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Sample records for putative leucine-rich repeat

  1. LMOh7858_0369, a gene encoding a putative leucine-rich repeat-containing protein, is required for virulence of Listeria monocytogenes.

    PubMed

    Zhang, Ting; Bae, Dongryeoul; Wang, Chinling

    2016-05-01

    Listeria monocytogenes possesses the highest number of leucine-rich repeat (LRR)-containing proteins among all Gram-positive bacteria; these LRR-containing molecules are known as the 'internalin' family. To understand the functions of largely uncharacterized LRR-containing molecules, we constructed seven deletion mutants in the L. monocytogenes H7858 strain targeting genes in this family and tested their virulence. Among the seven mutants, the ΔLMOh7858_0369 strain and the ΔLMOh7858_2546 strain showed significantly impaired invasiveness of HepG2 cells. We further tested the virulence of these two strains in the intravascular sepsis model using BALB/c mice. Interestingly, the ΔLMOh7858_0369 strain showed significant reduction in organ colonization, bacteremia and invasion of the brain compared with the parental wild-type strain. Host immune responses to listerial intravascular infection were measured at 24 and 72 h post-infection. Transcript levels of several proinflammatory cytokines and chemokines were significantly lower when induced by the ΔlmOh7858_0369 strain than when induced by the wild type. These results suggest that the putative LRR-containing protein encoded by LMOh7858_0369 might be a novel virulence factor of the L. monocytogenes H7858 strain. PMID:26976852

  2. Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins.

    PubMed

    Hu, Yueming; Huang, He; Hui, Xinjie; Cheng, Xi; White, Aaron P; Zhao, Zhendong; Wang, Yejun

    2016-08-01

    Leucine-rich repeat (LRR) proteins are widely distributed in bacteria, playing important roles in various protein-protein interaction processes. In Yersinia, the well-characterized type III secreted effector YopM also belongs to the LRR protein family and is encoded by virulence plasmids. However, little has been known about other LRR members encoded by Yersinia genomes or their evolution. In this study, the Yersinia LRR proteins were comprehensively screened, categorized, and compared. The LRR proteins encoded by chromosomes (LRR1 proteins) appeared to be more similar to each other and different from those encoded by plasmids (LRR2 proteins) with regard to repeat-unit length, amino acid composition profile, and gene expression regulation circuits. LRR1 proteins were also different from LRR2 proteins in that the LRR1 proteins contained an E3 ligase domain (NEL domain) in the C-terminal region or an NEL domain-encoding nucleotide relic in flanking genomic sequences. The LRR1 protein-encoding genes (LRR1 genes) varied dramatically and were categorized into 4 subgroups (a to d), with the LRR1a to -c genes evolving from the same ancestor and LRR1d genes evolving from another ancestor. The consensus and ancestor repeat-unit sequences were inferred for different LRR1 protein subgroups by use of a maximum parsimony modeling strategy. Structural modeling disclosed very similar repeat-unit structures between LRR1 and LRR2 proteins despite the different unit lengths and amino acid compositions. Structural constraints may serve as the driving force to explain the observed mutations in the LRR regions. This study suggests that there may be functional variation and lays the foundation for future experiments investigating the functions of the chromosomally encoded LRR proteins of Yersinia. PMID:27217422

  3. A nuclear factor containing the leucine-rich repeats expressed in murine cerebellar neurons.

    PubMed Central

    Matsuoka, K; Taoka, M; Satozawa, N; Nakayama, H; Ichimura, T; Takahashi, N; Yamakuni, T; Song, S Y; Isobe, T

    1994-01-01

    A nuclear protein, termed leucine-rich acidic nuclear protein (LANP), has been isolated from among rat cerebellar proteins whose expression was transiently increased during an early stage of postnatal development. The amino acid sequence, deduced from its cDNA, showed that LANP contains 247 amino acids consisting of two distinct structural domains: the N-terminal domain characterized by "leucine-rich repeat," which is found in many eukaryotic proteins and which potentially functions in mediating protein-protein interactions, and the C-terminal domain characterized by a cluster of acidic amino acids with a putative nuclear localization signal. Immunohistochemical study using an antibody against LANP revealed that the protein is localized mainly in nuclei of Purkinje cells. In the rat cerebellum on postnatal day 7, LANP mRNA was expressed moderately in the external granule and Purkinje cells and weakly in the internal granule cells. The expression in these cells, especially in Purkinje cells, increased in the second postnatal week and thereafter decreased to an adult level. The structural characteristics, localization, and the stage- and cell type-specific expression suggest a potential role of LANP in a signal transduction pathway that directs differentiation of cerebellar neurons. Images PMID:7937870

  4. Identification and mapping of nucleotide binding site-leucine rich repeat resistance gene analogs in bermudagrass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thirty-one bermudagrass (Cynodon spp.) disease resistance gene homologs (BRGH) were cloned and sequenced from diploid, triploid, and hexaploid bermudagrass using degenerate primers to target the nucleotide binding site (NBS) of the NBS- leucine rich repeat (LRR) resistance gene family. Alignment of ...

  5. Reevaluation of Phosphorylation Sites in the Parkinson Disease-associated Leucine-rich Repeat Kinase 2*

    PubMed Central

    Li, Xiaojie; Moore, Darren J.; Xiong, Yulan; Dawson, Ted M.; Dawson, Valina L.

    2010-01-01

    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an important cause of late-onset, autosomal dominant familial Parkinson disease and contribute to sporadic Parkinson disease. LRRK2 is a large complex protein with multiple functional domains, including a Roc-GTPase, protein kinase, and multiple protein-protein interaction domains. Previous studies have suggested an important role for kinase activity in LRRK2-induced neuronal toxicity and inclusion body formation. Disease-associated mutations in LRRK2 also tend to increase kinase activity. Thus, enhanced kinase activity may therefore underlie LRRK2-linked disease. Similar to the closely related mixed-lineage kinases, LRRK2 can undergo autophosphorylation in vitro. Three putative autophosphorylation sites (Thr-2031, Ser-2032, and Thr-2035) have been identified within the activation segment of the LRRK2 kinase domain based on sequence homology to mixed-lineage kinases. Phosphorylation at one or more of these sites is critical for the kinase activity of LRRK2. Sensitive phopho-specific antibodies to each of these three sites have been developed and validated by ELISA, dot-blot, and Western blot analysis. Using these antibodies, we have found that all three putative sites are phosphorylated in LRRK2, and Ser-2032 and Thr-2035 are the two important sites that regulate LRRK2 kinase activity. PMID:20595391

  6. Anatomical localization of leucine-rich repeat kinase 2 in mouse brain.

    PubMed

    Melrose, H; Lincoln, S; Tyndall, G; Dickson, D; Farrer, M

    2006-01-01

    Mutations in leucine-rich repeat kinase 2 (LRRK2) have recently been identified in autosomal dominant late-onset Parkinson's disease. Expression of LRRK2 has previously been reported in brain; however, no precise anatomical information is yet available. We have performed in situ hybridization and quantitative reverse transcription polymerase chain reaction to map LRRK2 mRNA expression in mouse brain. We find LRRK2 is highly expressed in the striatum, cortex and olfactory tubercle; however, little or no expression is found in the substantia nigra, where dopaminergic neurons preferentially degenerate in Parkinson's disease. These findings suggest that LRRK2 mRNA is expressed in dopamine-receptive areas rather than in the dopamine-synthesizing neurons. Consistent with a role LRRK2 in Parkinson's disease, dysfunction of leucine-rich repeat kinase 2 protein in dopamine-innervated areas may to lead to altered dopaminergic neurotransmission and degeneration of the nigro-striatal pathway. PMID:16504409

  7. Capping motifs stabilize the leucine-rich repeat protein PP32 and rigidify adjacent repeats.

    PubMed

    Dao, Thuy P; Majumdar, Ananya; Barrick, Doug

    2014-06-01

    Capping motifs are found to flank most β-strand-containing repeat proteins. To better understand the roles of these capping motifs in organizing structure and stability, we carried out folding and solution NMR studies on the leucine-rich repeat (LRR) domain of PP32, which is composed of five tandem LRR, capped by α-helical and β-hairpin motifs on the N- and C-termini. We were able to purify PP32 constructs lacking either cap and containing destabilizing substitutions. Removing the C-cap results in complete unfolding of PP32. Removing the N-cap has a much less severe effect, decreasing stability but retaining much of its secondary structure. In contrast, the dynamics and tertiary structure of the first two repeats are significantly perturbed, based on (1)H-(15)N relaxation studies, chemical shift perturbations, and residual dipolar couplings. However, more distal repeats (3 to C-cap) retain their native tertiary structure. In this regard, the N-cap drives the folding of adjacent repeats from what appears to be a molten-globule-like state. This interpretation is supported by extensive analysis using core packing substitutions in the full-length and N-cap-truncated PP32. This work highlights the importance of caps to the stability and structural integrity of β-strand-containing LRR proteins, and emphasizes the different contributions of the N- and C-terminal caps. PMID:24659532

  8. An evolutionary comparison of leucine-rich repeat containing G protein-coupled receptors reveals a novel LGR subtype.

    PubMed

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Van Loy, Tom; Vanden Broeck, Jozef

    2012-03-01

    Leucine-rich repeat containing G protein-coupled receptors or LGRs are receptors with important functions in development and reproduction. Belonging to this evolutionarily conserved group of receptors are the well-studied glycoprotein hormone receptors and relaxin receptors in mammals, as well as the bursicon receptor, which triggers cuticle hardening and tanning in freshly enclosed insects. In this study, the numerous LGR sequences in different animal phyla are analyzed and compared. Based on these data a phylogenetic tree was generated. This information sheds new light on structural and evolutionary aspects regarding this receptor group. Apart from vertebrates and insects, LGRs are also present in early chordates (Urochordata, Cephalochordata and Hyperoartia) and other arthropods (Arachnida and Branchiopoda) as well as in Mollusca, Echinodermata, Hemichordata, Nematoda, and even in ancient animal life forms, such as Cnidaria and Placozoa. Three distinct types of LGR exist, distinguishable by their number of leucine-rich repeats (LRRs), their type-specific hinge region and the presence or absence of an LDLa motif. Type C LGRs containing only one LDLa (C1 subtype) appear to be present in nearly all animal phyla. We here describe a second subtype, C2, containing multiple LDLa motifs, which was discovered in echinoderms, mollusks and in one insect species (Pediculus humanis corporis). In addition, eight putative LGRs can be predicted from the genome data of the placozoan species Trichoplax adhaerens. They may represent an ancient form of the LGRs, however, more genomic data will be required to confirm this hypothesis. PMID:22100731

  9. Potent, selective and orally bioavailable leucine-rich repeat kinase 2 (LRRK2) inhibitors.

    PubMed

    Greshock, Thomas J; Sanders, John M; Drolet, Robert E; Rajapakse, Hemaka A; Chang, Ronald K; Kim, Boyoung; Rada, Vanessa L; Tiscia, Heather E; Su, Hua; Lai, Ming-Tain; Sur, Sylvie M; Sanchez, Rosa I; Bilodeau, Mark T; Renger, John J; Kern, Jonathan T; McCauley, John A

    2016-06-01

    Familial Parkinson's disease cases have recently been associated with the leucine rich repeat kinase 2 (LRRK2) gene. It has been hypothesized that inhibition of the LRRK2 protein may have the potential to alter disease pathogenesis. A dihydrobenzothiophene series of potent, selective, orally bioavailable LRRK2 inhibitors were identified from a high-throughput screen of the internal Merck sample collection. Initial SAR studies around the core established the series as a tractable small molecule lead series of LRRK2 inhibitors for potential treatment of Parkinson's disease. It was also found that incorporation of a lactam into the core drastically improved the CNS and DMPK properties of these small molecules. PMID:27106707

  10. The Role of Leucine-Rich Repeat Containing Protein 10 (LRRC10) in Dilated Cardiomyopathy

    PubMed Central

    Brody, Matthew J.; Lee, Youngsook

    2016-01-01

    Leucine-rich repeat containing protein 10 (LRRC10) is a cardiomyocyte-specific member of the Leucine-rich repeat containing (LRRC) protein superfamily with critical roles in cardiac function and disease pathogenesis. Recent studies have identified LRRC10 mutations in human idiopathic dilated cardiomyopathy (DCM) and Lrrc10 homozygous knockout mice develop DCM, strongly linking LRRC10 to the molecular etiology of DCM. LRRC10 localizes to the dyad region in cardiomyocytes where it can interact with actin and α-actinin at the Z-disc and associate with T-tubule components. Indeed, this region is becoming increasingly recognized as a signaling center in cardiomyocytes, not only for calcium cycling, excitation-contraction coupling, and calcium-sensitive hypertrophic signaling, but also as a nodal signaling hub where the myocyte can sense and respond to mechanical stress. Disruption of a wide range of critical structural and signaling molecules in cardiomyocytes confers susceptibility to cardiomyopathies in addition to the more classically studied mutations in sarcomeric proteins. However, the molecular mechanisms underlying DCM remain unclear. Here, we review what is known about the cardiomyocyte functions of LRRC10, lessons learned about LRRC10 and DCM from the Lrrc10 knockout mouse model, and discuss ongoing efforts to elucidate molecular mechanisms whereby mutation or absence of LRRC10 mediates cardiac disease. PMID:27536250

  11. The Role of Leucine-Rich Repeat Containing Protein 10 (LRRC10) in Dilated Cardiomyopathy.

    PubMed

    Brody, Matthew J; Lee, Youngsook

    2016-01-01

    Leucine-rich repeat containing protein 10 (LRRC10) is a cardiomyocyte-specific member of the Leucine-rich repeat containing (LRRC) protein superfamily with critical roles in cardiac function and disease pathogenesis. Recent studies have identified LRRC10 mutations in human idiopathic dilated cardiomyopathy (DCM) and Lrrc10 homozygous knockout mice develop DCM, strongly linking LRRC10 to the molecular etiology of DCM. LRRC10 localizes to the dyad region in cardiomyocytes where it can interact with actin and α-actinin at the Z-disc and associate with T-tubule components. Indeed, this region is becoming increasingly recognized as a signaling center in cardiomyocytes, not only for calcium cycling, excitation-contraction coupling, and calcium-sensitive hypertrophic signaling, but also as a nodal signaling hub where the myocyte can sense and respond to mechanical stress. Disruption of a wide range of critical structural and signaling molecules in cardiomyocytes confers susceptibility to cardiomyopathies in addition to the more classically studied mutations in sarcomeric proteins. However, the molecular mechanisms underlying DCM remain unclear. Here, we review what is known about the cardiomyocyte functions of LRRC10, lessons learned about LRRC10 and DCM from the Lrrc10 knockout mouse model, and discuss ongoing efforts to elucidate molecular mechanisms whereby mutation or absence of LRRC10 mediates cardiac disease. PMID:27536250

  12. Positive selection in the leucine-rich repeat domain of Gro1 genes in Solanum species.

    PubMed

    Ruggieri, Valentino; Nunziata, Angelina; Barone, Amalia

    2014-12-01

    In pathogen resistant plants, solvent-exposed residues in the leucine-rich repeat (LRR) proteins are thought to mediate resistance by recognizing plant pathogen elicitors. In potato, the gene Gro1-4 confers resistance to Globodera rostochiensis. The investigation of variability in different copies of this gene represents a good model for the verification of positive selection mechanisms. Two datasets of Gro1 LRR sequences were constructed, one derived from the Gro1-4 gene, belonging to different cultivated and wild Solanum species, and the other belonging to paralogues of a resistant genotype. Analysis of nonsynonymous to synonymous substitution rates (K(a)/K(s)) highlighted 14 and six amino acids with K(a)/K(s) >1 in orthologue and paralogue datasets, respectively. Selection analysis revealed that the leucine-rich regions accumulate variability in a very specific way, and we found that some combinations of amino acids in these sites might be involved in pathogen recognition. The results confirm previous studies on positive selection in the LRR domain of R protein in Arabidopsis and other model plants and extend these to wild Solanum species. Moreover, positively selected sites in the Gro1 LRR domain show that coevolution mainly occurred in two regions on the internal surface of the three-dimensional horseshoe structure of the domain, albeit with different evolutionary forces between paralogues and orthologues. PMID:25572234

  13. Assembly of Neuronal Connectivity by Neurotrophic Factors and Leucine-Rich Repeat Proteins.

    PubMed

    Ledda, Fernanda; Paratcha, Gustavo

    2016-01-01

    Proper function of the nervous system critically relies on sophisticated neuronal networks interconnected in a highly specific pattern. The architecture of these connections arises from sequential developmental steps such as axonal growth and guidance, dendrite development, target determination, synapse formation and plasticity. Leucine-rich repeat (LRR) transmembrane proteins have been involved in cell-type specific signaling pathways that underlie these developmental processes. The members of this superfamily of proteins execute their functions acting as trans-synaptic cell adhesion molecules involved in target specificity and synapse formation or working in cis as cell-intrinsic modulators of neurotrophic factor receptor trafficking and signaling. In this review, we will focus on novel physiological mechanisms through which LRR proteins regulate neurotrophic factor receptor signaling, highlighting the importance of these modulatory events for proper axonal extension and guidance, tissue innervation and dendrite morphogenesis. Additionally, we discuss few examples linking this set of LRR proteins to neurodevelopmental and psychiatric disorders. PMID:27555809

  14. Assembly of Neuronal Connectivity by Neurotrophic Factors and Leucine-Rich Repeat Proteins

    PubMed Central

    Ledda, Fernanda; Paratcha, Gustavo

    2016-01-01

    Proper function of the nervous system critically relies on sophisticated neuronal networks interconnected in a highly specific pattern. The architecture of these connections arises from sequential developmental steps such as axonal growth and guidance, dendrite development, target determination, synapse formation and plasticity. Leucine-rich repeat (LRR) transmembrane proteins have been involved in cell-type specific signaling pathways that underlie these developmental processes. The members of this superfamily of proteins execute their functions acting as trans-synaptic cell adhesion molecules involved in target specificity and synapse formation or working in cis as cell-intrinsic modulators of neurotrophic factor receptor trafficking and signaling. In this review, we will focus on novel physiological mechanisms through which LRR proteins regulate neurotrophic factor receptor signaling, highlighting the importance of these modulatory events for proper axonal extension and guidance, tissue innervation and dendrite morphogenesis. Additionally, we discuss few examples linking this set of LRR proteins to neurodevelopmental and psychiatric disorders. PMID:27555809

  15. Ten Years and Counting: Moving Leucine-Rich Repeat Kinase 2 Inhibitors to the Clinic

    PubMed Central

    West, Andrew B

    2015-01-01

    The burden that Parkinson's disease (PD) exacts on the population continues to increase year after year. Though refinement of symptomatic treatments continues at a reasonable pace, no accepted therapies are available to slow or prevent disease progression. The leucine-rich repeat kinase 2 (LRRK2) gene was identified in PD genetic studies and offers new hope for novel therapeutic approaches. The evidence linking LRRK2 kinase activity to PD susceptibility is presented, as well as seminal discoveries relevant to the prosecution of LRRK2 kinase inhibition. Finally, suggestions are made for predictive preclinical modeling and successful first-in-human trials. © 2014 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society. PMID:25448543

  16. Metabolic labeling of leucine rich repeat kinases 1 and 2 with radioactive phosphate.

    PubMed

    Taymans, Jean-Marc; Gao, Fangye; Baekelandt, Veerle

    2013-01-01

    Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling, while LRRK2 is implicated in the pathogenesis of Parkinson's disease. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with (32)P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing (32)P-orthophosphate. The (32)P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography ((32)P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation. PMID:24084685

  17. Characterization of epiphycan, a small proteoglycan with a leucine-rich repeat core protein.

    PubMed

    Johnson, H J; Rosenberg, L; Choi, H U; Garza, S; Höök, M; Neame, P J

    1997-07-25

    The epiphysis of developing bones is a cartilaginous structure that is eventually replaced by bone during skeletal maturation. We have separated a dermatan sulfate proteoglycan, epiphycan, from decorin and biglycan by using dissociative extraction of bovine fetal epiphyseal cartilage, followed by sequential ion-exchange, gel permeation, hydrophobic, and Zn2+ chelate chromatographic steps. Epiphycan is a member of the small leucine-rich proteoglycan family, contains seven leucine-rich repeats (LRRs), is related to osteoglycin (osteoinductive factor) (Bentz, H., Nathan, R. M., Rosen, D. M., Armstrong, R. M., Thompson, A. Y., Segarini, P. R., Mathews, M. C., Dasch, J., Piez, K. A., and Seyedin, S. M. (1989) J. Biol. Chem. 264, 20805-20810), and appears to be the bovine equivalent of the chick proteoglycan PG-Lb (Shinomura, T., and Kimata, K. (1992) J. Biol. Chem. 267, 1265-1270). The intact proteoglycan had a median size of approximately 133 kDa. The core protein was 46 kDa by electrophoretic analysis, had a calculated size of 34,271 Da, and had two approximately equimolar N termini (APTLES ... and ETYDAT ... ) separated by 11 amino acids. There were at least three O-linked oligosaccharides in the N-terminal region of the protein, based on blank cycles in Edman degradation and corresponding serine or threonine residues in the translated cDNA sequence. The glycosaminoglycans ranged in size from 23 to 34 kDa were more heterogeneous than those in other dermatan sulfate small leucine-rich proteoglycans and were found in the acidic N-terminal region of the protein core, N-terminal to the LRRs. A four-cysteine cluster was present at the N terminus of the LRRs, and a disulfide-bonded cysteine pair was present at the C terminus of the protein core. The seventh LRR and an N-linked oligosaccharide were between the two C-terminal cysteines. An additional potential N-glycosylation site near the C terminus did not appear to be substituted at a significant level. PMID:9228042

  18. CORRELATION BETWEEN CYTOPLASMIC DOMAIN SEQUENCE AND AUTOPHOSPHORYLATION AMONG ARABIDOPSIS LEUCINE-RICH REPEAT RECEPTOR-LIKE KINASES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The leucine-rich repeat receptor-like kinases (LRR-RLKs) are implicated in signaling roles during plant growth, development and defense. A paradigm for receptor kinase activation involves dimerization and auto- or trans-phosphorylation within the cytoplasmic domain. Our goals are to identify intrace...

  19. Structural characterization of a novel subfamily of leucine-rich repeat proteins from the human pathogen Leptospira interrogans.

