene responsible for FMF, and PYPAF1. The ability of different recombinant PYPAF1 isoforms, expressed in HEK 293 cells, to regulate NF-,B signaling was subsequently assessed. Results No disease-causing mutation was found in MEFV. However, a nonsense mutation (p.Arg554X) was identified in PYPAF1; this defect resulted in a truncated protein lacking all leucine-rich repeats. Study of the wild-type and mutant PYPAF1 recombinant proteins revealed that PYPAF1 inhibited NF-,B proinflammatory pathways, and that the identified nonsense mutation impaired this property. Conclusion These molecular and clinical findings, together with the clinical manifestations in the patient, which call into question the current nosology of the hereditary recurrent fever syndromes, are consistent with the hypothesis that PYPAF1 acts as an inhibitor of NF-,B signaling. They also provide a clear elucidation of the functional consequences of this nonsense PYPAF1 mutation not previously described in the literature, which result in a partial loss of function and may thereby explain the pathophysiology of the autoinflammatory syndrome observed in this patient. [source] | |||||||||||||||||||||||||