    PubMed

    Miras, Isabelle; Saul, Frederick; Nowakowski, Mireille; Weber, Patrick; Haouz, Ahmed; Shepard, William; Picardeau, Mathieu

    2015-06-01

    Pathogenic Leptospira spp. are the agents of leptospirosis, an emerging zoonotic disease. Analyses of Leptospira genomes have shown that the pathogenic leptospires (but not the saprophytes) possess a large number of genes encoding proteins containing leucine-rich repeat (LRR) domains. In other pathogenic bacteria, proteins with LRR domains have been shown to be involved in mediating host-cell attachment and invasion, but their functions remain unknown in Leptospira. To gain insight into the potential function of leptospiral LRR proteins, the crystal structures of four LRR proteins that represent a novel subfamily with consecutive stretches of a 23-amino-acid LRR repeat motif have been solved. The four proteins analyzed adopt the characteristic α/β-solenoid horseshoe fold. The exposed residues of the inner concave surfaces of the solenoid, which constitute a putative functional binding site, are not conserved. The various leptospiral LRR proteins could therefore recognize distinct structural motifs of different host proteins and thus serve separate and complementary functions in the physiology of these bacteria. PMID:26057675

  20. Preferentially Expressed Antigen in Melanoma (PRAME) and the PRAME Family of Leucine-Rich Repeat Proteins.

    PubMed

    Hermes, Nora; Kewitz, Stefanie; Staege, Martin S

    2016-01-01

    Preferentially expressed antigen in melanoma (PRAME) is the best characterized member of the PRAME family of leucine-rich repeat (LRR) proteins. Mammalian genomes contain multiple members of the PRAME family whereas in other vertebrate genomes only one PRAME-like LRR protein was identified. PRAME is a cancer/testis antigen that is expressed at very low levels in normal adult tissues except testis but at high levels in a variety of cancer cells. In contrast to most other cancer/testis antigens, PRAME is expressed not only in solid tumors but also in leukemia cells. Expression of PRAME and other members of the PRAME family is regulated epigenetically. PRAME interacts with varying pathways that might be directly involved in the malignant phenotype of cancer cells. For instance, PRAME is able to dominantly repress retinoic acid signaling in these cells. On the other hand, PRAME-derived peptides can be recognized as epitopes by cytotoxic T cells and PRAME represents an attractive target for immunological treatment strategies. PMID:26694250

  1. Function and dysfunction of leucine-rich repeat kinase 2 (LRRK2): Parkinson's disease and beyond.

    PubMed

    Bae, Jae Ryul; Lee, Byoung Dae

    2015-05-01

    Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD). As such, functions and dysfunctions of LRRK2 in PD have been the subject of extensive investigation. In addition to PD, increasing evidence is suggesting that LRRK2 is associated with a wide range of diseases. Genome-wide association studies have implicated LRRK2 in Crohn's disease (CD) and leprosy, and the carriers with pathogenic mutations of LRRK2 show increased risk to develop particular types of cancer. LRRK2 mutations are rarely found in Alzheimer's disease (AD), but LRRK2 might play a part in tauopathies. The association of LRRK2 with the pathogenesis of apparently unrelated diseases remains enigmatic, but it might be related to the yet unknown diverse functions of LRRK2. Here, we reviewed current knowledge on the link between LRRK2 and several diseases, including PD, AD, CD, leprosy, and cancer, and discussed the possibility of targeting LRRK2 in such diseases. PMID:25703537

  2. Leucine-rich pentatricopeptide-repeat containing protein regulates mitochondrial transcription.

    PubMed

    Sondheimer, Neal; Fang, Ji-Kang; Polyak, Erzsebet; Falk, Marni J; Avadhani, Narayan G

    2010-09-01

    Mitochondrial function depends upon the coordinated expression of the mitochondrial and nuclear genomes. Although the basal factors that carry out the process of mitochondrial transcription are known, the regulation of this process is incompletely understood. To further our understanding of mitochondrial gene regulation, we identified proteins that bound to the previously described point of termination for the major mRNA-coding transcript H2. One was the leucine-rich pentatricopeptide-repeat containing protein (LRPPRC), which has been linked to the French-Canadian variant of Leigh syndrome. Cells with reduced expression of LRPPRC had a reduction in oxygen consumption. The expression of mitochondrial mRNA and tRNA was dependent upon LRPPRC levels, but reductions in LRPPRC did not affect the expression of mitochondrial rRNA. Reduction of LRPPRC levels interfered with mitochondrial transcription in vitro but did not affect the stability of mitochondrial mRNAs or alter the expression of nuclear genes responsible for mitochondrial transcription in vivo. These findings demonstrate the control of mitochondrial mRNA synthesis by a protein that has an established role in regulating nuclear transcription and a link to mitochondrial disease. PMID:20677761

  3. Role of extracytoplasmic leucine rich repeat proteins in plant defence mechanisms.

    PubMed

    Shanmugam, V

    2005-01-01

    Plant-pathogen interactions involve highly complex series of reactions in disease development. Plants are endowed with both, resistance and defence genes. The activation of defence genes after contact with avirulence gene products of pathogens depends on signals transduced by leucine-rich repeats (LRRs) contained in resistance genes. Additionally, LRRs play roles for various actions following ligand recognition. Polygalacturonase inhibiting proteins (PGIPs), the only plant LRR protein with known ligands, are pectinase inhibitors, bound by ionic interactions to the extracellular matrix (ECM) of plant cells. They have a high affinity for fungal endopolygalacturonases (endoPGs). PGIP genes are organised in families encoding proteins with similar physical characteristics but different specificities. They are induced by infection and stress related signals. The molecular basis of PG-PGIP interaction serves as a model to understand the evolution of plant LRR proteins in recognising non-self-molecules. Extensins form a different class of structural proteins with repetitive sequences. They are also regulated by wounding and pathogen infection. Linkage of extensins with LRR motifs is highly significant in defending host tissues against pathogen invasion. Overexpression of PGIPs or expression of several PGIPs in a plant tissue, and perhaps manipulation of extensin expression could be possible strategies for disease management. PMID:15782942

  4. Neuronal leucine-rich repeat 1 negatively regulates anaplastic lymphoma kinase in neuroblastoma

    PubMed Central

    Satoh, Shunpei; Takatori, Atsushi; Ogura, Atsushi; Kohashi, Kenichi; Souzaki, Ryota; Kinoshita, Yoshiaki; Taguchi, Tomoaki; Hossain, Md. Shamim; Ohira, Miki; Nakamura, Yohko; Nakagawara, Akira

    2016-01-01

    In neuroblastoma (NB), one of the most common paediatric solid tumours, activation of anaplastic lymphoma kinase (ALK) is often associated with poor outcomes. Although genetic studies have identified copy number alteration and nonsynonymous mutations of ALK, the regulatory mechanism of ALK signalling at protein levels is largely elusive. Neuronal leucine-rich repeat 1 (NLRR1) is a type 1 transmembrane protein that is highly expressed in unfavourable NB and potentially influences receptor tyrosine kinase signalling. Here, we showed that NLRR1 and ALK exhibited a mutually exclusive expression pattern in primary NB tissues by immunohistochemistry. Moreover, dorsal root ganglia of Nlrr1+/+ and Nlrr1−/− mice displayed the opposite expression patterns of Nlrr1 and Alk. Of interest, NLRR1 physically interacted with ALK in vitro through its extracellular region. Notably, the NLRR1 ectodomain impaired ALK phosphorylation and proliferation of ALK-mutated NB cells. A newly identified cleavage of the NLRR1 ectodomain also supported NLRR1-mediated ALK signal regulation in trans. Thus, we conclude that NLRR1 appears to be an extracellular negative regulator of ALK signalling in NB and neuronal development. Our findings may be beneficial to comprehend NB heterogeneity and to develop a novel therapy against unfavourable NB. PMID:27604320

  5. A framework for interpreting the leucine-rich repeats of the Listeria internalins

    PubMed Central

    Marino, Michael; Braun, Laurence; Cossart, Pascale; Ghosh, Partho

    2000-01-01

    The surface protein InlB of the bacterial pathogen Listeria monocytogenes is required for inducing phagocytosis in various nonphagocytic mammalian cell types in vitro. InlB causes tyrosine phosphorylation of host cell adaptor proteins, activation of phosphoinositide 3-kinase, and rearrangements of the actin cytoskeleton. These events lead to phagocytic uptake of the bacterium by the host cell. InlB belongs to the internalin family of Listeria proteins, which also includes InlA, another surface protein involved in host cell invasion. The internalins are the largest class of bacterial proteins containing leucine-rich repeats (LRR), a motif associated with protein–protein interactions. The LRR motif is found in a functionally diverse array of proteins, including those involved in the plant immune system and in the mammalian innate immune response. Structural and functional interpretations of the sequences of internalin family members are presented in light of the recently determined x-ray crystal structure of the InlB LRR domain. PMID:10922035

  6. Neuronal leucine-rich repeat 1 negatively regulates anaplastic lymphoma kinase in neuroblastoma.

    PubMed

    Satoh, Shunpei; Takatori, Atsushi; Ogura, Atsushi; Kohashi, Kenichi; Souzaki, Ryota; Kinoshita, Yoshiaki; Taguchi, Tomoaki; Hossain, Md Shamim; Ohira, Miki; Nakamura, Yohko; Nakagawara, Akira

    2016-01-01

    In neuroblastoma (NB), one of the most common paediatric solid tumours, activation of anaplastic lymphoma kinase (ALK) is often associated with poor outcomes. Although genetic studies have identified copy number alteration and nonsynonymous mutations of ALK, the regulatory mechanism of ALK signalling at protein levels is largely elusive. Neuronal leucine-rich repeat 1 (NLRR1) is a type 1 transmembrane protein that is highly expressed in unfavourable NB and potentially influences receptor tyrosine kinase signalling. Here, we showed that NLRR1 and ALK exhibited a mutually exclusive expression pattern in primary NB tissues by immunohistochemistry. Moreover, dorsal root ganglia of Nlrr1+/+ and Nlrr1-/- mice displayed the opposite expression patterns of Nlrr1 and Alk. Of interest, NLRR1 physically interacted with ALK in vitro through its extracellular region. Notably, the NLRR1 ectodomain impaired ALK phosphorylation and proliferation of ALK-mutated NB cells. A newly identified cleavage of the NLRR1 ectodomain also supported NLRR1-mediated ALK signal regulation in trans. Thus, we conclude that NLRR1 appears to be an extracellular negative regulator of ALK signalling in NB and neuronal development. Our findings may be beneficial to comprehend NB heterogeneity and to develop a novel therapy against unfavourable NB. PMID:27604320

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

    PubMed Central

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

    2011-01-01

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

  8. The Protein Synthesis Inhibitor Blasticidin S Enters Mammalian Cells via Leucine-rich Repeat-containing Protein 8D

    PubMed Central

    Lee, Clarissa C.; Freinkman, Elizaveta; Sabatini, David M.; Ploegh, Hidde L.

    2014-01-01

    Leucine-rich repeat-containing 8 (LRRC8) proteins have been identified as putative receptors involved in lymphocyte development and adipocyte differentiation. They remain poorly characterized, and no specific function has been assigned to them. There is no consensus on how this family of proteins might function because homology searches suggest that members of the LRRC8 family act not as plasma membrane receptors, but rather as channels that mediate cell-cell signaling. Here we provide experimental evidence that supports a role for LRRC8s in the transport of small molecules. We show that LRRC8D is a mammalian protein required for the import of the antibiotic blasticidin S. We characterize localization and topology of LRRC8A and LRRC8D and demonstrate that LRRC8D interacts with LRRC8A, LRRC8B, and LRRC8C. Given the suggested involvement in solute transport, our results support a model in which LRRC8s form one or more complexes that may mediate cell-cell communication by transporting small solutes. PMID:24782309

  9. Characterization of a novel anther-specific gene encoding a leucine-rich repeat protein in petunia.

    PubMed

    Yue, Y Z; Sun, J; Huang, X; Peng, H; Liu, G F; Hu, H R

    2014-01-01

    In Petunia x hybrida 'Fantasy Red', a leucine-rich repeat (LRR) gene referred to as PhLRR, was identified in a flower bud cDNA library. The open reading frame sequence of PhLRR was 1251 bp, encoding a putative 46.2-kDa protein of 416 amino acids. The PhLRR protein showed high similarity to members of polygalacturonase inhibitor proteins (PGIPs), contained 11 conserved LRR domains, and was an extracellular localization protein. Phylogenetic analysis showed that PhLRR belonged to the same PGIPs subfamily as SHY, indicating that PhLRR may be involved in the development of pollen-like SHY. Expression analysis revealed that PhLRR was abundantly expressed during early stages of flower bud and anther development, while it was not detected in any other examined organs, such as sepals, petals, pistils, roots, stems, leaves, or open flowers. Furthermore, many cis-acting elements (such as AGAAA and GTGA) related to anther-specific gene expression were identified in the PhLRR gene promoter region, indicating that the promoter is also anther-specific. These results suggested that PhLRR is a novel anther-specific gene that may be essential for the early development of anthers. PMID:25501199

  10. Mutational Analysis of the Arabidopsis Nucleotide Binding Site–Leucine-Rich Repeat Resistance Gene RPS2

    PubMed Central

    Tao, Yi; Yuan, Fenghua; Leister, R. Todd; Ausubel, Frederick M.; Katagiri, Fumiaki

    2000-01-01

    Disease resistance proteins containing a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region compose the largest class of disease resistance proteins. These so-called NBS-LRR proteins confer resistance against a wide variety of phytopathogens. To help elucidate the mechanism by which NBS-LRR proteins recognize and transmit pathogen-derived signals, we analyzed mutant versions of the Arabidopsis NBS-LRR protein RPS2. The RPS2 gene confers resistance against Pseudomonas syringae strains carrying the avirulence gene avrRpt2. The activity of RPS2 derivatives in response to AvrRpt2 was measured by using a functional transient expression assay or by expressing the mutant proteins in transgenic plants. Directed mutagenesis revealed that the NBS and an N-terminal leucine zipper (LZ) motif were critical for RPS2 function. Mutations near the N terminus, including an LZ mutation, resulted in proteins that exhibited a dominant negative effect on wild-type RPS2. Scanning the RPS2 molecule with a small in-frame internal deletion demonstrated that RPS2 does not have a large dispensable region. Overexpression of RPS2 in the transient assay in the absence of avrRpt2 also led to an apparent resistant response, presumably a consequence of a low basal activity of RPS2. The NBS and LZ were essential for this overdose effect, whereas the entire LRR was dispensable. RPS2 interaction with a 75-kD protein (p75) required an N-terminal portion of RPS2 that is smaller than the region required for the overdose effect. These findings illuminate the pathogen recognition mechanisms common among NBS-LRR proteins. PMID:11148296

  11. Leucine-rich repeat kinase 2 deficiency is protective in rhabdomyolysis-induced kidney injury.

    PubMed

    Boddu, Ravindra; Hull, Travis D; Bolisetty, Subhashini; Hu, Xianzhen; Moehle, Mark S; Daher, João Paulo Lima; Kamal, Ahmed Ibrahim; Joseph, Reny; George, James F; Agarwal, Anupam; Curtis, Lisa M; West, Andrew B

    2015-07-15

    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson's disease, and LRRK2 is also linked to Crohn's and Hansen's disease. LRRK2 is expressed in many organs in mammals but is particularly abundant in the kidney. We find that LRRK2 protein is predominantly localized to collecting duct cells in the rat kidney, with much lower expression in other kidney cells. While genetic knockout (KO) of LRRK2 expression is well-tolerated in mice and rats, a unique age-dependent pathology develops in the kidney. The cortex and medulla of LRRK2 KO rat kidneys become darkly pigmented in early adulthood, yet aged animals display no overt signs of kidney failure. Accompanying the dark pigment we find substantial macrophage infiltration in LRRK2 KO kidneys, suggesting the presence of chronic inflammation that may predispose to kidney disease. Unexpectedly, the dark kidneys of the LRRK2 KO rats are highly resistant to rhabdomyolysis-induced acute kidney injury compared with wild-type rats. Biochemical profiling of the LRRK2 KO kidneys using immunohistochemistry, proteomic and lipidomic analyses show a massive accumulation of hemoglobin and lipofuscin in renal tubules that account for the pigmentation. The proximal tubules demonstrate a corresponding up-regulation of the cytoprotective protein heme oxygenase-1 (HO-1) which is capable of mitigating acute kidney injury. The unusual kidney pathology of LRRK2 KO rats highlights several novel physiological roles for LRRK2 and provides indirect evidence for HO-1 expression as a protective mechanism in acute kidney injury in LRRK2 deficiency. PMID:25904107

  12. Point mutation in a leucine-rich repeat of platelet glycoprotein Ib alpha resulting in the Bernard-Soulier syndrome.

    PubMed Central

    Ware, J; Russell, S R; Marchese, P; Murata, M; Mazzucato, M; De Marco, L; Ruggeri, Z M

    1993-01-01

    Leucine-rich repeats are a conserved structural motif, of yet undefined significance, found in a group of proteins from different species. Among these are the four components of the human platelet glycoprotein Ib-IX-V complex, a membrane receptor that performs an essential role in the thrombogenic function of platelets by interacting with the adhesive protein, von Willebrand factor. We have found that a single amino acid substitution (Ala156-->Val) within one of the six leucine-rich repeats in the alpha-subunit of glycoprotein Ib results in a variant form of the congenital bleeding disorder, Bernard-Soulier syndrome, characterized by giant dysfunctional platelets. Genetic studies of the propositus and his family members were complemented by immunological and functional analysis of expressed recombinant GP Ib alpha fragments to demonstrate that the observed mutation is the cause of defective von Willebrand factor binding. These studies define the molecular basis of the Bernard-Soulier syndrome within this family and demonstrate that structural integrity of a leucine-rich repeat is necessary for normal function of the glycoprotein Ib-IX-V receptor complex and, possibly, for normal platelet morphology. Images PMID:7690774

  13. Deletion of internal structured repeats increases the stability of a leucine-rich repeat protein, YopM

    PubMed Central

    Barrick, Doug

    2011-01-01

    Mapping the stability distributions of proteins in their native folded states provides a critical link between structure, thermodynamics, and function. Linear repeat proteins have proven more amenable to this kind of mapping than globular proteins. C-terminal deletion studies of YopM, a large, linear leucine-rich repeat (LRR) protein, show that stability is distributed quite heterogeneously, yet a high level of cooperativity is maintained [1]. Key components of this distribution are three interfaces that strongly stabilize adjacent sequences, thereby maintaining structural integrity and promoting cooperativity. To better understand the distribution of interaction energy around these critical interfaces, we studied internal (rather than terminal) deletions of three LRRs in this region, including one of these stabilizing interfaces. Contrary to our expectation that deletion of structured repeats should be destabilizing, we find that internal deletion of folded repeats can actually stabilize the native state, suggesting that these repeats are destabilizing, although paradoxically, they are folded in the native state. We identified two residues within this destabilizing segment that deviate from the consensus sequence at a position that normally forms a stacked leucine ladder in the hydrophobic core. Replacement of these nonconsensus residues with leucine is stabilizing. This stability enhancement can be reproduced in the context of nonnative interfaces, but it requires an extended hydrophobic core. Our results demonstrate that different LRRs vary widely in their contribution to stability, and that this variation is context-dependent. These two factors are likely to determine the types of rearrangements that lead to folded, functional proteins, and in turn, are likely to restrict the pathways available for the evolution of linear repeat proteins. PMID:21764506

  14. Drosophila chaoptin, a member of the leucine-rich repeat family, is a photoreceptor cell-specific adhesion molecule.

    PubMed Central

    Krantz, D E; Zipursky, S L

    1990-01-01

    Drosophila chaoptin, required for photoreceptor cell morphogenesis, is a member of the leucine-rich repeat family of proteins. On the basis of biochemical and genetic analyses we previously proposed that chaoptin might function as a cell adhesion molecule. To test this hypothesis, chaoptin cDNA driven by the hsp 70 promoter was transfected into non-self-adherent Drosophila Schneider line 2 (S2) cells. Following heat shock induction of chaoptin expression, the transfected S2 cells formed multicellular aggregates. Mixing experiments of chaoptin expressing and non-expressing cells suggest that chaoptin expressing cells adhere homotypically. Previously it was shown that chaoptin is exclusively localized to photoreceptor cells. Thus, chaoptin is a cell-type-specific adhesion molecule. Biochemical analyses presented in this paper demonstrate that chaoptin is linked to the extracellular surface of the plasma membrane by covalent attachment to glycosyl-phosphatidylinositol. We propose that chaoptin and several other members of the leucine-rich repeat family of proteins define a new class of cell adhesion molecules. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 8. PMID:2189727

  15. Evolutionary Dynamics of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) Subfamily in Angiosperms1[OPEN

    PubMed Central

    Dufayard, Jean-François; Chantret, Nathalie

    2016-01-01

    Gene duplications are an important factor in plant evolution, and lineage-specific expanded (LSE) genes are of particular interest. Receptor-like kinases expanded massively in land plants, and leucine-rich repeat receptor-like kinases (LRR-RLK) constitute the largest receptor-like kinases family. Based on the phylogeny of 7,554 LRR-RLK genes from 31 fully sequenced flowering plant genomes, the complex evolutionary dynamics of this family was characterized in depth. We studied the involvement of selection during the expansion of this family among angiosperms. LRR-RLK subgroups harbor extremely contrasting rates of duplication, retention, or loss, and LSE copies are predominantly found in subgroups involved in environmental interactions. Expansion rates also differ significantly depending on the time when rounds of expansion or loss occurred on the angiosperm phylogenetic tree. Finally, using a dN/dS-based test in a phylogenetic framework, we searched for selection footprints on LSE and single-copy LRR-RLK genes. Selective constraint appeared to be globally relaxed at LSE genes, and codons under positive selection were detected in 50% of them. Moreover, the leucine-rich repeat domains, and specifically four amino acids in them, were found to be the main targets of positive selection. Here, we provide an extensive overview of the expansion and evolution of this very large gene family. PMID:26773008

  16. Leucine-rich repeat-containing G-protein-coupled receptor 5 is associated with invasion, metastasis, and could be a potential therapeutic target in human gastric cancer

    PubMed Central

    Xi, H Q; Cai, A Z; Wu, X S; Cui, J X; Shen, W S; Bian, S B; Wang, N; Li, J Y; Lu, C R; Song, Z; Wei, B; Chen, L

    2014-01-01

    Background: Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5), which is identified as a novel intestinal stem cell marker, is overexpressed in various tumours. In this study, we explore Lgr5 expression in gastric carcinoma and analyse its role in invasion, metastasis, and prognosis in carcinoma. Methods: A combination of immunohistochemistry, western blotting, and quantitative reverse transcription–polymerase chain reaction were used to detect mRNA and protein expression levels of Lgr5 and matrix metalloproteinase 2 (MMP2). Small interfering RNA against Lgr5 was designed, synthesised, and transfected into AGS cells. The effects of Lgr5 siRNA on cell invasion were detected by transwell invasion chamber assay and wound healing assay. Results: Leucine-rich repeat-containing G-protein-coupled receptor 5 expression was significantly higher in gastric carcinomas than in normal mucosa. Leucine-rich repeat-containing G-protein-coupled receptor 5 expression positively correlated with the depth of invasion, lymph node metastasis, distance of metastasis, and MMP2 expression levels. Multivariate analysis showed that Lgr5 had an independent effect on survival, and that it positively correlated with MMP2. Leucine-rich repeat-containing G-protein-coupled receptor 5 siRNAs inhibited Lgr5 mRNA and protein expression. Transwell assays indicated that these siRNAs resulted in significantly fewer cells migrating through the polycarbonate membrane, and wound healing assay also indicated that siRNAs decreased the migration of cells. Inhibition of Lgr5 resulted in a significant decrease in MMP2 and β-catenin levels compared with those in controls. Conclusions: Leucine-rich repeat-containing G-protein-coupled receptor 5 was correlated with invasion and metastasis. Leucine-rich repeat-containing G-protein-coupled receptor 5 inhibition could serve as a novel therapeutic approach. PMID:24594994

  17. An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens

    PubMed Central

    Kalunke, Raviraj M.; Tundo, Silvio; Benedetti, Manuel; Cervone, Felice; De Lorenzo, Giulia; D'Ovidio, Renato

    2015-01-01

    Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases secreted by microbial pathogens and insects. These ubiquitous inhibitors have a leucine-rich repeat structure that is strongly conserved in monocot and dicot plants. Previous reviews have summarized the importance of PGIP in plant defense and the structural basis of PG-PGIP interaction; here we update the current knowledge about PGIPs with the recent findings on the composition and evolution of pgip gene families, with a special emphasis on legume and cereal crops. We also update the information about the inhibition properties of single pgip gene products against microbial PGs and the results, including field tests, showing the capacity of PGIP to protect crop plants against fungal, oomycetes and bacterial pathogens. PMID:25852708

  18. Leucine-rich repeat kinase 2 interacts with p21-activated kinase 6 to control neurite complexity in mammalian brain.

    PubMed

    Civiero, Laura; Cirnaru, Maria Daniela; Beilina, Alexandra; Rodella, Umberto; Russo, Isabella; Belluzzi, Elisa; Lobbestael, Evy; Reyniers, Lauran; Hondhamuni, Geshanthi; Lewis, Patrick A; Van den Haute, Chris; Baekelandt, Veerle; Bandopadhyay, Rina; Bubacco, Luigi; Piccoli, Giovanni; Cookson, Mark R; Taymans, Jean-Marc; Greggio, Elisa

    2015-12-01

    Leucine-rich repeat kinase 2 (LRRK2) is a causative gene for Parkinson's disease, but the physiological function and the mechanism(s) by which the cellular activity of LRRK2 is regulated are poorly understood. Here, we identified p21-activated kinase 6 (PAK6) as a novel interactor of the GTPase/ROC domain of LRRK2. p21-activated kinases are serine-threonine kinases that serve as targets for the small GTP binding proteins Cdc42 and Rac1 and have been implicated in different morphogenetic processes through remodeling of the actin cytoskeleton such as synapse formation and neuritogenesis. Using an in vivo neuromorphology assay, we show that PAK6 is a positive regulator of neurite outgrowth and that LRRK2 is required for this function. Analyses of post-mortem brain tissue from idiopathic and LRRK2 G2019S carriers reveal an increase in PAK6 activation state, whereas knock-out LRRK2 mice display reduced PAK6 activation and phosphorylation of PAK6 substrates. Taken together, these results support a critical role of LRRK2 GTPase domain in cytoskeletal dynamics in vivo through the novel interactor PAK6, and provide a valuable platform to unravel the mechanism underlying LRRK2-mediated pathophysiology. We propose p21-activated kinase 6 (PAK6) as a novel interactor of leucine-rich repeat kinase 2 (LRRK2), a kinase involved in Parkinson's disease (PD). In health, PAK6 regulates neurite complexity in the brain and LRRK2 is required for its function, (a) whereas PAK6 is aberrantly activated in LRRK2-linked PD brain (b) suggesting that LRRK2 toxicity is mediated by PAK6. PMID:26375402

  19. Thermodynamics, kinetics, and salt-dependence of folding of YopM, a large leucine-rich repeat protein

    PubMed Central

    Kloss, Ellen; Barrick, Doug

    2011-01-01

    Small globular proteins have many contacts between residues that are distant in primary sequence. These contacts create a complex network between sequence-distant segments of secondary structure, which may be expected to promote the cooperative folding of globular proteins. Although repeat proteins, which are made up of tandem modular units, lack sequence-distant contacts, several of considerable length have been shown to undergo cooperative two-state folding. To explore the limits of cooperativity in repeat proteins, we have studied the unfolding of YopM, a leucine-rich repeat (LRR) protein of over 400 residues. Despite its large size and modular architecture (15 repeats), YopM equilibrium unfolding is highly cooperative, and shows a very strong dependence on urea concentration. In contrast, kinetic studies of YopM folding indicate a mechanism that includes one or more transient intermediates. The urea dependence of the folding and unfolding rates suggests a relatively small transition state ensemble. As with the urea dependence, we have found an extreme dependence of the free energy of unfolding on salt concentration. This salt dependence likely results from general screening of a large number of unfavorable columbic interactions in the folded state, rather than from specific cation binding. PMID:18793647

  20. Wound induced Beta vulgaris polygalacturonase-inhibiting protein genes encode a longer leucine-rich repeat domain and inhibit fungal polygalacturonases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defense. Sugar beet (Beta vulgaris L.) PGIP genes, BvPGIP1, BvPGIP2 and BvPGIP3, were isolated from two breeding lines, F1016 and F1010. Full-length cDNA sequences of the three BvPGIP genes encod...

  1. Functional Analysis and Phosphorylation Site Mapping of Leucine-Rich Repeat Receptor-Like Kinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The completed genome sequences of Arabidopsis thaliana and rice have revealed very large multi-gene families encoding predicted proteins with an organization of functional domains similar to that of animal receptor kinases, including a putative extracellular ligand-binding domain, a single-pass tran...

  2. F-box and Leucine-rich Repeat Protein 5 (FBXL5): sensing intracellular iron and oxygen

    PubMed Central

    Ruiz, Julio C.; Bruick, Richard K.

    2014-01-01

    Though essential for many vital biological processes, excess iron results in the formation of damaging reactive oxygen species (ROS). Therefore, iron metabolism must be tightly regulated. F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, regulates cellular and systemic iron homeostasis by facilitating iron regulatory protein 2 (IRP2) degradation. FBXL5 possesses an N-terminal hemerythrin (Hr)-like domain that mediates its own differential stability by switching between two different conformations to communicate cellular iron availability. In addition, the FBXL5-Hr domain also senses O2 availability, albeit by a distinct mechanism. Mice lacking FBXL5 fail to sense intracellular iron levels and die in utero due to iron overload and exposure to damaging levels of oxidative stress. By closely monitoring intracellular levels of iron and oxygen, FBLX5 prevents the formation of conditions that favor ROS formation. These findings suggest that FBXL5 is essential for the maintenance of iron homeostasis and is a key sensor of bioavailable iron. Here, we describe the iron and oxygen sensing mechanisms of the FBXL5 Hr-like domain and its role in mediating ROS biology. PMID:24508277

  3. F-box and leucine-rich repeat protein 5 (FBXL5): sensing intracellular iron and oxygen.

    PubMed

    Ruiz, Julio C; Bruick, Richard K

    2014-04-01

    Though essential for many vital biological processes, excess iron results in the formation of damaging reactive oxygen species (ROS). Therefore, iron metabolism must be tightly regulated. F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, regulates cellular and systemic iron homeostasis by facilitating iron regulatory protein 2 (IRP2) degradation. FBXL5 possesses an N-terminal hemerythrin (Hr)-like domain that mediates its own differential stability by switching between two different conformations to communicate cellular iron availability. In addition, the FBXL5-Hr domain also senses O2 availability, albeit by a distinct mechanism. Mice lacking FBXL5 fail to sense intracellular iron levels and die in utero due to iron overload and exposure to damaging levels of oxidative stress. By closely monitoring intracellular levels of iron and oxygen, FBLX5 prevents the formation of conditions that favor ROS formation. These findings suggest that FBXL5 is essential for the maintenance of iron homeostasis and is a key sensor of bioavailable iron. Here, we describe the iron and oxygen sensing mechanisms of the FBXL5 Hr-like domain and its role in mediating ROS biology. PMID:24508277

  4. Role of leucine-rich pentatricopeptide repeat motif-containing protein (LRPPRC) for anti-apoptosis and tumourigenesis in cancers.

    PubMed

    Tian, Tian; Ikeda, Jun-ichiro; Wang, Yi; Mamat, Suhana; Luo, Wenjuan; Aozasa, Katsuyuki; Morii, Eiichi

    2012-10-01

    Due to accelerated energy consumption, enhanced function of mitochondria in tumour cells compared to normal cells is prerequisite for tumour development. Leucine-rich pentatricopeptide repeat motif-containing protein (LRPPRC) regulates the expression of all mitochondrial DNA-encoded mRNAs, thus plays an important role in the mitochondrial function. LRPPRC is abundantly expressed in the side population of lung adenocarcinoma cell lines, where cancer stem cells are enriched. However, the role of LRPPRC in tumour development remained to be clarified in detail. Here, the expression of LRPPRC was examined in various types of tumours, such as lung adenocarcinoma, oesophageal squamous cell carcinoma, stomach, colon, mammary and endometrial adenocarcinoma, and lymphoma. Immunohistochemistry revealed that all kinds of examined tumours abundantly expressed LRPPRC. In contrast, surrounding non-neoplastic cells hardly expressed LRPPRC. The knocked-down expression of LRPPRC in lung adenocarcinoma cells did not affect amount of side population and activity of aldehyde dehydrogenase 1, known to be highly expressed in cancer stem cells of the lung. However, the knocked-down expression of LRPPRC reduced the abilities for anti-apoptosis, invasion and in vitro colony formation in lung adenocarcinoma, as well as Hodgkin lymphoma cells. Double staining of LRPPRC with active caspase-3 in clinical samples of lung adenocarcinoma revealed that apoptotic cells were hardly observed in LRPPRC-expressing tumours. These findings indicate that LRPPRC played an important role in tumourigenesis through the resistance to apoptosis and high invasive activity. PMID:22326293

  5. TRIP: a novel double stranded RNA binding protein which interacts with the leucine rich repeat of flightless I.

    PubMed Central

    Wilson, S A; Brown, E C; Kingsman, A J; Kingsman, S M

    1998-01-01

    A northwestern screen of a CHO-K1 cell line cDNA library with radiolabelled HIV-1 TAR RNA identified a novel TAR RNA interacting protein, TRIP. The human trip cDNA was also cloned and its expression is induced by phorbol esters. The N-terminus of TRIP shows high homology to the coiled coil domain of FLAP, a protein which binds the leucine-rich repeat (LRR) of Flightless I (FLI) and the interaction of TRIP with the FLI LRR has been confirmed in vitro . TRIP does not bind single stranded DNA or RNA significantly and binds double stranded DNA weakly. In contrast, TRIP binds double stranded RNA with high affinity and two molecules of TRIP bind the TAR stem. The RNA binding domain has been identified and encompasses a lysine-rich motif. A TRIP-GFP fusion is localised in the cytoplasm and excluded from the nucleus. FLI has a C-terminal gelsolin-like domain which binds actin and therefore the association of TRIP with the FLI LRR may provide a link between the actin cytoskeleton and RNA in mammalian cells. PMID:9671805

  6. TRIP: a novel double stranded RNA binding protein which interacts with the leucine rich repeat of flightless I.

    PubMed

    Wilson, S A; Brown, E C; Kingsman, A J; Kingsman, S M

    1998-08-01

    A northwestern screen of a CHO-K1 cell line cDNA library with radiolabelled HIV-1 TAR RNA identified a novel TAR RNA interacting protein, TRIP. The human trip cDNA was also cloned and its expression is induced by phorbol esters. The N-terminus of TRIP shows high homology to the coiled coil domain of FLAP, a protein which binds the leucine-rich repeat (LRR) of Flightless I (FLI) and the interaction of TRIP with the FLI LRR has been confirmed in vitro . TRIP does not bind single stranded DNA or RNA significantly and binds double stranded DNA weakly. In contrast, TRIP binds double stranded RNA with high affinity and two molecules of TRIP bind the TAR stem. The RNA binding domain has been identified and encompasses a lysine-rich motif. A TRIP-GFP fusion is localised in the cytoplasm and excluded from the nucleus. FLI has a C-terminal gelsolin-like domain which binds actin and therefore the association of TRIP with the FLI LRR may provide a link between the actin cytoskeleton and RNA in mammalian cells. PMID:9671805

  7. Pleckstrin homology domain leucine-rich repeat protein phosphatases set the amplitude of receptor tyrosine kinase output

    PubMed Central

    Reyes, Gloria; Niederst, Matt; Cohen-Katsenelson, Ksenya; Stender, Joshua D.; Kunkel, Maya T.; Chen, Muhan; Brognard, John; Sierecki, Emma; Gao, Tianyan; Nowak, Dawid G.; Trotman, Lloyd C.; Glass, Christopher K.; Newton, Alexandra C.

    2014-01-01

    Growth factor receptor levels are aberrantly high in diverse cancers, driving the proliferation and survival of tumor cells. Understanding the molecular basis for this aberrant elevation has profound clinical implications. Here we show that the pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) suppresses receptor tyrosine kinase (RTK) signaling output by a previously unidentified epigenetic mechanism unrelated to its previously described function as the hydrophobic motif phosphatase for the protein kinase AKT, protein kinase C, and S6 kinase. Specifically, we show that nuclear-localized PHLPP suppresses histone phosphorylation and acetylation, in turn suppressing the transcription of diverse growth factor receptors, including the EGF receptor. These data uncover a much broader role for PHLPP in regulation of growth factor signaling beyond its direct inactivation of AKT: By suppressing RTK levels, PHLPP dampens the downstream signaling output of two major oncogenic pathways, the PI3 kinase/AKT and the Rat sarcoma (RAS)/ERK pathways. Our data are consistent with a model in which PHLPP modifies the histone code to control the transcription of RTKs. PMID:25201979

  8. Paracrine regulation of growth factor signaling by shed leucine-rich repeats and immunoglobulin-like domains 1

    SciTech Connect

    Yi, Wei; Holmlund, Camilla; Nilsson, Jonas; Inui, Shigeki; Lei, Ting; Itami, Satoshi; Henriksson, Roger; Hedman, Hakan

    2011-02-15

    Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a recently discovered negative regulator of growth factor signaling. The LRIG1 integral membrane protein has been demonstrated to regulate various oncogenic receptor tyrosine kinases, including epidermal growth factor (EGF) receptor (EGFR), by cell-autonomous mechanisms. Here, we investigated whether LRIG1 ectodomains were shed, and if LRIG1 could regulate cell proliferation and EGF signaling in a paracrine manner. Cells constitutively shed LRIG1 ectodomains in vitro, and shedding was modulated by known regulators of metalloproteases, including the ADAM17 specific inhibitor TAPI-2. Furthermore, shedding was enhanced by ectopic expression of Adam17. LRIG1 ectodomains appeared to be shed in vivo, as well, as demonstrated by immunoblotting of mouse and human tissue lysates. Ectopic expression of LRIG1 in lymphocytes suppressed EGF signaling in co-cultured fibroblastoid cells, demonstrating that shed LRIG1 ectodomains can function in a paracrine fashion. Purified LRIG1 ectodomains suppressed EGF signaling without any apparent downregulation of EGFR levels. Taken together, the results show that the LRIG1 ectodomain can be proteolytically shed and can function as a non-cell-autonomous regulator of growth factor signaling. Thus, LRIG1 or its ectodomain could have therapeutic potential in the treatment of growth factor receptor-dependent cancers.

  9. GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats.

    PubMed Central

    Flick, J S; Johnston, M

    1991-01-01

    Growth of the yeast Saccharomyces cerevisiae on glucose leads to repression of transcription of many genes required for alternative carbohydrate metabolism. The GRR1 gene appears to be of central importance to the glucose repression mechanism, because mutations in GRR1 result in a pleiotropic loss of glucose repression (R. Bailey and A. Woodword, Mol. Gen. Genet. 193:507-512, 1984). We have isolated the GRR1 gene and determined that null mutants are viable and display a number of growth defects in addition to the loss of glucose repression. Surprisingly, grr1 mutations convert SUC2, normally a glucose-repressed gene, into a glucose-induced gene. GRR1 encodes a protein of 1,151 amino acids that is expressed constitutively at low levels in yeast cells. GRR1 protein contains 12 tandem repeats of a sequence similar to leucine-rich motifs found in other proteins that may mediate protein-protein interactions. Indeed, cell fractionation studies are consistent with this view, suggesting that GRR1 protein is tightly associated with a particulate protein fraction in yeast extracts. The combined genetic and molecular data are consistent with the idea that GRR1 protein is a primary response element in the glucose repression pathway and is required for the generation or interpretation of the signal that induces glucose repression. Images PMID:1922034

  10. Identification of protein phosphatase 2A as an interacting protein of leucine-rich repeat kinase 2.

    PubMed

    Athanasopoulos, Panagiotis S; Jacob, Wright; Neumann, Sebastian; Kutsch, Miriam; Wolters, Dirk; Tan, Eng K; Bichler, Zoë; Herrmann, Christian; Heumann, Rolf

    2016-06-01

    Mutations in the gene coding for the multi-domain protein leucine-rich repeat kinase 2 (LRRK2) are the leading cause of genetically inherited Parkinson's disease (PD). Two of the common found mutations are the R1441C and G2019S. In this study we identified protein phosphatase 2A (PP2A) as an interacting partner of LRRK2. We were able to demonstrate that the Ras of complex protein (ROC) domain is sufficient to interact with the three subunits of PP2A in human neuroblastoma SH-SY5Y cells and in HeLa cells. The alpha subunit of PP2A is interacting with LRRK2 in the perinuclear region of HeLa cells. Silencing the catalytic subunit of PP2A by shRNA aggravated cellular degeneration induced by the pathogenic R1441C-LRRK2 mutant expressed in neuroblastoma SH-SY5Y cells. A similar enhancement of apoptotic nuclei was observed by downregulation of the catalytic subunit of PP2A in cultured cortical cells derived from neurons overexpressing the pathogenic mutant G2019S-LRRK2. Conversely, pharmacological activation of PP2A by sodium selenate showed a partial neuroprotection from R1441C-LRRK2-induced cellular degeneration. All these data suggest that PP2A is a new interacting partner of LRRK2 and reveal the importance of PP2A as a potential therapeutic target in PD. PMID:26894577

  11. Leucine-rich repeat kinase 2 regulates Sec16A at ER exit sites to allow ER–Golgi export

    PubMed Central

    Cho, Hyun Jin; Yu, Jia; Xie, Chengsong; Rudrabhatla, Parvathi; Chen, Xi; Wu, Junbing; Parisiadou, Loukia; Liu, Guoxiang; Sun, Lixin; Ma, Bo; Ding, Jinhui; Liu, Zhihua; Cai, Huaibin

    2014-01-01

    Leucine-rich repeat kinase 2 (LRRK2) has been associated with Parkinson’s disease (PD) and other disorders. However, its normal physiological functions and pathogenic properties remain elusive. Here we show that LRRK2 regulates the anterograde ER–Golgi transport through anchoring Sec16A at the endoplasmic reticulum exit sites (ERES). LRRK2 interacted and co-localized with Sec16A, a key protein in the formation of ERES. Lrrk2 depletion caused a dispersion of Sec16A from ERES and impaired ER export. In neurons, LRRK2 and Sec16A showed extensive co-localization at the dendritic ERES (dERES) that locally regulate the transport of proteins to the dendritic spines. A loss of Lrrk2 affected the association of Sec16A with dERES and impaired the activity-dependent targeting of glutamate receptors onto the cell/synapse surface. Furthermore, the PD-related LRRK2 R1441C missense mutation in the GTPase domain interfered with the interaction of LRRK2 with Sec16A and also affected ER–Golgi transport, while LRRK2 kinase activity was not required for these functions. Therefore, our findings reveal a new physiological function of LRRK2 in ER–Golgi transport, suggesting ERES dysfunction may contribute to the pathogenesis of PD. PMID:25201882

  12. Leucine-rich repeat kinase 2 functionally interacts with microtubules and kinase-dependently modulates cell migration.

    PubMed

    Caesar, Mareike; Zach, Susanne; Carlson, Coby B; Brockmann, Kathrin; Gasser, Thomas; Gillardon, Frank

    2013-06-01

    Recent studies indicate that the Parkinson's disease-linked leucine-rich repeat kinase 2 (LRRK2) modulates cytoskeletal functions by regulating actin and tubulin dynamics, thereby affecting neurite outgrowth. By interactome analysis we demonstrate that the binding of LRRK2 to tubulins is significantly enhanced by pharmacological LRRK2 inhibition in cells. Co-incubation of LRRK2 with microtubules increased the LRRK2 GTPase activity in a cell-free assay. Destabilization of microtubules causes a rapid decrease in cellular LRRK2(S935) phosphorylation indicating a decreased LRRK2 kinase activity. Moreover, both human LRRK2(G2019S) fibroblasts and mouse LRRK2(R1441G) fibroblasts exhibit alterations in cell migration in culture. Treatment of mouse fibroblasts with the selective LRRK2 inhibitor LRRK2-IN1 reduces cell motility. These findings suggest that LRRK2 and microtubules mutually interact both in non-neuronal cells and in neurons, which might contribute to our understanding of its pathogenic effects in Parkinson's disease. PMID:23318930

  13. Re-examination of the dimerization state of leucine-rich repeat kinase 2: predominance of the monomeric form.

    PubMed

    Ito, Genta; Iwatsubo, Takeshi

    2012-02-01

    Mutations in the LRRK2 (leucine-rich repeat kinase 2) gene have been identified in PARK8, a major form of autosomal-dominantly inherited familial Parkinson's disease, although the biochemical properties of LRRK2 are not fully understood. It has been proposed that LRRK2 predominantly exists as a homodimer on the basis of the observation that LRRK2, with a theoretical molecular mass of 280 kDa, migrates at 600 kDa (p600 LRRK2) on native polyacrylamide gels. In the present study, we biochemically re-examined the nature of p600 LRRK2 and found that p600 LRRK2 was fractionated with a single peak at ~272 kDa by ultracentrifugation on a glycerol gradient. In addition, p600 LRRK2 behaved similarly to monomeric proteins upon two-dimensional electrophoretic separation. These results suggested a monomeric composition of p600 LRRK2 within cells. The p600 LRRK2 exhibited kinase activity as well as GTP-binding activity, and forced dimerization of LRRK2 neither upregulated its kinase activity nor altered its subcellular localization. Collectively, we conclude that the monomer form of LRRK2 is predominant within cells, and that dimerization is dispensable for its enzymatic activity. PMID:22047502

  14. Structural features of helical secondary structures and leucine-rich repeat superhelix in proteins as revealed by HELFIT analyses

    NASA Astrophysics Data System (ADS)

    Matsushima, Norio; Enkhbayar, Purevjav

    2012-09-01

    The HELFIT program determines the helical parameters - pitch, residues per turn (n), radius, and handedness - and p = rmsd / (N - 1)1/2 estimating helical regularity, where "rmsd" is the root mean square deviation from the best fit helix or superhelix and "N" is helix/superhelix length. Helical secondary structures - α-helix and 310-helix - and solenoid structures of leucine rich repeats (LRRs) in The Protein Data Bank (PDB) were analyzed by the HELFIT program. The results indicate that the definition of 310-helices in terms of average, uniform dihedral angles is not appropriate and that it is inherently unstable for a polypeptide to form an extended, regular 310-helix. The 310-helices observed in proteins are better referred to parahelices. A modification of the α-helix, termed the ω-helix, that has four residues in one turn of a helix, has been identified only in synthetic polypeptides. The results also demonstrate that the right-handed ω-helix occur really in proteins. The solenoid structures of LRR domains in brasinosteroid insensitive 1 (BRI1), internalin J (InlJ), and internalin A (InlA) are well represented by a superhelix rather than by a circular arc.

  15. Uncovering the dynamic evolution of nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes in Brassicaceae.

    PubMed

    Zhang, Yan-Mei; Shao, Zhu-Qing; Wang, Qiang; Hang, Yue-Yu; Xue, Jia-Yu; Wang, Bin; Chen, Jian-Qun

    2016-02-01

    Plant genomes harbor dozens to hundreds of nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes; however, the long-term evolutionary history of these resistance genes has not been fully understood. This study focuses on five Brassicaceae genomes and the Carica papaya genome to explore changes in NBS-LRR genes that have taken place in this Rosid II lineage during the past 72 million years. Various numbers of NBS-LRR genes were identified from Arabidopsis lyrata (198), A. thaliana (165), Brassica rapa (204), Capsella rubella (127), Thellungiella salsuginea (88), and C. papaya (51). In each genome, the identified NBS-LRR genes were found to be unevenly distributed among chromosomes and most of them were clustered together. Phylogenetic analysis revealed that, before and after Brassicaceae speciation events, both toll/interleukin-1 receptor-NBS-LRR (TNL) genes and non-toll/interleukin-1 receptor-NBS-LRR (nTNL) genes exhibited a pattern of first expansion and then contraction, suggesting that both subclasses of NBS-LRR genes were responding to pathogen pressures synchronically. Further, by examining the gain/loss of TNL and nTNL genes at different evolutionary nodes, this study revealed that both events often occurred more drastically in TNL genes. Finally, the phylogeny of nTNL genes suggested that this NBS-LRR subclass is composed of two separate ancient gene types: RPW8-NBS-LRR and Coiled-coil-NBS-LRR. PMID:25926337

  16. Internalin of Listeria monocytogenes with an intact leucine-rich repeat region is sufficient to promote internalization.

    PubMed Central

    Lecuit, M; Ohayon, H; Braun, L; Mengaud, J; Cossart, P

    1997-01-01

    Listeria monocytogenes can use two different surface proteins, internalin (InlA) and InlB, to invade mammalian cells. The exact role of these invasiveness factors in vivo remains to be determined. In cultured cells, InlA is necessary to promote Listeria entry into human epithelial cells, such as Caco-2 cells, whereas InlB is necessary to promote Listeria internalization in several other cell types, including hepatocytes, fibroblasts, and epithelioid cells, such as Vero, HeLa, CHO, or Hep-2 cells. We have recently reported that the InlA receptor on Caco-2 cells is the cell adhesion molecule E-cadherin and demonstrated that nonpermissive fibroblasts become permissive for internalin-mediated entry when transfected with the gene coding for LCAM, the chicken homolog of the human E-cadherin gene. In this study, we demonstrate for the first time that the internalin protein alone is sufficient to promote internalization into cells expressing its receptor. Indeed, internalin confers invasiveness to both Enterococcus faecalis and internalin-coated latex beads. As shown by transmission electron microscopy, these beads were phagocytosed via a "zipper" mechanism similar to that observed during the internalin-E-cadherin-mediated entry of Listeria. Moreover, a functional analysis of internalin demonstrates that its amino-terminal region, encompassing the leucine-rich repeat (LRR) region and the inter-repeat (IR) region, is necessary and sufficient to promote bacterial entry into cells expressing its receptor. Several lines of evidence suggest that the LRR region would interact directly with E-cadherin, whereas the IR region would be required for a proper folding of the LRR region. PMID:9393831

  17. Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase

    SciTech Connect

    Knaap, E. van der; Sauter, M.; Kende, H. . DOE Plant Research Lab.); Song, W.Y.; Ruan, D.L.; Ronald, P.C. . Dept. of Plant Pathology)

    1999-06-01

    The authors identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

  18. Comparative genetics of nucleotide binding site-leucine rich repeat resistance gene homologues in the genomes of two dicotyledons: tomato and arabidopsis.

    PubMed Central

    Pan, Q; Liu, Y S; Budai-Hadrian, O; Sela, M; Carmel-Goren, L; Zamir, D; Fluhr, R

    2000-01-01

    The presence of a single resistance (R) gene allele can determine plant disease resistance. The protein products of such genes may act as receptors that specifically interact with pathogen-derived factors. Most functionally defined R-genes are of the nucleotide binding site-leucine rich repeat (NBS-LRR) supergene family and are present as large multigene families. The specificity of R-gene interactions together with the robustness of plant-pathogen interactions raises the question of their gene number and diversity in the genome. Genomic sequences from tomato showing significant homology to genes conferring race-specific resistance to pathogens were identified by systematically "scanning" the genome using a variety of primer pairs based on ubiquitous NBS motifs. Over 70 sequences were isolated and 10% are putative pseudogenes. Mapping of the amplified sequences on the tomato genetic map revealed their organization as mixed clusters of R-gene homologues that showed in many cases linkage to genetically characterized tomato resistance loci. Interspecific examination within Lycopersicon showed the existence of a null allele. Consideration of the tomato and potato comparative genetic maps unveiled conserved syntenic positions of R-gene homologues. Phylogenetic clustering of R-gene homologues within tomato and other Solanaceae family members was observed but not with R-gene homologues from Arabidopsis thaliana. Our data indicate remarkably rapid evolution of R-gene homologues during diversification of plant families. PMID:10790405

  19. Isolation of a cDNA encoding 5T4 oncofetal trophoblast glycoprotein. An antigen associated with metastasis contains leucine-rich repeats.

    PubMed

    Myers, K A; Rahi-Saund, V; Davison, M D; Young, J A; Cheater, A J; Stern, P L

    1994-03-25

    The monoclonal antibody 5T4 defines a human oncotrophoblast antigen expressed by a variety of carcinomas but with a restricted pattern of expression in normal adult tissues. The 5T4 antigen has been isolated from term placenta as a 72-kDa glycoprotein consisting of a 42-kDa core protein with extensive N-linked glycosylation. A cDNA has been isolated from a human placental library using pools of oligonucleotides based on amino acid sequence obtained from purified 5T4 molecules. The predicted open reading frame encodes a protein of 420 amino acids with a molecular mass of 46 kDa and 8 potential N-glycosylation sites. There are N- and C-terminal hydrophobic segments corresponding to putative signal and membrane anchorage sequences, respectively. Northern analysis has demonstrated a major 2.5-kilobase mRNA present in cell lines serologically reactive with the monoclonal antibody 5T4. Comparison of the 5T4 protein sequence with current sequence data bases has identified the presence of leucine-rich repeats, which are found in a variety of proteins from yeast, insects, and mammals. The 5T4 antigen expression is strongly associated with metastasis in colorectal and gastric cancer, and, hence, the possible functions of the gene product and its relationship to tumor growth and progression are discussed. PMID:8132670

  20. Characterization of microsporidia-induced developmental arrest and a transmembrane leucine-rich repeat protein in Caenorhabditis elegans.

    PubMed

    Luallen, Robert J; Bakowski, Malina A; Troemel, Emily R

    2015-01-01

    Microsporidia comprise a highly diverged phylum of intracellular, eukaryotic pathogens, with some species able to cause life-threatening illnesses in immunocompromised patients. To better understand microsporidian infection in animals, we study infection of the genetic model organism Caenorhabditis elegans and a species of microsporidia, Nematocida parisii, which infects Caenorhabditis nematodes in the wild. We conducted a targeted RNAi screen for host C. elegans genes important for infection and growth of N. parisii, using nematode larval arrest as an assay for infection. Here, we present the results of this RNAi screen, and our analyses on one of the RNAi hits from the screen that was ultimately not corroborated by loss of function mutants. This hit was an RNAi clone against F56A8.3, a conserved gene that encodes a transmembrane protein containing leucine-rich repeats (LRRs), a domain found in numerous pathogen receptors from other systems. This RNAi clone caused C. elegans to be resistant to infection by N. parisii, leading to reduced larval arrest and lower pathogen load. Characterization of the endogenous F56A8.3 protein revealed that it is expressed in the intestine, localized to the membrane around lysosome-related organelles (LROs), and exists in two different protein isoforms in C. elegans. We used the CRISPR-Cas9 system to edit the F56A8.3 locus and created both a frameshift mutant resulting in a truncated protein and a complete knockout mutant. Neither of these mutants was able to recapitulate the infection phenotypes of the RNAi clone, indicating that the RNAi-mediated phenotypes are due to an off-target effect of the RNAi clone. Nevertheless, this study describes microsporidia-induced developmental arrest in C. elegans, presents results from an RNAi screen for host genes important for microsporidian infection, and characterizes aspects of the conserved F56A8.3 gene and its protein product. PMID:25874557

  1. Leucine-Rich Repeat Kinase 2 Modulates Neuroinflammation and Neurotoxicity in Models of Human Immunodeficiency Virus 1-Associated Neurocognitive Disorders

    PubMed Central

    Marker, Daniel F.; Fitzgerald, Tim; Barbieri, Justin; Kim, Christopher S.; Miller-Rhodes, Patrick; Lu, Shao-Ming; Dewhurst, Stephen; Gelbard, Harris A.

    2015-01-01

    Leucine-rich repeat kinase 2 (LRRK2) is the single most common genetic cause of both familial and sporadic Parkinson's disease (PD), both of which share pathogenetic and neurologic similarities with human immunodeficiency virus 1 (HIV-1)-associated neurocognitive disorders (HAND). Pathologic LRRK2 activity may also contribute to neuroinflammation, because microglia lacking LRRK2 exposed to proinflammatory stimuli have attenuated responses. Because microglial activation is a hallmark of HIV-1 neuropathology, we have investigated the role of LRRK2 activation using in vitro and in vivo models of HAND. We hypothesize that LRRK2 is a key modulator of microglial inflammatory responses, which play a pathogenic role in both HAND and PD, and that these responses may cause or exacerbate neuronal damage in these diseases. The HIV-1 Tat protein is a potent neurotoxin produced during HAND that induces activation of primary microglia in culture and long-lasting neuroinflammation and neurotoxicity when injected into the CNS of mice. We found that LRRK2 inhibition attenuates Tat-induced pS935–LRRK2 expression, proinflammatory cytokine and chemokine expression, and phosphorylated p38 and Jun N-terminal kinase signaling in primary microglia. In our murine model, cortical Tat injection in LRRK2 knock-out (KO) mice results in significantly diminished neuronal damage, as assessed by microtubule-associated protein 2 (MAP2), class III β-tubulin TUJ1, synapsin-1, VGluT, and cleaved caspase-3 immunostaining. Furthermore, Tat-injected LRRK2 KO animals have decreased infiltration of peripheral neutrophils, and the morphology of microglia from these mice were similar to that of vehicle-injected controls. We conclude that pathologic activation of LRRK2 regulates a significant component of the neuroinflammation associated with HAND. PMID:25834052

  2. The leucine-rich pentatricopeptide repeat-containing protein (LRPPRC) does not activate transcription in mammalian mitochondria.

    PubMed

    Harmel, Julia; Ruzzenente, Benedetta; Terzioglu, Mügen; Spåhr, Henrik; Falkenberg, Maria; Larsson, Nils-Göran

    2013-05-31

    Regulation of mtDNA expression is critical for controlling oxidative phosphorylation capacity and has been reported to occur at several different levels in mammalian mitochondria. LRPPRC (leucine-rich pentatricopeptide repeat-containing protein) has a key role in this regulation and acts at the post-transcriptional level to stabilize mitochondrial mRNAs, to promote mitochondrial mRNA polyadenylation, and to coordinate mitochondrial translation. However, recent studies have suggested that LRPPRC may have an additional intramitochondrial role by directly interacting with the mitochondrial RNA polymerase POLRMT to stimulate mtDNA transcription. In this study, we have further examined the intramitochondrial roles for LRPPRC by creating bacterial artificial chromosome transgenic mice with moderately increased LRPPRC expression and heterozygous Lrpprc knock-out mice with moderately decreased LRPPRC expression. Variation of LRPPRC levels in mice in vivo, occurring within a predicted normal physiological range, strongly affected the levels of an unprocessed mitochondrial precursor transcript (ND5-cytochrome b) but had no effect on steady-state levels of mitochondrial transcripts or de novo transcription of mtDNA. We further assessed the role of LRPPRC in mitochondrial transcription by performing size exclusion chromatography and immunoprecipitation experiments in human cell lines and mice, but we found no interaction between LRPPRC and POLRMT. Furthermore, addition of purified LRPPRC to a recombinant human in vitro transcription system did not activate mtDNA transcription. On the basis of these data, we conclude that LRPPRC does not directly regulate mtDNA transcription but rather acts as a post-transcriptional regulator of mammalian mtDNA expression. PMID:23599432

  3. Leucine-rich Repeats of Bacterial Surface Proteins Serve as Common Pattern Recognition Motifs of Human Scavenger Receptor gp340*

    PubMed Central

    Loimaranta, Vuokko; Hytönen, Jukka; Pulliainen, Arto T.; Sharma, Ashu; Tenovuo, Jorma; Strömberg, Nicklas; Finne, Jukka

    2009-01-01

    Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340. PMID:19465482

  4. The Leucine-rich Pentatricopeptide Repeat-containing Protein (LRPPRC) Does Not Activate Transcription in Mammalian Mitochondria*

    PubMed Central

    Harmel, Julia; Ruzzenente, Benedetta; Terzioglu, Mügen; Spåhr, Henrik; Falkenberg, Maria; Larsson, Nils-Göran

    2013-01-01

    Regulation of mtDNA expression is critical for controlling oxidative phosphorylation capacity and has been reported to occur at several different levels in mammalian mitochondria. LRPPRC (leucine-rich pentatricopeptide repeat-containing protein) has a key role in this regulation and acts at the post-transcriptional level to stabilize mitochondrial mRNAs, to promote mitochondrial mRNA polyadenylation, and to coordinate mitochondrial translation. However, recent studies have suggested that LRPPRC may have an additional intramitochondrial role by directly interacting with the mitochondrial RNA polymerase POLRMT to stimulate mtDNA transcription. In this study, we have further examined the intramitochondrial roles for LRPPRC by creating bacterial artificial chromosome transgenic mice with moderately increased LRPPRC expression and heterozygous Lrpprc knock-out mice with moderately decreased LRPPRC expression. Variation of LRPPRC levels in mice in vivo, occurring within a predicted normal physiological range, strongly affected the levels of an unprocessed mitochondrial precursor transcript (ND5-cytochrome b) but had no effect on steady-state levels of mitochondrial transcripts or de novo transcription of mtDNA. We further assessed the role of LRPPRC in mitochondrial transcription by performing size exclusion chromatography and immunoprecipitation experiments in human cell lines and mice, but we found no interaction between LRPPRC and POLRMT. Furthermore, addition of purified LRPPRC to a recombinant human in vitro transcription system did not activate mtDNA transcription. On the basis of these data, we conclude that LRPPRC does not directly regulate mtDNA transcription but rather acts as a post-transcriptional regulator of mammalian mtDNA expression. PMID:23599432

  5. Genomic and Post-Translational Modification Analysis of Leucine-Rich-Repeat Receptor-Like Kinases in Brassica rapa

    PubMed Central

    Dhandapani, Vignesh; Yu, Xiaona; Choi, Su Ryun; Oh, Man-Ho; Lim, Yong Pyo

    2015-01-01

    Among several receptor-like kinases (RLKs), leucine-rich-repeat receptor-like kinases (LRR-RLKs) are a major group of genes that play crucial roles in growth, development and stress responses in plant systems. Given that they have several functional roles, it is important to investigate their roles in Brassica rapa. In the present study, 303 LRR-RLKs were identified in the genome of B. rapa and comparative phylogenetic analysis of 1213 combined LRR-RLKs of B. rapa, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa helped us to categorize the gene family into 15 subfamilies based on their sequence and structural similarities. The chromosome localizations of 293 genes allowed the prediction of duplicates, and motif conservation and intron/exon patterns showed differences among the B. rapa LRR-RLK (BrLRR-RLK) genes. Additionally, computational function annotation and expression analysis was used to predict their possible functional roles in the plant system. Biochemical results for 11 selected genes showed variations in phosphorylation activity. Interestingly, BrBAK1 showed strong auto-phosphorylation and trans-phosphorylation on its tyrosine and threonine residues compared with AtBAK1 in previous studies. The AtBAK1 receptor kinase is involved in plant growth and development, plant innate immunity, and programmed cell death, and our results suggest that BrBAK1 might also be involved in the same functions. Another interesting result was that BrBAK1, BrBRI1, BrPEPR1 and BrPEPR2 showed activity with both anti-phosphotyrosine and anti-phosphothreonine antibodies, indicating that they might have dual-specificity kinase activity. This study provides comprehensive results for the BrLRR-RLKs, revealing expansion of the gene family through gene duplications, structural similarities and variations among the genes, and potential functional roles according to gene ontology, transcriptome profiling and biochemical analysis. PMID:26588465

  6. Characterization of Microsporidia-Induced Developmental Arrest and a Transmembrane Leucine-Rich Repeat Protein in Caenorhabditis elegans

    PubMed Central

    Luallen, Robert J.; Bakowski, Malina A.; Troemel, Emily R.

    2015-01-01

    Microsporidia comprise a highly diverged phylum of intracellular, eukaryotic pathogens, with some species able to cause life-threatening illnesses in immunocompromised patients. To better understand microsporidian infection in animals, we study infection of the genetic model organism Caenorhabditis elegans and a species of microsporidia, Nematocida parisii, which infects Caenorhabditis nematodes in the wild. We conducted a targeted RNAi screen for host C. elegans genes important for infection and growth of N. parisii, using nematode larval arrest as an assay for infection. Here, we present the results of this RNAi screen, and our analyses on one of the RNAi hits from the screen that was ultimately not corroborated by loss of function mutants. This hit was an RNAi clone against F56A8.3, a conserved gene that encodes a transmembrane protein containing leucine-rich repeats (LRRs), a domain found in numerous pathogen receptors from other systems. This RNAi clone caused C. elegans to be resistant to infection by N. parisii, leading to reduced larval arrest and lower pathogen load. Characterization of the endogenous F56A8.3 protein revealed that it is expressed in the intestine, localized to the membrane around lysosome-related organelles (LROs), and exists in two different protein isoforms in C. elegans. We used the CRISPR-Cas9 system to edit the F56A8.3 locus and created both a frameshift mutant resulting in a truncated protein and a complete knockout mutant. Neither of these mutants was able to recapitulate the infection phenotypes of the RNAi clone, indicating that the RNAi-mediated phenotypes are due to an off-target effect of the RNAi clone. Nevertheless, this study describes microsporidia-induced developmental arrest in C. elegans, presents results from an RNAi screen for host genes important for microsporidian infection, and characterizes aspects of the conserved F56A8.3 gene and its protein product. PMID:25874557

  7. A Direct Interaction between Leucine-rich Repeat Kinase 2 and Specific β-Tubulin Isoforms Regulates Tubulin Acetylation*

    PubMed Central

    Law, Bernard M. H.; Spain, Victoria A.; Leinster, Veronica H. L.; Chia, Ruth; Beilina, Alexandra; Cho, Hyun J.; Taymans, Jean-Marc; Urban, Mary K.; Sancho, Rosa M.; Ramírez, Marian Blanca; Biskup, Saskia; Baekelandt, Veerle; Cai, Huaibin; Cookson, Mark R.; Berwick, Daniel C.; Harvey, Kirsten

    2014-01-01

    Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease. PMID:24275654

  8. Genomic and Post-Translational Modification Analysis of Leucine-Rich-Repeat Receptor-Like Kinases in Brassica rapa.

    PubMed

    Rameneni, Jana Jeevan; Lee, Yeon; Dhandapani, Vignesh; Yu, Xiaona; Choi, Su Ryun; Oh, Man-Ho; Lim, Yong Pyo

    2015-01-01

    Among several receptor-like kinases (RLKs), leucine-rich-repeat receptor-like kinases (LRR-RLKs) are a major group of genes that play crucial roles in growth, development and stress responses in plant systems. Given that they have several functional roles, it is important to investigate their roles in Brassica rapa. In the present study, 303 LRR-RLKs were identified in the genome of B. rapa and comparative phylogenetic analysis of 1213 combined LRR-RLKs of B. rapa, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa helped us to categorize the gene family into 15 subfamilies based on their sequence and structural similarities. The chromosome localizations of 293 genes allowed the prediction of duplicates, and motif conservation and intron/exon patterns showed differences among the B. rapa LRR-RLK (BrLRR-RLK) genes. Additionally, computational function annotation and expression analysis was used to predict their possible functional roles in the plant system. Biochemical results for 11 selected genes showed variations in phosphorylation activity. Interestingly, BrBAK1 showed strong auto-phosphorylation and trans-phosphorylation on its tyrosine and threonine residues compared with AtBAK1 in previous studies. The AtBAK1 receptor kinase is involved in plant growth and development, plant innate immunity, and programmed cell death, and our results suggest that BrBAK1 might also be involved in the same functions. Another interesting result was that BrBAK1, BrBRI1, BrPEPR1 and BrPEPR2 showed activity with both anti-phosphotyrosine and anti-phosphothreonine antibodies, indicating that they might have dual-specificity kinase activity. This study provides comprehensive results for the BrLRR-RLKs, revealing expansion of the gene family through gene duplications, structural similarities and variations among the genes, and potential functional roles according to gene ontology, transcriptome profiling and biochemical analysis. PMID:26588465

  9. Distinct Secondary Structures of the Leucine-Rich Repeat Proteoglycans Decorin and Biglycan: Glycosylation-Dependent Conformational Stability

    NASA Technical Reports Server (NTRS)

    Krishnan, Priya; Hocking, Anne M.; Scholtz, J. Martin; Pace, C. Nick; Holik, Kimberly K.; McQuillan, David J.

    1998-01-01

    Biglycan and decorin, closely related small leucine-rich repeat proteoglycans, have been overexpressed in eukaryotic cers and two major glycoforms isolated under native conditions: a proteoglycan substituted with glycosaminoglycan chains; and a core protein form secreted devoid of glycosaminoglycans. A comparative biophysical study of these glycoforms has revealed that the overall secondary structures of biglycan and decorin are different. Far-UV Circular Dichroism (CD) spectroscopy of decorin and biglycan proteoglycans indicates that, although they are predominantly Beta-sheet, biglycan has a significantly higher content of alpha-helical structure. Decorin proteoglycan and core protein are very similar, whereas the biglycan core protein exhibits closer similarity to the decorin glycoforms than to. the biglycan proteoglycan form. However, enzymatic removal of the chondroitin sulfate chains from biglycan proteoglycan does not induce a shift to the core protein structure, suggesting that the fmal form is influenced by polysaccharide addition only during biosynthesis. Fluorescence emission spectroscopy demonstrated that the single tryptophan residue, which is at a conserved position at the C-terminal domain of both biglycan and decorin, is found in similar microenvironments. This indicates that at least in this specific domain, the different glycoforms do exhibit apparent conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity, which indicates that the emission from tryptophan in the native state is quenched. Comparison of urea-induced protein unfolding curves provided further evidence that decorin and biglycan assume different structures in solution. Decorin proteoglycan and core protein unfold in a manner similar to a classic two-state model, in which there is a steep transition to an unfolded state between 1-2 M urea. The biglycan core protein also shows a similar steep transition. However, biglycan

  10. Mutational analysis identifies leucine-rich repeat insertions crucial for pigeon toll-like receptor 7 recognition and signaling.

    PubMed

    Xiong, Dan; Song, Li; Jiao, Yang; Kang, Xilong; Chen, Xiang; Geng, Shizhong; Pan, Zhiming; Jiao, Xinan

    2015-11-15

    Toll-like receptor 7 (TLR7) is responsible for recognizing viral single-stranded RNA and antiviral imidazoquinoline compounds, leading to the activation of the innate immune response. In this study, mutated pigeon TLR7 fragments, in which the insertion at position 10 of leucine-rich repeat 10 (LRR10) or at position 15 of LRR2/11/13/14 was deleted, were amplified with an overlap-PCR method, and inserted into the expression vector pCMV. The immune functions of the TLR7 mutants were determined with an NF-κB luciferase assay of transfected cells. The deletion of the insertions absolutely abolished TLR7-NF-κB signaling. With quantitative real-time PCR and sandwich enzyme-linked immunosorbent assay, we observed that stimulation with R848 failed to induce the expression of interleukin 8 (IL-8) in any of the mutant-TLR7-transfected cells, consistent with their lack of NF-κB activity. However, the expression of interferon α (IFN-α) and tumor necrosis factor α (TNF-α) was significantly upregulated in the Del10IN10 and Del14IN15 groups. Remarkably, the levels of pigeon TLR7 expression were significantly increased in all the TLR7-mutated groups. Therefore, we speculate that another part of the deficient TLR7 mediates the induction of IFN-α and TNF-α by increasing the expression of TLR7 as compensation. However, the increased expression of TLR7 in the Del11IN15 group failed to induce the production of IFN-α, IL-8, or TNF-α, indicating that a false compensation occurred when the crucial LRR insertion was deleted. PMID:26553562

  11. Antibodies to the leucine-rich repeat region of internalin block entry of Listeria monocytogenes into cells expressing E-cadherin.

    PubMed

    Mengaud, J; Lecuit, M; Lebrun, M; Nato, F; Mazie, J C; Cossart, P

    1996-12-01

    Internalin, a surface protein essential for entry of Listeria monocytogenes EGD into epithelial cells, was used as an antigen to raise nine monoclonal antibodies. These monoclonal antibodies recognized seven distinct epitopes which were located in three different regions of the protein. Three of them inhibited internalin-mediated entry and recognized the amino-terminal leucine-rich repeat region of the protein, suggesting that this region is essential for entry. PMID:8945603

  12. Kinase domain inhibition of leucine rich repeat kinase 2 (LRRK2) using a [1,2,4]triazolo[4,3-b]pyridazine scaffold.

    PubMed

    Galatsis, Paul; Henderson, Jaclyn L; Kormos, Bethany L; Han, Seungil; Kurumbail, Ravi G; Wager, Travis T; Verhoest, Patrick R; Noell, G Stephen; Chen, Yi; Needle, Elie; Berger, Zdenek; Steyn, Stefanus J; Houle, Christopher; Hirst, Warren D

    2014-09-01

    Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson's disease (PD). The most common mutant, G2019S, increases kinase activity, thus LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the structure, potential ligand-protein binding interactions, and pharmacological profiling of potent and highly selective kinase inhibitors based on a triazolopyridazine chemical scaffold. PMID:25113930

  13. Global expression analysis of nucleotide binding site-leucine rich repeat-encoding and related genes in Arabidopsis

    PubMed Central

    Tan, Xiaoping; Meyers, Blake C; Kozik, Alexander; West, Marilyn AL; Morgante, Michele; St Clair, Dina A; Bent, Andrew F; Michelmore, Richard W

    2007-01-01

    Background Nucleotide binding site-leucine rich repeat (NBS-LRR)-encoding genes comprise the largest class of plant disease resistance genes. The 149 NBS-LRR-encoding genes and the 58 related genes that do not encode LRRs represent approximately 0.8% of all ORFs so far annotated in Arabidopsis ecotype Col-0. Despite their prevalence in the genome and functional importance, there was little information regarding expression of these genes. Results We analyzed the expression patterns of ~170 NBS-LRR-encoding and related genes in Arabidopsis Col-0 using multiple analytical approaches: expressed sequenced tag (EST) representation, massively parallel signature sequencing (MPSS), microarray analysis, rapid amplification of cDNA ends (RACE) PCR, and gene trap lines. Most of these genes were expressed at low levels with a variety of tissue specificities. Expression was detected by at least one approach for all but 10 of these genes. The expression of some but not the majority of NBS-LRR-encoding and related genes was affected by salicylic acid (SA) treatment; the response to SA varied among different accessions. An analysis of previously published microarray data indicated that ten NBS-LRR-encoding and related genes exhibited increased expression in wild-type Landsberg erecta (Ler) after flagellin treatment. Several of these ten genes also showed altered expression after SA treatment, consistent with the regulation of R gene expression during defense responses and overlap between the basal defense response and salicylic acid signaling pathways. Enhancer trap analysis indicated that neither jasmonic acid nor benzothiadiazole (BTH), a salicylic acid analog, induced detectable expression of the five NBS-LRR-encoding genes and one TIR-NBS-encoding gene tested; however, BTH did induce detectable expression of the other TIR-NBS-encoding gene analyzed. Evidence for alternative mRNA polyadenylation sites was observed for many of the tested genes. Evidence for alternative splicing

  14. Hypothesis: Do miRNAs Targeting the Leucine-Rich Repeat Kinase 2 Gene (LRRK2) Influence Parkinson's Disease Susceptibility?

    PubMed

    Yılmaz, Şenay Görücü; Geyik, Sırma; Neyal, Ayşe Münife; Soko, Nyarai D; Bozkurt, Hakan; Dandara, Collet

    2016-04-01

    Parkinson's disease (PD) is a frequently occurring neurodegenerative motor disorder adversely impacting global health. There is a paucity of biomarkers and diagnostics that can forecast susceptibility to PD. A new research frontier for PD pathophysiology is the study of variations in microRNA (miRNA) expression whereby miRNAs serve as "upstream regulators" of gene expression in relation to functioning of the dopamine neuronal pathways. Leucine-Rich Repeat Kinase 2 (LRRK2) is a frequently studied gene in PD. Little is known about the ways in which expression of miRNAs targeting LRKK2 impact PD susceptibility. In a sample of 204 unrelated subjects (102 persons with PD and 102 healthy controls), we report here candidate miRNA expression in whole blood samples as measured by real-time PCR (hsa-miR-4671-3p, hsa-miR-335-3p, hsa-miR-561-3p, hsa-miR-579-3p, and hsa-miR-3143) that target LRRK2. Using step-wise logistic regression, and controlling for covariates such as age, gender, PD disease severity, concomitant medications, and co-morbidity, we found that the combination of has-miR-335-3p, has-miR-561-3p, and has-miR-579-3p account for 50% of the variation in regards to PD susceptibility (p < 0.0001). Notably, the hsa-miR-561-3p expression was the most robust predictor of PD in both univariate and multivariate analyses (p < 0.001). Moreover, the biological direction (polarity) of the association was plausible in that the candidate miRNAs displayed a diminished expression in patients. This is consistent with the hypothesis that decreased levels of miRNAs targeting LRRK2 might result in a gain of function for LRRK2, and by extension, loss of neuronal viability. To the best of our knowledge, this is the first clinical association study of the above candidate miRNAs' expression in PD using peripheral samples. These observations may guide future clinical diagnostics research on PD. PMID:27093107

  15. Requirement of the Cytosolic Interaction between PATHOGENESIS-RELATED PROTEIN10 and LEUCINE-RICH REPEAT PROTEIN1 for Cell Death and Defense Signaling in Pepper[W

    PubMed Central

    Choi, Du Seok; Hwang, In Sun; Hwang, Byung Kook

    2012-01-01

    Plants recruit innate immune receptors such as leucine-rich repeat (LRR) proteins to recognize pathogen attack and activate defense genes. Here, we identified the pepper (Capsicum annuum) pathogenesis-related protein10 (PR10) as a leucine-rich repeat protein1 (LRR1)–interacting partner. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirmed the specific interaction between LRR1 and PR10 in planta. Avirulent Xanthomonas campestris pv vesicatoria infection induces PR10 expression associated with the hypersensitive cell death response. Transient expression of PR10 triggers hypersensitive cell death in pepper and Nicotiana benthamiana leaves, which is amplified by LRR1 coexpression as a positive regulator. LRR1 promotes the ribonuclease activity and phosphorylation of PR10, leading to enhanced cell death signaling. The LRR1-PR10 complex is formed in the cytoplasm, resulting in its secretion into the apoplastic space. Engineered nuclear confinement of both proteins revealed that the cytoplasmic localization of the PR10-LRR1 complex is essential for cell death–mediated defense signaling. PR10/LRR1 silencing in pepper compromises resistance to avirulent X. campestris pv vesicatoria infection. By contrast, PR10/LRR1 overexpression in Arabidopsis thaliana confers enhanced resistance to Pseudomonas syringae pv tomato and Hyaloperonospora arabidopsidis. Together, these results suggest that the cytosolic LRR-PR10 complex is responsible for cell death–mediated defense signaling. PMID:22492811

  16. The leucine-rich repeats of LINGO-1 are not required for self-interaction or interaction with the amyloid precursor protein.

    PubMed

    Stein, Thomas; Walmsley, Adrian Robert

    2012-02-10

    LINGO-1 (leucine rich repeat and Ig domain containing Nogo receptor interacting protein-1) is a central nervous system transmembrane protein which simultaneously interacts with the Nogo-66 receptor and p75(NTR) or TROY on neurons to form a receptor complex responsible for myelin-mediated neurite outgrowth inhibition. On oligodendroglial cells, LINGO-1 interacts with p75(NTR) to constitutively inhibit multiple aspects of oligodendrocyte differentiation. Recently, LINGO-1 was identified as an in vivo interacting partner of the amyloid precursor protein (APP) and, correspondingly, cellular LINGO-1 expression was found to augment the release of the Abeta peptide, the potential causative agent of Alzheimer's disease. In addition, the recombinant LINGO-1 ectodomain has been shown to self-interact in solution and after crystallisation. Here, we have used deletional mutagenesis to identify the regions on LINGO-1 that are involved in homo- and heterotypic interactions. We have found that the N-terminal region containing the leucine-rich repeats along with the transmembrane and cytoplasmic domains of LINGO-1 are not required for self-interaction or interaction with APP. PMID:22133804

  17. A Proline/Arginine-Rich End Leucine-Rich Repeat Protein (PRELP) Variant Is Uniquely Expressed in Chronic Lymphocytic Leukemia Cells

    PubMed Central

    Mikaelsson, Eva; Österborg, Anders; Jeddi-Tehrani, Mahmood; Kokhaei, Parviz; Ostadkarampour, Mahyar; Hadavi, Reza; Gholamin, Mehran; Akhondi, Mehdi; Shokri, Fazel; Rabbani, Hodjattallah; Mellstedt, Håkan

    2013-01-01

    Proline/arginine-rich end leucine-rich repeat protein (PRELP) belongs to the small leucine-rich proteoglycan (SLRP) family, normally expressed in extracellular matrix of collagen-rich tissues. We have previously reported on another SLRP, fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). PRELP is structurally similar to FMOD with adjacent localization on chromosome 1 (1q32.1). As cluster-upregulation of genes may occur in malignancies, the aim of our study was to analyze PRELP expression in CLL. PRELP was expressed (RT-PCR) in all CLL patients (30/30), as well as in some patients with mantle cell lymphoma (3/5), but not in healthy donor leukocytes (0/20) or tumor samples from other hematological malignancies (0/35). PRELP was also detected in CLL cell-lines (4/4) but not in cell-lines from other hematological tumors (0/9). PRELP protein was detected in all CLL samples but not in normal leukocytes. Deglycosylation experiments revealed a CLL-unique 38 kDa core protein, with an intact signal peptide. This 38 kDa protein was, in contrast to the normal 55 kDa size, not detected in serum which, in combination with the uncleaved signal peptide, suggests cellular retention. The unique expression of a 38 kDa PRELP in CLL cells may suggest involvement in the pathobiology of CLL and merits further studies. PMID:23826326

  18. A proline/arginine-rich end leucine-rich repeat protein (PRELP) variant is uniquely expressed in chronic lymphocytic leukemia cells.

    PubMed

    Mikaelsson, Eva; Österborg, Anders; Jeddi-Tehrani, Mahmood; Kokhaei, Parviz; Ostadkarampour, Mahyar; Hadavi, Reza; Gholamin, Mehran; Akhondi, Mehdi; Shokri, Fazel; Rabbani, Hodjattallah; Mellstedt, Håkan

    2013-01-01

    Proline/arginine-rich end leucine-rich repeat protein (PRELP) belongs to the small leucine-rich proteoglycan (SLRP) family, normally expressed in extracellular matrix of collagen-rich tissues. We have previously reported on another SLRP, fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). PRELP is structurally similar to FMOD with adjacent localization on chromosome 1 (1q32.1). As cluster-upregulation of genes may occur in malignancies, the aim of our study was to analyze PRELP expression in CLL. PRELP was expressed (RT-PCR) in all CLL patients (30/30), as well as in some patients with mantle cell lymphoma (3/5), but not in healthy donor leukocytes (0/20) or tumor samples from other hematological malignancies (0/35). PRELP was also detected in CLL cell-lines (4/4) but not in cell-lines from other hematological tumors (0/9). PRELP protein was detected in all CLL samples but not in normal leukocytes. Deglycosylation experiments revealed a CLL-unique 38 kDa core protein, with an intact signal peptide. This 38 kDa protein was, in contrast to the normal 55 kDa size, not detected in serum which, in combination with the uncleaved signal peptide, suggests cellular retention. The unique expression of a 38 kDa PRELP in CLL cells may suggest involvement in the pathobiology of CLL and merits further studies. PMID:23826326

  19. The central leucine-rich repeat region of chicken TLR16 dictates unique ligand specificity and species-specific interaction with TLR2.

    PubMed

    Keestra, A Marijke; de Zoete, Marcel R; van Aubel, Rémon A M H; van Putten, Jos P M

    2007-06-01

    The ligand specificity of human TLR (hTLR) 2 is determined through the formation of functional heterodimers with either hTLR1 or hTLR6. The chicken carries two TLR (chTLR) 2 isoforms, type 1 and type 2 (chTLR2t1 and chTLR2t2), and one putative TLR1/6/10 homologue (chTLR16) of unknown function. In this study, we report that transfection of HeLa cells with the various chicken receptors yields potent NF-kappaB activation for the receptor combination of chTLR2t2 and chTLR16 only. The sensitivity of this complex was strongly enhanced by human CD14. The functional chTLR16/chTLR2t2 complex responded toward both the hTLR2/6-specific diacylated peptide S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) and the hTLR2/1 specific triacylated peptide tripalmitoyl-S-(bis(palmitoyloxy)propyl)-Cys-Ser-(Lys)(3)-Lys (Pam(3)CSK(4)), indicating that chTLR16 covers the functions of both mammalian TLR1 and TLR6. Dissection of the species specificity of TLR2 and its coreceptors showed functional chTLR16 complex formation with chTLR2t2 but not hTLR2. Conversely, chTLR2t2 did not function in combination with hTLR1 or hTLR6. The use of constructed chimeric receptors in which the defined domains of chTLR16 and hTLR1 or hTLR6 had been exchanged revealed that the transfer of leucine-rich repeats (LRR) 6-16 of chTLR16 into hTLR6 was sufficient to confer dual ligand specificity to the human receptor and to establish species-specific interaction with chTLR2t2. Collectively, our data indicate that diversification of the central LRR region of the TLR2 coreceptors during evolution has put constraints on both their ligand specificity and their ability to form functional complexes with TLR2. PMID:17513760

  20. Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease

    PubMed Central

    Beilina, Alexandria; Rudenko, Iakov N.; Kaganovich, Alice; Civiero, Laura; Chau, Hien; Kalia, Suneil K.; Kalia, Lorraine V.; Lobbestael, Evy; Chia, Ruth; Ndukwe, Kelechi; Ding, Jinhui; Nalls, Mike A.; Olszewski, Maciej; Hauser, David N.; Kumaran, Ravindran; Lozano, Andres M.; Baekelandt, Veerle; Greene, Lois E.; Taymans, Jean-Marc; Greggio, Elisa; Cookson, Mark R.; Nalls, Mike A.; Plagnol, Vincent; Martinez, Maria; Hernandez, Dena G; Sharma, Manu; Sheerin, Una-Marie; Saad, Mohamad; Simón-Sánchez, Javier; Schulte, Claudia; Lesage, Suzanne; Sveinbjörnsdóttir, Sigurlaug; Arepalli, Sampath; Barker, Roger; Ben-Shlomo, Yoav; Berendse, Henk W; Berg, Daniela; Bhatia, Kailash; de Bie, Rob M A; Biffi, Alessandro; Bloem, Bas; Bochdanovits, Zoltan; Bonin, Michael; Bras, Jose M; Brockmann, Kathrin; Brooks, Janet; Burn, David J; Charlesworth, Gavin; Chen, Honglei; Chong, Sean; Clarke, Carl E; Cookson, Mark R; Cooper, J Mark; Corvol, Jean Christophe; Counsell, Carl; Damier, Philippe; Dartigues, Jean-François; Deloukas, Panos; Deuschl, Günther; Dexter, David T; van Dijk, Karin D; Dillman, Allissa; Durif, Frank; Dürr, Alexandra; Edkins, Sarah; Evans, Jonathan R; Foltynie, Thomas; Gao, Jianjun; Gardner, Michelle; Gibbs, J Raphael; Goate, Alison; Gray, Emma; Guerreiro, Rita; Gústafsson, Ómar; Harris, Clare; van Hilten, Jacobus J; Hofman, Albert; Hollenbeck, Albert; Holton, Janice; Hu, Michele; Huang, Xuemei; Huber, Heiko; Hudson, Gavin; Hunt, Sarah E; Huttenlocher, Johanna; Illig, Thomas; München, Helmholtz Zentrum; Jónsson, Pálmi V; Lambert, Jean-Charles; Langford, Cordelia; Lees, Andrew; Lichtner, Peter; München, Helmholtz Zentrum; Limousin, Patricia; Lopez, Grisel; Lorenz, Delia; McNeill, Alisdair; Moorby, Catriona; Moore, Matthew; Morris, Huw R; Morrison, Karen E; Mudanohwo, Ese; O’Sullivan, Sean S; Pearson, Justin; Perlmutter, Joel S; Pétursson, Hjörvar; Pollak, Pierre; Post, Bart; Potter, Simon; Ravina, Bernard; Revesz, Tamas; Riess, Olaf; Rivadeneira, Fernando; Rizzu, Patrizia; Ryten, Mina; Sawcer, Stephen; Schapira, Anthony; Scheffer, Hans; Shaw, Karen; Shoulson, Ira; Sidransky, Ellen; Smith, Colin; Spencer, Chris C A; Stefánsson, Hreinn; Steinberg, Stacy; Stockton, Joanna D; Strange, Amy; Talbot, Kevin; Tanner, Carlie M; Tashakkori-Ghanbaria, Avazeh; Tison, François; Trabzuni, Daniah; Traynor, Bryan J; Uitterlinden, André G; Velseboer, Daan; Vidailhet, Marie; Walker, Robert; van de Warrenburg, Bart; Wickremaratchi, Mirdhu; Williams, Nigel; Williams-Gray, Caroline H; Winder-Rhodes, Sophie; Stefánsson, Kári; Hardy, John; Heutink, Peter; Brice, Alexis; Gasser, Thomas; Singleton, Andrew B; Wood, Nicholas W; Chinnery, Patrick F; Arepalli, Sampath; Cookson, Mark R; Dillman, Allissa; Ferrucci, Luigi; Gibbs, J Raphael; Hernandez, Dena G; Johnson, Robert; Longo, Dan L; Majounie, Elisa; Nalls, Michael A; O’Brien, Richard; Singleton, Andrew B; Traynor, Bryan J; Troncoso, Juan; van der Brug, Marcel; Zielke, H Ronald; Zonderman, Alan B

    2014-01-01

    Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein–protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G–associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy–lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms. PMID:24510904

  1. Interaction of Prevotella intermedia Strain 17 Leucine-Rich Repeat Domain Protein AdpF with Eukaryotic Cells Promotes Bacterial Internalization

    PubMed Central

    Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K.; Miyazaki, Hiroshi

    2014-01-01

    Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells. PMID:24711565

  2. Leucine-rich repeat kinase 2 binds to neuronal vesicles through protein interactions mediated by its C-terminal WD40 domain.

    PubMed

    Piccoli, Giovanni; Onofri, Franco; Cirnaru, Maria Daniela; Kaiser, Christoph J O; Jagtap, Pravinkumar; Kastenmüller, Andreas; Pischedda, Francesca; Marte, Antonella; von Zweydorf, Felix; Vogt, Andreas; Giesert, Florian; Pan, Lifeng; Antonucci, Flavia; Kiel, Christina; Zhang, Mingjie; Weinkauf, Sevil; Sattler, Michael; Sala, Carlo; Matteoli, Michela; Ueffing, Marius; Gloeckner, Christian Johannes

    2014-06-01

    Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function. PMID:24687852

  3. A Scan Without Evidence Is Not Evidence of Absence: Scans Without Evidence of Dopaminergic Deficit in a Symptomatic Leucine-Rich repeat Kinase 2 Mutation Carrier

    PubMed Central

    Wile, Daryl J.; Dinelle, Katie; Vafai, Nasim; McKenzie, Jessamyn; Tsui, Joseph K.; Schaffer, Paul; Ding, Yu-Shin; Farrer, Matthew; Sossi, Vesna; Stoessl, A. Jon

    2016-01-01

    Introduction The basis for SWEDD is unclear, with most cases representing PD mimics but some later developing PD with a dopaminergic deficit. Methods We studied a patient initially diagnosed with SWEDD (based on 18F-dopa PET) who developed unequivocal PD associated with a leucine-rich repeat kinase 2 p.G2019S mutation. Repeat multitracer PET was performed at 17 years’ disease duration, including (+)[11C]dihydrotetrabenazine, [11C](N,N-dimethyl-2-(2-amino-4-cyanophenylthio) benzylamine (which binds the serotonin transporter), and 18F-dopa. Results The patient showed bilateral striatal dopaminergic denervation (right putamen 28% of age-matched normal, left putamen 33%). 18F-dopa uptake was decreased, particularly on the left (mean 31% of normal vs. 45% on the more affected right side). Serotonin transporter binding was relatively preserved in the putamen (right mean 90% of normal, left 81%) and several cortical regions. Conclusions SWEDD can occur in genetically determined PD and may, in some cases, be the result of compensatory nondopaminergic mechanisms operating in early disease. PMID:26685774

  4. Comparative Geometrical Analysis of Leucine-Rich Repeat Structures in the Nod-Like and Toll-Like Receptors in Vertebrate Innate Immunity.

    PubMed

    Matsushima, Norio; Miyashita, Hiroki; Enkhbayar, Purevjav; Kretsinger, Robert H

    2015-01-01

    The NOD-like receptors (NLRs) and Toll-like receptors (TLRs) are pattern recognition receptors that are involved in the innate, pathogen pattern recognition system. The TLR and NLR receptors contain leucine-rich repeats (LRRs) that are responsible for ligand interactions. In LRRs short β-strands stack parallel and then the LRRs form a super helical arrangement of repeating structural units (called a coil of solenoids). The structures of the LRR domains of NLRC4, NLRP1, and NLRX1 in NLRs and of TLR1-5, TLR6, TLR8, TLR9 in TLRs have been determined. Here we report nine geometrical parameters that characterize the LRR domains; these include four helical parameters from HELFIT analysis. These nine parameters characterize well the LRR structures in NLRs and TLRs; the LRRs of NLR adopts a right-handed helix. In contrast, the TLR LRRs adopt either a left-handed helix or are nearly flat; RP105 and CD14 also adopt a left-handed helix. This geometrical analysis subdivides TLRs into four groups consisting of TLR3/TLR8/TLR9, TLR1/TLR2/TRR6, TLR4, and TLR5; these correspond to the phylogenetic tree based on amino acid sequences. In the TLRs an ascending lateral surface that consists of loops connecting the β-strand at the C-terminal side is involved in protein, protein/ligand interactions, but not the descending lateral surface on the opposite side. PMID:26295267

  5. Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

    PubMed Central

    Piccoli, Giovanni; Onofri, Franco; Cirnaru, Maria Daniela; Kaiser, Christoph J. O.; Jagtap, Pravinkumar; Kastenmüller, Andreas; Pischedda, Francesca; Marte, Antonella; von Zweydorf, Felix; Vogt, Andreas; Giesert, Florian; Pan, Lifeng; Antonucci, Flavia; Kiel, Christina; Zhang, Mingjie; Weinkauf, Sevil; Sattler, Michael; Sala, Carlo; Matteoli, Michela; Ueffing, Marius

    2014-01-01

    Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function. PMID:24687852

  6. F-box and leucine-rich repeat protein 5 (FBXL5) is required for maintenance of cellular and systemic iron homeostasis.

    PubMed

    Ruiz, Julio C; Walker, Scott D; Anderson, Sheila A; Eisenstein, Richard S; Bruick, Richard K

    2013-01-01

    Maintenance of cellular iron homeostasis requires post-transcriptional regulation of iron metabolism genes by iron regulatory protein 2 (IRP2). The hemerythrin-like domain of F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, senses iron and oxygen availability and facilitates IRP2 degradation in iron replete cells. Disruption of the ubiquitously expressed murine Fbxl5 gene results in a failure to sense increased cellular iron availability, accompanied by constitutive IRP2 accumulation and misexpression of IRP2 target genes. FBXL5-null mice die during embryogenesis, although viability is restored by simultaneous deletion of the IRP2, but not IRP1, gene. Mice containing a single functional Fbxl5 allele behave like their wild type littermates when fed an iron-sufficient diet. However, unlike wild type mice that manifest decreased hematocrit and hemoglobin levels when fed a low-iron diet, Fbxl5 heterozygotes maintain normal hematologic values due to increased iron absorption. The responsiveness of IRP2 to low iron is specifically enhanced in the duodena of the heterozygotes and is accompanied by increased expression of the divalent metal transporter-1. These results confirm the role of FBXL5 in the in vivo maintenance of cellular and systemic iron homeostasis and reveal a privileged role for the intestine in their regulation by virtue of its unique FBXL5 iron sensitivity. PMID:23135277

  7. F-box and Leucine-rich Repeat Protein 5 (FBXL5) Is Required for Maintenance of Cellular and Systemic Iron Homeostasis*

    PubMed Central

    Ruiz, Julio C.; Walker, Scott D.; Anderson, Sheila A.; Eisenstein, Richard S.; Bruick, Richard K.

    2013-01-01

    Maintenance of cellular iron homeostasis requires post-transcriptional regulation of iron metabolism genes by iron regulatory protein 2 (IRP2). The hemerythrin-like domain of F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, senses iron and oxygen availability and facilitates IRP2 degradation in iron replete cells. Disruption of the ubiquitously expressed murine Fbxl5 gene results in a failure to sense increased cellular iron availability, accompanied by constitutive IRP2 accumulation and misexpression of IRP2 target genes. FBXL5-null mice die during embryogenesis, although viability is restored by simultaneous deletion of the IRP2, but not IRP1, gene. Mice containing a single functional Fbxl5 allele behave like their wild type littermates when fed an iron-sufficient diet. However, unlike wild type mice that manifest decreased hematocrit and hemoglobin levels when fed a low-iron diet, Fbxl5 heterozygotes maintain normal hematologic values due to increased iron absorption. The responsiveness of IRP2 to low iron is specifically enhanced in the duodena of the heterozygotes and is accompanied by increased expression of the divalent metal transporter-1. These results confirm the role of FBXL5 in the in vivo maintenance of cellular and systemic iron homeostasis and reveal a privileged role for the intestine in their regulation by virtue of its unique FBXL5 iron sensitivity. PMID:23135277

  8. A role for the human nucleotide-binding domain, leucine-rich repeat-containing family member NLRC5 in antiviral responses.

    PubMed

    Neerincx, Andreas; Lautz, Katja; Menning, Maureen; Kremmer, Elisabeth; Zigrino, Paola; Hösel, Marianna; Büning, Hildegard; Schwarzenbacher, Robert; Kufer, Thomas A

    2010-08-20

    Proteins of the nucleotide-binding domain, leucine-rich repeat (NLR)-containing family recently gained attention as important components of the innate immune system. Although over 20 of these proteins are present in humans, only a few members including the cytosolic pattern recognition receptors NOD1, NOD2, and NLRP3 have been analyzed extensively. These NLRs were shown to be pivotal for mounting innate immune response toward microbial invasion. Here we report on the characterization of human NLRC5 and provide evidence that this NLR has a function in innate immune responses. We found that NLRC5 is a cytosolic protein expressed predominantly in hematopoetic cells. NLRC5 mRNA and protein expression was inducible by the double-stranded RNA analog poly(I.C) and Sendai virus. Overexpression of NLRC5 failed to trigger inflammatory responses such as the NF-kappaB or interferon pathways in HEK293T cells. However, knockdown of endogenous NLRC5 reduced Sendai virus- and poly(I.C)-mediated type I interferon pathway-dependent responses in THP-1 cells and human primary dermal fibroblasts. Taken together, this defines a function for NLRC5 in anti-viral innate immune responses. PMID:20538593

  9. Arsenite Stress Down-regulates Phosphorylation and 14-3-3 Binding of Leucine-rich Repeat Kinase 2 (LRRK2), Promoting Self-association and Cellular Redistribution*

    PubMed Central

    Mamais, Adamantios; Chia, Ruth; Beilina, Alexandra; Hauser, David N.; Hall, Christine; Lewis, Patrick A.; Cookson, Mark R.; Bandopadhyay, Rina

    2014-01-01

    Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson disease, but the mechanisms whereby LRRK2 is regulated are unknown. Phosphorylation of LRRK2 at Ser910/Ser935 mediates interaction with 14-3-3. Pharmacological inhibition of its kinase activity abolishes Ser910/Ser935 phosphorylation and 14-3-3 binding, and this effect is also mimicked by pathogenic mutations. However, physiological situations where dephosphorylation occurs have not been defined. Here, we show that arsenite or H2O2-induced stresses promote loss of Ser910/Ser935 phosphorylation, which is reversed by phosphatase inhibition. Arsenite-induced dephosphorylation is accompanied by loss of 14-3-3 binding and is observed in wild type, G2019S, and kinase-dead D2017A LRRK2. Arsenite stress stimulates LRRK2 self-association and association with protein phosphatase 1α, decreases kinase activity and GTP binding in vitro, and induces translocation of LRRK2 to centrosomes. Our data indicate that signaling events induced by arsenite and oxidative stress may regulate LRRK2 function. PMID:24942733

  10. An atypical soybean leucine-rich repeat receptor-like kinase, GmLRK1, may be involved in the regulation of cell elongation.

    PubMed

    Kim, Sunghan; Kim, Su-Jin; Shin, Yun-Jeong; Kang, Ji-Hye; Kim, Mi-Ran; Nam, Kyoung Hee; Lee, Myeong-Sok; Lee, Suk-Ha; Kim, Yul-Ho; Hong, Soon-Kwan; Verma, Desh Pal S; Chun, Jong-Yoon; Cheon, Choong-Ill

    2009-03-01

    A leucine-rich repeat receptor-like kinase (LRR-RLK) encoded by one of the genes highly expressed in a specific stage of soybean seed development, referred to as GmLRK1, was identified and characterized. Examination of its kinase domain indicated that GmLRK1 may be a catalytically inactive atypical receptor kinase. An autophosphorylation assay confirmed that GmLRK1 is incapable of autophosphorylation in vitro. However, the phosphorylation of GmRLK1 could be induced after incubation with plant protein extracts, suggesting that some plant proteins may interact with GmLRK1 and phosphorylate the protein in vivo. Analyses of the expression profiles of GmLRK1 and its Arabidopsis ortholog At2g36570 revealed that they may be involved in regulation of more fundamental metabolic and/or developmental pathways, rather than a specialized developmental program such as seed development. Our results further indicate that the GmLRK1 and At2g36570 may play a role in the regulation of certain cellular processes that lead to cell elongation and expansion. PMID:19115064

  11. The root knot nematode resistance gene Mi from tomato is a member of the leucine zipper, nucleotide binding, leucine-rich repeat family of plant genes.

    PubMed Central

    Milligan, S B; Bodeau, J; Yaghoobi, J; Kaloshian, I; Zabel, P; Williamson, V M

    1998-01-01

    The Mi locus of tomato confers resistance to root knot nematodes. Tomato DNA spanning the locus was isolated as bacterial artificial chromosome clones, and 52 kb of contiguous DNA was sequenced. Three open reading frames were identified with similarity to cloned plant disease resistance genes. Two of them, Mi-1.1 and Mi-1.2, appear to be intact genes; the third is a pseudogene. A 4-kb mRNA hybridizing with these genes is present in tomato roots. Complementation studies using cloned copies of Mi-1.1 and Mi-1.2 indicated that Mi-1.2, but not Mi-1.1, is sufficient to confer resistance to a susceptible tomato line with the progeny of transformants segregating for resistance. The cloned gene most similar to Mi-1.2 is Prf, a tomato gene required for resistance to Pseudomonas syringae. Prf and Mi-1.2 share several structural motifs, including a nucleotide binding site and a leucine-rich repeat region, that are characteristic of a family of plant proteins, including several that are required for resistance against viruses, bacteria, fungi, and now, nematodes. PMID:9707531

  12. Genome-Wide Comparative Analyses Reveal the Dynamic Evolution of Nucleotide-Binding Leucine-Rich Repeat Gene Family among Solanaceae Plants

    PubMed Central

    Seo, Eunyoung; Kim, Seungill; Yeom, Seon-In; Choi, Doil

    2016-01-01

    Plants have evolved an elaborate innate immune system against invading pathogens. Within this system, intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors are known play critical roles in effector-triggered immunity (ETI) plant defense. We performed genome-wide identification and classification of NLR-coding sequences from the genomes of pepper, tomato, and potato using fixed criteria. We then compared genomic duplication and evolution features. We identified intact 267, 443, and 755 NLR-encoding genes in tomato, potato, and pepper genomes, respectively. Phylogenetic analysis and classification of Solanaceae NLRs revealed that the majority of NLR super family members fell into 14 subgroups, including a TIR-NLR (TNL) subgroup and 13 non-TNL subgroups. Specific subgroups have expanded in each genome, with the expansion in pepper showing subgroup-specific physical clusters. Comparative analysis of duplications showed distinct duplication patterns within pepper and among Solanaceae plants suggesting subgroup- or species-specific gene duplication events after speciation, resulting in divergent evolution. Taken together, genome-wide analysis of NLR family members provide insights into their evolutionary history in Solanaceae. These findings also provide important foundational knowledge for understanding NLR evolution and will empower broader characterization of disease resistance genes to be used for crop breeding. PMID:27559340

  13. Genome-Wide Comparative Analyses Reveal the Dynamic Evolution of Nucleotide-Binding Leucine-Rich Repeat Gene Family among Solanaceae Plants.

    PubMed

    Seo, Eunyoung; Kim, Seungill; Yeom, Seon-In; Choi, Doil

    2016-01-01

    Plants have evolved an elaborate innate immune system against invading pathogens. Within this system, intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors are known play critical roles in effector-triggered immunity (ETI) plant defense. We performed genome-wide identification and classification of NLR-coding sequences from the genomes of pepper, tomato, and potato using fixed criteria. We then compared genomic duplication and evolution features. We identified intact 267, 443, and 755 NLR-encoding genes in tomato, potato, and pepper genomes, respectively. Phylogenetic analysis and classification of Solanaceae NLRs revealed that the majority of NLR super family members fell into 14 subgroups, including a TIR-NLR (TNL) subgroup and 13 non-TNL subgroups. Specific subgroups have expanded in each genome, with the expansion in pepper showing subgroup-specific physical clusters. Comparative analysis of duplications showed distinct duplication patterns within pepper and among Solanaceae plants suggesting subgroup- or species-specific gene duplication events after speciation, resulting in divergent evolution. Taken together, genome-wide analysis of NLR family members provide insights into their evolutionary history in Solanaceae. These findings also provide important foundational knowledge for understanding NLR evolution and will empower broader characterization of disease resistance genes to be used for crop breeding. PMID:27559340

  14. The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein*

    PubMed Central

    Fenyk, Stepan; Townsend, Philip D.; Dixon, Christopher H.; Spies, Gerhard B.; de San Eustaquio Campillo, Alba; Slootweg, Erik J.; Westerhof, Lotte B.; Gawehns, Fleur K. K.; Knight, Marc R.; Sharples, Gary J.; Goverse, Aska; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2015-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR. PMID:26306038

  15. Effect of a Leucine-rich Repeat Kinase 2 Variant on Motor and Non-motor Symptoms in Chinese Parkinson’s Disease Patients

    PubMed Central

    Sun, Qian; Wang, Tian; Jiang, Tian-Fang; Huang, Pei; Li, Dun-Hui; Wang, Ying; Xiao, Qin; Liu, Jun; Chen, Sheng-Di

    2016-01-01

    The G2385R variant of the leucine-rich repeat kinase 2 (LRRK2) is strongly associated with Parkinson’s disease (PD) in Asian populations. However, it is still unclear whether the clinical phenotype of PD patients with the G2385R variant can be distinguished from that of patients with idiopathic PD. In this study, we investigated motor and non-motor symptoms of LRRK2 G2385R variant carriers in a Chinese population. We genotyped 1031 Chinese PD patients for the G2385R variant of the LRRK2 gene, and examined the demographic and clinical characteristics of LRRK2 G2385R variant carrier and non-carrier PD patients. LRRK2 G2385R variant carriers were more likely to present the postural instability and gait difficulty dominant (PIGD) phenotype. This variant was also significantly associated with motor fluctuations and the levodopa equivalent dose (LED). G2385R variant carriers had higher REM sleep behavior disorder (RBD) screening questionnaire (RBDSQ) score and more RBD symptoms compared with non-carriers. We concluded that the G2385R variant could be a risk factor for the PIGD phenotype, motor fluctuations, LED values and RBD symptoms. PMID:27330837

  16. Effect of a Leucine-rich Repeat Kinase 2 Variant on Motor and Non-motor Symptoms in Chinese Parkinson's Disease Patients.

    PubMed

    Sun, Qian; Wang, Tian; Jiang, Tian-Fang; Huang, Pei; Li, Dun-Hui; Wang, Ying; Xiao, Qin; Liu, Jun; Chen, Sheng-Di

    2016-05-01

    The G2385R variant of the leucine-rich repeat kinase 2 (LRRK2) is strongly associated with Parkinson's disease (PD) in Asian populations. However, it is still unclear whether the clinical phenotype of PD patients with the G2385R variant can be distinguished from that of patients with idiopathic PD. In this study, we investigated motor and non-motor symptoms of LRRK2 G2385R variant carriers in a Chinese population. We genotyped 1031 Chinese PD patients for the G2385R variant of the LRRK2 gene, and examined the demographic and clinical characteristics of LRRK2 G2385R variant carrier and non-carrier PD patients. LRRK2 G2385R variant carriers were more likely to present the postural instability and gait difficulty dominant (PIGD) phenotype. This variant was also significantly associated with motor fluctuations and the levodopa equivalent dose (LED). G2385R variant carriers had higher REM sleep behavior disorder (RBD) screening questionnaire (RBDSQ) score and more RBD symptoms compared with non-carriers. We concluded that the G2385R variant could be a risk factor for the PIGD phenotype, motor fluctuations, LED values and RBD symptoms. PMID:27330837

  17. Changes in matrix metalloprotease activity and progranulin levels may contribute to the pathophysiological function of mutant leucine-rich repeat kinase 2.

    PubMed

    Caesar, Mareike; Felk, Sandra; Zach, Susanne; Brønstad, Gunnar; Aasly, Jan O; Gasser, Thomas; Gillardon, Frank

    2014-07-01

    Increasing evidence suggests that Parkinson's disease (PD)-linked Leucine-rich repeat kinase 2 (LRRK2) has a role in peripheral and brain-resident immune cells. Furthermore, dysregulation of the anti-inflammatory, neurotrophic protein progranulin (PGRN) has been demonstrated in several chronic neurodegenerative diseases. Here we show that PGRN levels are significantly reduced in conditioned medium of LRRK2(R1441G) mutant mouse fibroblasts, leukocytes, and microglia, whereas levels of proinflammatory factors, like interleukin-1β and keratinocyte-derived chemokine, were significantly increased. Decreased PGRN levels were also detected in supernatants of cultured human fibroblasts isolated from presymptomatic LRRK2(G2019S) mutation carriers, while mitochondrial function was unaffected. Furthermore, medium levels of matrix metalloprotease (MMP) 2 increased, whereas MMP 9 decreased in LRRK2(R1441G) mutant microglia. Increased proteolytic cleavage of the MMP substrates ICAM-5 and α-synuclein in synaptoneurosomes from LRRK2(R1441G) mutant mouse brain indicates increased net synaptic MMP activity. PGRN levels were decreased in the cerebrospinal fluid of presymptomatic LRRK2 mutant mice, whereas PGRN levels were increased in aged symptomatic mutant mice. Notably, PGRN levels were also increased in the cerebrospinal fluid of PD patients carrying LRRK2 mutations, but not in idiopathic PD patients and in healthy control donors. Our data suggest that proinflammatory activity of peripheral and brain-resident immune cells may particularly contribute to the early stages of Parkinson's disease caused by LRRK2 mutations. PMID:24652679

  18. Comparison of leucine-rich repeat-containing G protein-coupled receptor 5 expression in different cancer and normal cell lines

    PubMed Central

    ALIZADEH-NAVAEI, REZA; RAFIEI, ALIREZA; ABEDIAN-KENARI, SAEID; ASGARIAN-OMRAN, HOSSEIN; VALADAN, REZA; HEDAYATIZADEH-OMRAN, AKBAR

    2016-01-01

    Evaluating the expression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) may be useful for predicting the best models and achieving more accurate results in cancer research. Therefore, the aim of the present study was to analyze the LGR5 expression levels in different cell lines. Eight commonly used cell lines were assessed (COS-7, NIH3T3, HEK293, VERO, HeLa, BHK, HepG2 and AGS). All the cell lines were cultured in RPMI-1640 medium contain 10% fetal calf serum at 37°C in humidified conditions with 5% CO2. According to the western blotting results, LGR5 was expressed in all cell lines. Densitometry results of LGR5 expression in the different cell lines showed that high LGR5 expression levels were apparent in BHK, AGS, VERO and NIH3T3 cell lines compared with the other cell lines. The results indicate that for the normal and cancer cell lines, BNK and AGS may be a better choice, respectively, for in vitro cancer studies. PMID:27347416

  19. Cloning, Expression, and Sequencing of a Cell Surface Antigen Containing a Leucine-Rich Repeat Motif from Bacteroides forsythus ATCC 43037

    PubMed Central

    Sharma, Ashu; Sojar, Hakimuddin T.; Glurich, Ingrid; Honma, Kiyonobu; Kuramitsu, Howard K.; Genco, Robert J.

    1998-01-01

    Bacteroides forsythus is a recently recognized human periodontopathogen associated with advanced, as well as recurrent, periodontitis. However, very little is known about the mechanism of pathogenesis of this organism. The present study was undertaken to identify the surface molecules of this bacterium that may play roles in its adherence to oral tissues or triggering of a host immune response(s). The gene (bspA) encoding a cell surface-associated protein of B. forsythus with an apparent molecular mass of 98 kDa was isolated by immunoscreening of a B. forsythus gene library constructed in a lambda ZAP II vector. The encoded 98-kDa protein (BspA) contains 14 complete repeats of 23 amino acid residues that show partial homology to leucine-rich repeat motifs. A recombinant protein containing the repeat region was expressed in Escherichia coli, purified, and utilized for antibody production, as well as in vitro binding studies. The purified recombinant protein bound strongly to fibronectin and fibrinogen in a dose-dependent manner and further inhibited the binding of B. forsythus cells to these extracellular matrix (ECM) components. In addition, adult patients with B. forsythus-associated periodontitis expressed specific antibodies against the BspA protein. We report here the cloning and expression of an immunogenic cell surface-associated protein (BspA) of B. forsythus and speculate that it mediates the binding of bacteria to ECM components and clotting factors (fibronectin and fibrinogen, respectively), which may be important in the colonization of the oral cavity by this bacterium and is also a target for the host immune response. PMID:9826345

  20. Structural determinants at the interface of the ARC2 and leucine-rich repeat domains control the activation of the plant immune receptors Rx1 and Gpa2.

    PubMed

    Slootweg, Erik J; Spiridon, Laurentiu N; Roosien, Jan; Butterbach, Patrick; Pomp, Rikus; Westerhof, Lotte; Wilbers, Ruud; Bakker, Erin; Bakker, Jaap; Petrescu, Andrei-José; Smant, Geert; Goverse, Aska

    2013-07-01

    Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins. PMID:23660837

  1. Conserved charged residues in the leucine-rich repeat domain of the Ran GTPase activating protein are required for Ran binding and GTPase activation.

    PubMed Central

    Haberland, J; Gerke, V

    1999-01-01

    GTPase activating proteins (GAPs) for Ran, a Ras-related GTPase participating in nucleocytoplasmic transport, have been identified in different species ranging from yeast to man. All RanGAPs are characterized by a conserved domain consisting of eight leucine-rich repeats (LRRs) interrupted at two positions by so-called separating regions, the latter being unique for RanGAPs within the family of LRR proteins. The cytosolic RanGAP activity is essential for the Ran GTPase cycle which in turn provides directionality in nucleocytoplasmic transport, but the structural basis for the interaction between Ran and its GAP has not been elucidated. In order to gain a better understanding of this interaction we generated a number of mutant RanGAPs carrying amino acid substitutions in the LRR domain and analysed their complex formation with Ran as well as their ability to stimulate the intrinsic GTPase activity of the G protein. We show that conserved charged residues present in the separating regions of the LRR domain are indispensable for efficient Ran binding and GAP activity. These separating regions contain three conserved arginines which could possibly serve as catalytic residues similar to the arginine fingers identified in GAPs for other small GTPases. However, mutations in two of these arginines do not affect the GAP activity and replacement of the third conserved arginine (Arg91 in human RanGAP) severely interferes not only with GAP activity but also with Ran binding. This indicates that RanGAP-stimulated GTP hydrolysis on Ran does not involve a catalytic arginine residue but requires certain charged residues of the LRR domain of the GAP for mediating the protein-protein interaction. PMID:10527945

  2. Large-Scale Analyses of Angiosperm Nucleotide-Binding Site-Leucine-Rich Repeat Genes Reveal Three Anciently Diverged Classes with Distinct Evolutionary Patterns.

    PubMed

    Shao, Zhu-Qing; Xue, Jia-Yu; Wu, Ping; Zhang, Yan-Mei; Wu, Yue; Hang, Yue-Yu; Wang, Bin; Chen, Jian-Qun

    2016-04-01

    Nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes make up the largest plant disease resistance gene family (R genes), with hundreds of copies occurring in individual angiosperm genomes. However, the expansion history of NBS-LRR genes during angiosperm evolution is largely unknown. By identifying more than 6,000 NBS-LRR genes in 22 representative angiosperms and reconstructing their phylogenies, we present a potential framework of NBS-LRR gene evolution in the angiosperm. Three anciently diverged NBS-LRR classes (TNLs, CNLs, and RNLs) were distinguished with unique exon-intron structures and DNA motif sequences. A total of seven ancient TNL, 14 CNL, and two RNL lineages were discovered in the ancestral angiosperm, from which all current NBS-LRR gene repertoires were evolved. A pattern of gradual expansion during the first 100 million years of evolution of the angiosperm clade was observed for CNLs. TNL numbers remained stable during this period but were eventually deleted in three divergent angiosperm lineages. We inferred that an intense expansion of both TNL and CNL genes started from the Cretaceous-Paleogene boundary. Because dramatic environmental changes and an explosion in fungal diversity occurred during this period, the observed expansions of R genes probably reflect convergent adaptive responses of various angiosperm families. An ancient whole-genome duplication event that occurred in an angiosperm ancestor resulted in two RNL lineages, which were conservatively evolved and acted as scaffold proteins for defense signal transduction. Overall, the reconstructed framework of angiosperm NBS-LRR gene evolution in this study may serve as a fundamental reference for better understanding angiosperm NBS-LRR genes. PMID:26839128

  3. Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

    SciTech Connect

    Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.; Kobe, Bostjan

    2015-03-16

    Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex. We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.

  4. Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

    DOE PAGESBeta

    Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.; Kobe, Bostjan

    2015-03-16

    Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex.more » We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.« less

  5. Regulation and Function of the Nucleotide Binding Domain Leucine-Rich Repeat-Containing Receptor, Pyrin Domain-Containing-3 Inflammasome in Lung Disease.

    PubMed

    Lee, Seonmin; Suh, Gee-Young; Ryter, Stefan W; Choi, Augustine M K

    2016-02-01

    Inflammasomes are specialized inflammatory signaling platforms that govern the maturation and secretion of proinflammatory cytokines, such as IL-1β and IL-18, through the regulation of caspase-1-dependent proteolytic processing. Several nucleotide binding domain leucine-rich repeat-containing receptor (NLR) family members (i.e., NLR family, pyrin domain containing [NLRP] 1, NLRP3, and NLR family, caspase recruitment domain containing-4 [NLRC4]) as well as the pyrin and hemopoietic expression, interferon-inducibility, nuclear localization domain-containing family member, absent in melanoma 2, can form inflammasome complexes in human cells. In particular, the NLRP3 inflammasome is activated in response to cellular stresses through a two-component pathway, involving Toll-like receptor 4-ligand interaction (priming) followed by a second signal, such as ATP-dependent P2X purinoreceptor 7 receptor activation. Emerging studies suggest that the NLRP3 inflammasome can exert pleiotropic effects in human diseases with potentially both pro- and antipathogenic sequelae. Whereas NLRP3 inflammasome activation can serve as a vital component of host defense against invading bacteria and pathogens, excessive activation of the inflammasome can lead to inflammation-associated tissue injury in the setting of chronic disease. In addition, pyroptosis, an inflammasome-associated mode of cell death, contributes to host defense. Recent research has described the regulation and function of the NLRP3 inflammasome in various pulmonary diseases, including acute lung injury and acute respiratory distress syndrome, sepsis, respiratory infections, chronic obstructive pulmonary disease, asthma, pulmonary hypertension, cystic fibrosis, and idiopathic pulmonary fibrosis. The NLRP3 and related inflammasomes, and their regulated cytokines or receptors, may represent novel diagnostic or therapeutic targets in pulmonary diseases and other diseases in which inflammation contributes to pathogenesis. PMID

  6. Kinetic, mechanistic, and structural modeling studies of truncated wild-type leucine-rich repeat kinase 2 and the G2019S mutant.

    PubMed

    Liu, Min; Kang, Stephanie; Ray, Soumya; Jackson, Justin; Zaitsev, Alexandra D; Gerber, Scott A; Cuny, Gregory D; Glicksman, Marcie A

    2011-11-01

    Leucine-rich repeat kinase 2 (LRRK2), a large and complex protein that possesses two enzymatic properties, kinase and GTPase, is one of the major genetic factors in Parkinson's disease (PD). Here, we characterize the kinetic and catalytic mechanisms of truncated wild-type (t-wt) LRRK2 and its most common mutant, G2019S (t-G2019S), with a structural interpretation of the kinase domain. First, the substitution of threonine with serine in the LRRKtide peptide results in a much less efficient substrate as demonstrated by a 26-fold decrease in k(cat) and a 6-fold decrease in binding affinity. The significant decrease in k(cat) is attributed to a slow chemical transfer step as evidenced by the inverse solvent kinetic isotope effect in the proton inventory and pL (pH or pD)-dependent studies. The shape of the proton inventory and pL profile clearly signals the involvement of a general base (pK(a) = 7.5) in the catalysis with a low fractionation factor in the ground state. We report for the first time that the increased kinase activity of the G2019S mutant is substrate-dependent. Homology modeling of the kinase domain (open and closed forms) and structural analysis of the docked peptide substrates suggest that electrostatic interactions play an important role in substrate recognition, which is affected by G2019S and may directly influence the kinetic properties of the enzyme. Finally, the GTPase activity of the t-G2019S mutant was characterized, and the mutation modestly decreases GTPase activity without significantly affecting GTP binding affinity. PMID:21961647

  7. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

    PubMed

    Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

    2016-01-15

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

  8. Transcriptional responses to loss or gain of function of the leucine-rich repeat kinase 2 (LRRK2) gene uncover biological processes modulated by LRRK2 activity

    PubMed Central

    Nikonova, Elena V.; Xiong, Yulan; Tanis, Keith Q.; Dawson, Valina L.; Vogel, Robert L.; Finney, Eva M.; Stone, David J.; Reynolds, Ian J.; Kern, Jonathan T.; Dawson, Ted M.

    2012-01-01

    Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common genetic cause of Parkinson's disease (PD) and cause both autosomal dominant familial and sporadic PD. Currently, the physiological and pathogenic activities of LRRK2 are poorly understood. To decipher the biological functions of LRRK2, including the genes and pathways modulated by LRRK2 kinase activity in vivo, we assayed genome-wide mRNA expression in the brain and peripheral tissues from LRRK2 knockout (KO) and kinase hyperactive G2019S (G2019S) transgenic mice. Subtle but significant differences in mRNA expression were observed relative to wild-type (WT) controls in the cortex, striatum and kidney of KO animals, but only in the striatum in the G2019S model. In contrast, robust, consistent and highly significant differences were identified by the direct comparison of KO and G2019S profiles in the cortex, striatum, kidney and muscle, indicating opposite effects on mRNA expression by the two models relative to WT. Ribosomal and glycolytic biological functions were consistently and significantly up-regulated in LRRK2 G2019S compared with LRRK2 KO tissues. Genes involved in membrane-bound organelles, oxidative phosphorylation, mRNA processing and the endoplasmic reticulum were down-regulated in LRRK2 G2019S mice compared with KO. We confirmed the expression patterns of 35 LRRK2-regulated genes using quantitative reverse transcription polymerase chain reaction. These findings provide the first description of the transcriptional responses to genetically modified LRRK2 activity and provide preclinical target engagement and/or pharmacodynamic biomarker strategies for LRRK2 and may inform future therapeutic strategies for LRRK2-associated PD. PMID:21972245

  9. Identification and developmental expression of leucine-rich repeat-containing G protein-coupled receptor 6 (lgr6) in the medaka fish, Oryzias latipes.

    PubMed

    Deguchi, Tomonori; Kawasaki, Takashi; Ohnishi, Hiroe; Yuba, Shunsuke; Takahashi, Toshio

    2012-07-01

    G protein-coupled receptors are critical regulators of diverse developmental processes such as oocyte maturation, fertilization, gastrulation, and organogenesis. To further study the molecular mechanisms underlying these processes, we cloned and characterized the orphan leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6), a stem cell marker in mammalian hair follicles, in medaka fish, Oryzias latipes. To examine the expression pattern of lgr6, we performed whole-mount in situ hybridization (WISH) during embryogenesis. The expression of lgr6 was first detected as a band in the anterior part of the posterior brain vesicle in 0.5-1 day post fertilization (dpf) embryos. This band disappeared by 2 dpf, but new signals appeared in the otic vesicles bordering the original band and also detected in the nasal placode and posterior lateral line primordia. At later stages (3-5 dpf), lgr6 was widely expressed in the brain, otic vesicle, neuromasts, root of the pectoral fin, cranial cartilage, and gut. Then, we conducted more detailed expression analysis of lgr6 in adult gut using WISH and immunohistochemical staining. Lgr6-positive cells were detected in the crypt-like proliferative zone and in parts of the villus. We also performed RT-PCR of mRNAs from different tissues. The lgr6 mRNA was found highest in the kidney and gill. The transcript was also present in the brain, heart, liver, spleen, intestine, skeletal muscle, testis, and ovary, similar to that of mammalian LGR6. These results suggest that medaka lgr6 plays an important role in organ development during embryogenesis and serves as a good molecular marker for future studies of postembryonic organ-specific development in mammals. PMID:22576653

  10. Lower expression of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) association with poor prognosis of gastric cancer

    PubMed Central

    Hou, Yachao; Deng, Jingyu; Zhang, Li; Xie, Xingming; Guo, Xiaofan; Sun, Changyu; Zhang, Rupeng; Liang, Han

    2015-01-01

    Background: The aim of this study was to investigate the expression of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) in gastric cancer (GC), and its potential influence on the prognosis of GC patients. Methods: At present study, we examined the immunohistochemical expression of PHLPP1 on tissue microarrays (TMAs) containing 135 gastric adenocarcinoma tissues and 135 matched adjacent non-tumor tissues. In addition, both semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blotting analysis (WB) were adopted to detect of the expression of PHLPP1 in the GC cell lines (AGS, SUN-1, KATO-III, BGC-823, MGC-803, SGC-7901, and HGC-27) and the normal gastric cell line GES-1. Survival analysis was used to investigate the efficiency of the prognostic evaluation of PHLPP1 expression in GC patients. Results: Positive expression rate of PHLPP1 in the primary GC tissues was significantly lower than that in adjacent non-tumor tissues (55.6% vs. 87.4%, P<0.001). Both gene transcription (mRNA) and Protein expression of PHLPP1 in the GC cell lines were significantly lower than those in the GES-1 cell line, respectively. The Kaplan-Meier analysis showed that patients presented PHLPP1 negative expression in the GC tissues had significantly lower overall survival rate than those presented PHLPP1 positive expression in the GC tissues (P=0.008). With the multivariate survival analysis (Cox regression), PHLPP1 expression in the GC tissue was identified as an independent predictor of the survival of patients. Conclusions: This study indicated that aberrant PHLPP1 expression was observed in GC tissues, which was significantly associated with the poor prognostic outcomes of GC patients. PMID:26884964

  11. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

    PubMed Central

    Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2016-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

  12. Report of leucine-rich repeats (LRRs) from Scylla serrata: Ontogeny, molecular cloning, characterization and expression analysis following ligand stimulation, and upon bacterial and viral infections.

    PubMed

    Vidya, R; Makesh, M; Purushothaman, C S; Chaudhari, A; Gireesh-Babu, P; Rajendran, K V

    2016-09-15

    Leucine-rich repeat (LRR) proteins are present in all living organisms, and their participation in signal transduction and defense mechanisms has been elucidated in humans and mosquitoes. LRRs possibly involve in protein-protein interactions also and show differential expression pattern upon challenge with pathogens. In the present study, a new LRR gene was identified in mud crab, Scylla serrata. LRR gene mRNA levels in different developmental stages and various tissues of S. serrata were analysed. Further, the response of the gene against different ligands, Gram-negative bacterium, and white spot syndrome virus (WSSV) was investigated in vitro and in vivo. Full-length cDNA sequence of S. serrata LRR (SsLRR) was found to be 2290 nucleotide long with an open reading frame of 1893bp. SsLRR encodes for a protein containing 630 deduced amino acids with 17 conserved LRR domains and exhibits significant similarity with crustacean LRRs so that these could be clustered into a branch in the phylogenetic tree. SsLRR mRNA transcripts were detected in all the developmental stages (egg, Zoea1-5, megalopa and crab instar), haemocytes and various tissues such as, stomach, gill, muscle, hepatopancreas, hematopoietic organ, heart, epithelial layer and testis by reverse-transcriptase PCR. SsLRR transcripts in cultured haemocytes showed a 2-fold increase in expression at 1.5 and 12h upon Poly I:C induction. WSSV challenge resulted in significant early up-regulation at 3h in-vitro and late up-regulation at 72h in-vivo. Peptidoglycan (PGN)-induction resulted in marginal up-regulation of SsLRR at timepoints, 6, 12 and 24h (fold change below 1.5) and no significant change in the expression at early timepoints. LPS-stimulation, on the other hand, showed either down-regulation or normal level of expression at all timepoints. However, a delayed 5-fold up-regulation was observed in vivo against Vibrio parahaemolyticus infection at 72hpi. The constitutive expression of the LRR gene in all the

  13. The Leucine-Rich Repeat Receptor-Like Kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 and the Cytochrome P450 PHYTOALEXIN DEFICIENT3 Contribute to Innate Immunity to Aphids in Arabidopsis1[C][W][OPEN

    PubMed Central

    Prince, David C.; Drurey, Claire; Zipfel, Cyril; Hogenhout, Saskia A.

    2014-01-01

    The importance of pathogen-associated molecular pattern-triggered immunity (PTI) against microbial pathogens has been recently demonstrated. However, it is currently unclear if this layer of immunity mediated by surface-localized pattern recognition receptors (PRRs) also plays a role in basal resistance to insects, such as aphids. Here, we show that PTI is an important component of plant innate immunity to insects. Extract of the green peach aphid (GPA; Myzus persicae) triggers responses characteristic of PTI in Arabidopsis (Arabidopsis thaliana). Two separate eliciting GPA-derived fractions trigger induced resistance to GPA that is dependent on the leucine-rich repeat receptor-like kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)/SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE3, which is a key regulator of several leucine-rich repeat-containing PRRs. BAK1 is required for GPA elicitor-mediated induction of reactive oxygen species and callose deposition. Arabidopsis bak1 mutant plants are also compromised in immunity to the pea aphid (Acyrthosiphon pisum), for which Arabidopsis is normally a nonhost. Aphid-derived elicitors induce expression of PHYTOALEXIN DEFICIENT3 (PAD3), a key cytochrome P450 involved in the biosynthesis of camalexin, which is a major Arabidopsis phytoalexin that is toxic to GPA. PAD3 is also required for induced resistance to GPA, independently of BAK1 and reactive oxygen species production. Our results reveal that plant innate immunity to insects may involve early perception of elicitors by cell surface-localized PRRs, leading to subsequent downstream immune signaling. PMID:24586042

  14. The role of the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene in cytochrome oxidase assembly: mutation causes lowered levels of COX (cytochrome c oxidase) I and COX III mRNA.

    PubMed

    Xu, Fenghao; Morin, Charles; Mitchell, Grant; Ackerley, Cameron; Robinson, Brian H

    2004-08-15

    Leigh syndrome French Canadian (LSFC) is a variant of cytochrome oxidase deficiency found in Québec and caused by mutations in the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene. Northern blots showed that the LRPPRC mRNA levels seen in skeletal muscle>heart>placenta>kidney>liver>lung=brain were proportionally almost opposite in strength to the severity of the enzymic cytochrome oxidase defect. The levels of COX (cytochrome c oxidase) I and COX III mRNA visible on Northern blots were reduced in LSFC patients due to the common (A354V, Ala354-->Val) founder mutation. The amount of LRPPRC protein found in both fibroblast and liver mitochondria from LSFC patients was consistently reduced to <30% of control levels. Import of [(35)S]methionine LRPPRC into rat liver mitochondria was slower for the mutant (A354V) protein. A titre of LRPPRC protein was also found in nuclear fractions that could not be easily accounted for by mitochondrial contamination. [35S]Methionine labelling of mitochondrial translation products showed that the translation of COX I, and perhaps COX III, was specifically reduced in the presence of the mutation. These results suggest that the gene product of LRPPRC, like PET 309p, has a role in the translation or stability of the mRNA for mitochondrially encoded COX subunits. A more diffuse distribution of LRPPRC in LSFC cells compared with controls was evident when viewed by immunofluorescence microscopy, with less LRPPRC present in peripheral mitochondria. PMID:15139850

  15. A novel neurofibromin (NF1) interaction with the leucine-rich pentatricopeptide repeat motif-containing protein links neurofibromatosis type 1 and the French Canadian variant of Leigh's syndrome in a common molecular complex.

    PubMed

    Arun, Vedant; Wiley, Joseph C; Kaur, Harpreet; Kaplan, David R; Guha, Abhijit

    2013-04-01

    Loss-of-function mutations and deletions in the neurofibromin tumor suppressor gene (NF1) cause neurofibromatosis type 1 (NF-1), the most common inherited syndrome of the nervous system in humans, with a birth incidence of 1:3,000. The most visible features of NF-1 are the neoplastic manifestations caused by the loss of Ras-GTPase-activating protein (Ras-GAP) activity mediated through the GAP-related domain (GRD) of neurofibromin (NF1), the protein encoded by NF1. However, the syndrome is also characterized by cognitive dysfunction and a number of developmental abnormalities. The molecular etiology of many of these nonneoplastic phenotypes remains unknown. Here we show that the tubulin-binding domain (TBD) of NF1 is a binding partner of the leucine-rich pentatricopeptide repeat motif-containing (LRPPRC) protein. These two proteins complex with Kinesin 5B, hnRNP A2, Staufen1, and Myelin Basic Protein (MBP) mRNA, likely in RNA granules. This interaction is of interest in that it links NF-1 with Leigh's syndrome, French Canadian variant (LSFC), an autosomal recessive neurodegenerative disorder that arises from mutations in the LRPPRC gene. Our findings provide clues to how loss or mutation of NF1 and LRPPRC may contribute to the manifestations of NF-1 and LSFC. PMID:23361976

  16. PRELP (proline/arginine-rich end leucine-rich repeat protein) promotes osteoblastic differentiation of preosteoblastic MC3T3-E1 cells by regulating the β-catenin pathway.

    PubMed

    Li, Haiying; Cui, Yazhou; Luan, Jing; Zhang, Xiumei; Li, Chengzhi; Zhou, Xiaoyan; Shi, Liang; Wang, Huaxin; Han, Jinxiang

    2016-02-12

    Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a collagen-binding proteoglycan highly expressed in the developing bones. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-κB inhibitor. However, its role during osteoblast differentiation is still unclear. In this study, we confirmed that the expression of PRELP increased with the osteogenesis induction of preosteoblastic MC3T3-E1 cells. Down-regulation of PRELP expression by shRNA reduced ALP activity, mineralization and expression of osteogenic marker gene Runx2. Our microarray analysis data suggested that β-catenin may act as a hub gene in the PRELP-mediated gene network. We validated furtherly that PRELP knockdown could inhibit the level of connexin43, a key regulator of osteoblast differentiation by affecting β-catenin protein expression, and its nuclear translocation in MC3T3-E1 preosteoblasts. Therefore, this study established a new role of PRELP in modulating β-catenin/connexin43 pathway and osteoblast differentiation. PMID:26809092

  17. The Drosophila melanogaster flightless-I gene involved in gastrulation and muscle degeneration encodes gelsolin-like and leucine-rich repeat domains and is conserved in Caenorhabditis elegans and humans.

    PubMed Central

    Campbell, H D; Schimansky, T; Claudianos, C; Ozsarac, N; Kasprzak, A B; Cotsell, J N; Young, I G; de Couet, H G; Miklos, G L

    1993-01-01

    Mutations at the flightless-I locus (fliI) of Drosophila melanogaster cause flightlessness or, when severe, incomplete cellularization during early embryogenesis, with subsequent abnormalities in mesoderm invagination and in gastrulation. After chromosome walking, deficiency mapping, and transgenic analysis, we have isolated and characterized flightless-I cDNAs, enabling prediction of the complete amino acid sequence of the 1256-residue protein. Data base searches revealed a homologous gene in Caenorhabditis elegans, and we have isolated and characterized corresponding cDNAs. By using the polymerase chain reaction with nested sets of degenerate oligonucleotide primers based on conserved regions of the C. elegans and D. melanogaster proteins, we have cloned a homologous human cDNA. The predicted C. elegans and human proteins are, respectively, 49% and 58% identical to the D. melanogaster protein. The predicted proteins have significant sequence similarity to the actin-binding protein gelsolin and related proteins and, in addition, have an N-terminal domain consisting of a repetitive amphipathic leucine-rich motif. This repeat is found in D. melanogaster, Saccharomyces cerevisiae, and mammalian proteins known to be involved in cell adhesion and in binding to other proteins. The structure of the maternally expressed flightless-I protein suggests that it may play a key role in embryonic cellularization by interacting with both the cytoskeleton and other cellular components. The presence of a highly conserved homologue in nematodes, flies, and humans is indicative of a fundamental role for this protein in many metazoans. PMID:8248259

  18. MicroRNA let-7b induces lens epithelial cell apoptosis by targeting leucine-rich repeat containing G protein-coupled receptor 4 (Lgr4) in age-related cataract.

    PubMed

    Dong, Yuchen; Zheng, Yajuan; Xiao, Jun; Zhu, Chao; Zhao, Meisheng

    2016-06-01

    Owing to a rapidly aging population, vision impairment caused by age-related cataract has become very common. Age-related cataract has also become one of the principal causes of blindness, and apoptosis of lens epithelial cells contributes to non-congenital cataract development. Previous studies have reported that microRNA let-7b (let-7b) is upregulated in cataractous lens epithelial cells, and the expression level of let-7b is positively associated with N, C and P cataract scores. However, the role of let-7b in the development of age-related cataract remains unclear. Here, we observed that the expression level of let-7b in the anterior lens capsules of age-related cataract was significantly higher than that in the normal anterior lens capsules. We performed ultraviolet (UV) irradiation to induce lens epithelial cell apoptosis. The results showed that the expression level of let-7b in lens epithelial cells which were treated by UV irradiation was significantly higher than that in the control, and let-7b promoted UV irradiation-induced apoptosis. Furthermore, we showed that leucine-rich repeat containing G protein-coupled receptor 4 (Lgr4) was a direct target of let-7b, and let-7b modulated lens epithelial cell apoptosis by directly targeting Lgr4. These findings will offer new insights into our understanding of the molecular mechanisms underlying the pathogenesis of cataract. PMID:27179410

  19. The 213-amino-acid leucine-rich repeat region of the listeria monocytogenes InlB protein is sufficient for entry into mammalian cells, stimulation of PI 3-kinase and membrane ruffling.

    PubMed

    Braun, L; Nato, F; Payrastre, B; Mazié, J C; Cossart, P

    1999-10-01

    The Listeria monocytogenes InlB protein is a 630-amino-acid surface protein that mediates entry of the bacterium into a wide variety of cell types, including hepatocytes, fibroblasts and epithelial cells such as Vero, HEp-2 and HeLa cells. Invasion stimulates host proteins tyrosine phosphorylation, PI 3-kinase activity and rearrangements in the actin cytoskeleton. We previously showed that InlB is sufficient for entry of InlB-coated latex beads into cells and recent results indicate that purified InlB can stimulate PI 3-kinase activity and is thus the first bacterial agonist of this lipid kinase. In this study, we identified the region of InlB responsible for entry and stimulation of signal transduction events. Eight monoclonal antibodies directed against InlB were raised and, of those, five inhibited bacterial entry. These five antibodies recognized epitopes within the leucine-rich repeat (LRR) region and/or the inter-repeat (IR) region. InlB-staphylococcal protein A (SPA) fusion proteins and recombinant InlB derivatives were generated and tested for their capacity to mediate entry into cultured mammalian cells. All the InlB derivatives that carried the amino-terminal 213-amino-acid LRR region conferred invasiveness to the normally non-invasive bacterium L. innocua or to inert latex beads and the corresponding purified polypeptides inhibited bacterial entry. In addition, the 213-amino-acid LRR region was able to stimulate PI 3-kinase activity and changes in the actin cytoskeleton (membrane ruffling). These properties were not detected with purified internalin, another invasion protein of L. monocytogenes that displays LRRs similar to those of InlB. Taken together, these results show that the first 213 amino acids of InlB are critical for its specific properties. PMID:10540282

  20. Structural Determinants at the Interface of the ARC2 and Leucine-Rich Repeat Domains Control the Activation of the Plant Immune Receptors Rx1 and Gpa21[C][W][OA

    PubMed Central

    Slootweg, Erik J.; Spiridon, Laurentiu N.; Roosien, Jan; Butterbach, Patrick; Pomp, Rikus; Westerhof, Lotte; Wilbers, Ruud; Bakker, Erin; Bakker, Jaap; Petrescu, Andrei-José; Smant, Geert; Goverse, Aska

    2013-01-01

    Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins. PMID:23660837

  1. A novel mitochondrially-targeted apocynin derivative prevents hyposmia and loss of motor function in the leucine-rich repeat kinase 2 (LRRK2(R1441G)) transgenic mouse model of Parkinson's disease.

    PubMed

    Dranka, Brian P; Gifford, Alison; McAllister, Donna; Zielonka, Jacek; Joseph, Joy; O'Hara, Crystal L; Stucky, Cheryl L; Kanthasamy, Anumantha G; Kalyanaraman, Balaraman

    2014-11-01

    Recently, we demonstrated that dimeric apocynin prevented loss of motor function in the leucine-rich repeat kinase 2 (LRRK2(R1441G)) transgenic (tg) mouse (treated with 200mg/kg, three times per week) [B.P. Dranka et al., Neurosci. Lett. 549 (2013) 57-62]. Here we extend those studies by treating LRRK2(R1441G) mice with an orally-available, mitochondrially-targeted apocynin derivative. We hypothesized that the increased mitochondrial permeability of Mito-apocynin, due to the triphenylphosphonium moiety, would allow improvement of Parkinson's disease (PD) symptoms at lower doses than those required for diapocynin. Tests of motor coordination (pole test, Rotor-Rod) revealed a significant deficit in coordinated motor function in LRRK2(R1441G) mice by 15 months of age. Decreased performance on the pole test and Rotor-Rod in the LRRK2(R1441G) mice was prevented with Mito-apocynin treatment (3mg/kg, three times per week). Decreased olfactory function is an early indication of PD in human patients. LRRK2(R1441G) tg mice displayed deficits in sense of smell in both the hidden treat test, and a radial arm maze test. Interestingly, treatment with Mito-apocynin prevented this hyposmia, and animals retained normal ability to identify either a scented treat or a food pellet as well as wild type littermates. Together, these data demonstrate that the mitochondria-targeted apocynin analog is effective in preventing early PD-like symptoms in the LRRK2(R1441G) mouse model. PMID:25263790

  2. The Blast Resistance Gene Pi37 Encodes a Nucleotide Binding Site–Leucine-Rich Repeat Protein and Is a Member of a Resistance Gene Cluster on Rice Chromosome 1

    PubMed Central

    Lin, Fei; Chen, Shen; Que, Zhiqun; Wang, Ling; Liu, Xinqiong; Pan, Qinghua

    2007-01-01

    The resistance (R) gene Pi37, present in the rice cultivar St. No. 1, was isolated by an in silico map-based cloning procedure. The equivalent genetic region in Nipponbare contains four nucleotide binding site–leucine-rich repeat (NBS–LRR) type loci. These four candidates for Pi37 (Pi37-1, -2, -3, and -4) were amplified separately from St. No. 1 via long-range PCR, and cloned into a binary vector. Each construct was individually transformed into the highly blast susceptible cultivar Q1063. The subsequent complementation analysis revealed Pi37-3 to be the functional gene, while -1, -2, and -4 are probably pseudogenes. Pi37 encodes a 1290 peptide NBS–LRR product, and the presence of substitutions at two sites in the NBS region (V239A and I247M) is associated with the resistance phenotype. Semiquantitative expression analysis showed that in St. No. 1, Pi37 was constitutively expressed and only slightly induced by blast infection. Transient expression experiments indicated that the Pi37 product is restricted to the cytoplasm. Pi37-3 is thought to have evolved recently from -2, which in turn was derived from an ancestral -1 sequence. Pi37-4 is likely the most recently evolved member of the cluster and probably represents a duplication of -3. The four Pi37 paralogs are more closely related to maize rp1 than to any of the currently isolated rice blast R genes Pita, Pib, Pi9, Pi2, Piz-t, and Pi36. PMID:17947408

  3. The Novel Small Leucine-Rich Repeat Protein Podocan is a Negative Regulator of Migration and Proliferation of Smooth Muscle Cells, Modulates Neointima Formation and is Expressed in Human Atheroma

    PubMed Central

    Hutter, Randolph; Huang, Li; Speidl, Walter S.; Giannarelli, Chiara; Trubin, Paul; Bauriedel, Gerhard; Klotman, Mary E.; Fuster, Valentin; Badimon, Juan J.; Klotman, Paul E.

    2014-01-01

    Background SMC migration and proliferation critically influence the clinical course of vascular disease. We tested the effect of the novel small leucine-rich repeat protein podocan on SMC migration and proliferation using a podocan deficient mouse in combination with a model of arterial injury and aortic explant SMC culture. In addition, we examined the effect of overexpression of the human form of podocan on human SMC and tested for podocan expression in human atherosclerosis. In all these conditions we evaluated concomitantly the Wnt-TCF-pathway. Methods and Results Podocan was strongly and selectively expressed in arteries of WT mice after injury. Podocan−/− mice showed increased arterial lesion formation as compared to WT littermates in response to injury (P<0.05). Also, SMC proliferation was increased in arteries of podocan −/− mice compared to WT (P<0.05). In vitro, migration and proliferation were increased in podocan−/− SMC and were normalized by transfection with the WT podocan gene (P<0.05). In addition, upregulation of the Wnt-TCF-pathway was found in SMC of podocan−/− mice both in vitro and in vivo. On the other hand, podocan overexpression in human SMC significantly reduced SMC migration and proliferation inhibiting the Wnt-TCF-pathway. Podocan and a Wnt-TCF-pathway marker were differently expressed in human coronary restenotic versus primary lesions. Conclusions Podocan appears to be a potent negative regulator of the migration and proliferation of both murine and human SMC. The lack of podocan results in excessive arterial repair and prolonged SMC proliferation, which likely is mediated by the Wnt-TCF-pathway. PMID:24043300

  4. Vacuolar protein sorting 35 (Vps35) rescues locomotor deficits and shortened lifespan in Drosophila expressing a Parkinson’s disease mutant of Leucine-rich repeat kinase 2 (LRRK2)

    PubMed Central

    2014-01-01

    Background Parkinson’s disease (PD) is the most common movement neurodegenerative movement disorder. An incomplete understanding of the molecular pathways involved in its pathogenesis impedes the development of effective disease-modifying treatments. To address this gap, we have previously generated a Drosophila model of PD that overexpresses PD pathogenic mutant form of the second most common causative gene of PD, Leucine-Rich Repeat Kinase 2 (LRRK2). Findings We employed this model in a genetic modifier screen and identified a gene that encodes for a core subunit of retromer – a complex essential for the sorting and recycling of specific cargo proteins from endosomes to the trans-Golgi network and cell surface. We present evidence that overexpression of the Vps35 or Vps26 component of the cargo-recognition subunit of the retromer complex ameliorates the pathogenic mutant LRRK2 eye phenotype. Furthermore, overexpression of Vps35 or Vps26 significantly protects from the locomotor deficits observed in mutant LRRK2 flies, as assessed by the negative geotaxis assay, and rescues their shortened lifespan. Strikingly, overexpressing Vps35 alone protects from toxicity of rotenone, a neurotoxin commonly used to model parkinsonism, both in terms of lifespan and locomotor activity of the flies, and this protection is sustained and even augmented in the presence of mutant LRRK2. Finally, we demonstrate that knocking down expression of Vps35 in dopaminergic neurons causes a significant locomotor impairment. Conclusions From these results we conclude that LRRK2 plays a role in the retromer pathway and that this pathway is involved in PD pathogenesis. PMID:24915984

  5. Selective expression of Parkinson's disease-related Leucine-rich repeat kinase 2 G2019S missense mutation in midbrain dopaminergic neurons impairs dopamine release and dopaminergic gene expression.

    PubMed

    Liu, Guoxiang; Sgobio, Carmelo; Gu, Xinglong; Sun, Lixin; Lin, Xian; Yu, Jia; Parisiadou, Loukia; Xie, Chengsong; Sastry, Namratha; Ding, Jinhui; Lohr, Kelly M; Miller, Gary W; Mateo, Yolanda; Lovinger, David M; Cai, Huaibin

    2015-09-15

    Preferential dysfunction/degeneration of midbrain substantia nigra pars compacta (SNpc) dopaminergic (DA) neurons contributes to the main movement symptoms manifested in Parkinson's disease (PD). Although the Leucine-rich repeat kinase 2 (LRRK2) G2019S missense mutation (LRRK2 G2019S) is the most common causative genetic factor linked to PD, the effects of LRRK2 G2019S on the function and survival of SNpc DA neurons are poorly understood. Using a binary gene expression system, we generated transgenic mice expressing either wild-type human LRRK2 (WT mice) or the LRRK2 G2019S mutation (G2019S mice) selectively in the midbrain DA neurons. Here we show that overexpression of LRRK2 G2019S did not induce overt motor abnormalities or substantial SNpc DA neuron loss. However, the LRRK2 G2019S mutation impaired dopamine homeostasis and release in aged mice. This reduction in dopamine content/release coincided with the degeneration of DA axon terminals and decreased expression of DA neuron-enriched genes tyrosine hydroxylase (TH), vesicular monoamine transporter 2, dopamine transporter and aldehyde dehydrogenase 1. These factors are responsible for dopamine synthesis, transport and degradation, and their expression is regulated by transcription factor paired-like homeodomain 3 (PITX3). Levels of Pitx3 mRNA and protein were similarly decreased in the SNpc DA neurons of aged G2019S mice. Together, these findings suggest that PITX3-dependent transcription regulation could be one of the many potential mechanisms by which LRRK2 G2019S acts in SNpc DA neurons, resulting in downregulation of its downstream target genes critical for dopamine homeostasis and release. PMID:26123485

  6. Downregulation of a barley (Hordeum vulgare) leucine-rich repeat, non-arginine-aspartate receptor-like protein kinase reduces expression of numerous genes involved in plant pathogen defense.

    PubMed

    Parrott, David L; Huang, Li; Fischer, Andreas M

    2016-03-01

    Pattern recognition receptors represent a first line of plant defense against pathogens. Comparing the flag leaf transcriptomes of barley (Hordeum vulgare L.) near-isogenic lines varying in the allelic state of a locus controlling senescence, we have previously identified a leucine-rich repeat receptor-like protein kinase gene (LRR-RLK; GenBank accession: AK249842), which was strongly upregulated in leaves of early-as compared to late-senescing germplasm. Bioinformatic analysis indicated that this gene codes for a subfamily XII, non-arginine-aspartate (non-RD) LRR-RLK. Virus-induced gene silencing resulted in a two-fold reduction of transcript levels as compared to controls. Transcriptomic comparison of leaves from untreated plants, from plants treated with virus only without any plant sequences (referred to as 'empty virus' control), and from plants in which AK249842 expression was knocked down identified numerous genes involved in pathogen defense. These genes were strongly induced in 'empty virus' as compared to untreated controls, but their expression was significantly reduced (again compared to 'empty virus' controls) when AK249842 was knocked down, indicating that their expression partially depends on the LRR-RLK investigated here. Expression analysis, using datasets from BarleyBase/PLEXdb, demonstrated that AK249842 transcript levels are heavily influenced by the allelic state of the well-characterized mildew resistance a (Mla) locus, and that the gene is induced after powdery mildew and stem rust infection. Together, our data suggest that AK249842 is a barley pattern recognition receptor with a tentative role in defense against fungal pathogens, setting the stage for its full functional characterization. PMID:26820571

  7. Leucine-rich glioma-inactivated protein 1 antibody encephalitis

    PubMed Central

    Mayasi, Yunis; Takhtani, Deepak

    2014-01-01

    Objective: To describe a case of leucine-rich glioma-inactivated protein 1 (LGI1) antibody–associated encephalitis. Methods: The clinical and ancillary data and brain MRIs were gathered retrospectively by chart review. Relevant literature on similar cases was also reviewed. Results: The diagnosis of LGI1 antibody–associated autoimmune encephalitis was based on the typical clinical presentation of seizures, psychiatric symptoms, and memory loss as well as negative diagnostic testing for cancer; the diagnosis was confirmed by positive LGI1 antibody. The patient responded favorably to treatment with IV immunoglobulin and continues to do well. Conclusion: LGI1 antibody–associated encephalitis has increasingly been recognized as a primary autoimmune disorder with good prognosis and response to treatment. PMID:25520958

  8. Long-term remission with rituximab in refractory leucine-rich glioma inactivated 1 antibody encephalitis.

    PubMed

    Brown, J William L; Martin, Peter J; Thorpe, John W; Michell, Andrew W; Coles, Alasdair J; Cox, Amanda L; Vincent, Angela; Zandi, Michael S

    2014-06-15

    Autoimmune encephalitis associated with antibodies to leucine-rich glioma inactivated 1 (LGI1) is recently described and there is a lack of detailed reports on the treatment of relapsing or refractory cases and long-term outcomes. Two case reports are presented. Both cases had faciobrachial dystonic seizures (FBDS) and received rituximab after relapsing or refractory disease. Both cases achieved sustained clinical remission of up to 15 and 56 months respectively. Rituximab use allowed withdrawal of corticosteroids and was well tolerated. Randomized clinical trials are needed in LGI1 encephalitis and other autoimmune encephalitides. PMID:24703099

  9. An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We conducted a family-wide study to identify and characterize sites of autophosphorylation in 73 representative LRR RLKs of the 223 member LRR RLK family in Arabidopsis thaliana. His-tagged constructs of intact cytoplasmic domains (CDs) for 73 of 223 A. thaliana LRR RLKs were cloned into E. coli BL-...

  10. LEUCINE-RICH AMELOGENIN PEPTIDE INDUCES OSTEOGENESIS IN MOUSE EMBRYONIC STEM CELLS

    PubMed Central

    Warotayanont, Rungnapa; Zhu, Danhong; Snead, Malcolm L.; Zhou, Yan

    2008-01-01

    Leucine-rich amelogenin peptide (LRAP), an alternatively spliced amelogenin protein, possesses a signaling property shown to induce osteogenic differentiation. In the current study, we detected LRAP expression during osteogenesis of wild-type (WT) embryonic stem (ES) cells and observed the absence of LRAP expression in amelogenin-null (KO) ES cells. We explored the signaling effect of LRAP on wild-type ES cells, and the ability of LRAP to rescue the impaired osteogenesis phenotype observed in KO ES cells. Our data indicate that LRAP treatment of WT and KO ES cells induces a significant increase in mineral matrix formation, and significant increases in bone sialoprotein and osterix gene expression. In addition, the amelogenin KO phenotype is partially rescued by the addition of exogenous LRAP. These data suggest a unique function of LRAP during ES cell differentiation along osteogenic lineage. PMID:18086559

  11. Physical exercise and a leucine-rich diet modulate the muscle protein metabolism in Walker tumor-bearing rats.

    PubMed

    Salomão, Emilianne M; Toneto, Aline T; Silva, Gisele O; Gomes-Marcondes, Maria Cristina C

    2010-01-01

    Leucine-supplemented diet can recover lean body mass and preserve muscle protein mass. Additionally, physical exercise can be an excellent alternative to improve the rehabilitation of cancer patients. Knowing these facts, we examined the effects of a leucine-rich diet with or without physical aerobic exercise on muscle protein metabolism in Walker tumor-bearing rats. Young rats were divided into 4 groups that did or did not perform light aerobic exercise (swim training) and were on a leucine-rich diet or a control diet for 2 mo. After this time, these animals were implanted or not with tumors (subcutaneously) following groups for either control diet or leucine-rich diet fed rats: control, trained, tumor-bearing, and trained tumor-bearing. Twenty-one days after implantation, the tumor growth induced a decrease in the muscle protein synthesis and increased the catabolic process, which was associated with an increase in the expression of the ubiquitin-proteasome subunits (20S, 19S, and 11S). In contrast, the exercise program minimized the muscle degradation process and increased muscle myosin content. Additionally, leucine supplementation also modulated proteasome subunits, especially the 19S and 11S. In summary, the exercise has beneficial effects by reducing tumor growth, leading to an improvement in protein turnover especially when in conjunction with a leucine-rich diet. PMID:21058197

  12. An alternative domain containing a leucine-rich sequence regulates nuclear cytoplasmic localization of protein 4.1R.

    PubMed

    Luque, Carlos M; Pérez-Ferreiro, Carmen M; Pérez-Gonzalez, Alicia; Englmeier, Ludwig; Koffa, Maria D; Correas, Isabel

    2003-01-24

    In red blood cells, protein 4.1 (4.1R) is an 80-kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane. The picture is more complex in nucleated cells, in which many 4.1R isoforms, varying in size and intracellular location, have been identified. To contribute to the characterization of signals involved in differential intracellular localization of 4.1R, we have analyzed the role the exon 5-encoded sequence plays in 4.1R distribution. We show that exon 5 encodes a leucine-rich sequence that shares key features with nuclear export signals (NESs). This sequence adopts the topology employed for NESs of other proteins and conserves two hydrophobic residues that are shown to be critical for NES function. A 4.1R isoform expressing the leucine-rich sequence binds to the export receptor CRM1 in a RanGTP-dependent fashion, whereas this does not occur in a mutant whose two conserved hydrophobic residues are substituted. These two residues are also essential for 4.1R intracellular distribution, because the 4.1R protein containing the leucine-rich sequence localizes in the cytoplasm, whereas the mutant protein predominantly accumulates in the nucleus. We hypothesize that the leucine-rich sequence in 4.1R controls distribution and concomitantly function of a specific set of 4.1R isoforms. PMID:12427749

  13. Structural basis for leucine-rich nuclear export signal recognition by CRM1

    SciTech Connect

    Dong, Xiuhua; Biswas, Anindita; Süel, Katherine E.; Jackson, Laurie K.; Martinez, Rita; Gu, Hongmei; Chook, Yuh Min

    2009-07-10

    CRM1 (also known as XPO1 and exportin 1) mediates nuclear export of hundreds of proteins through the recognition of the leucine-rich nuclear export signal (LR-NES). Here we present the 2.9 {angstrom} structure of CRM1 bound to snurportin 1 (SNUPN). Snurportin 1 binds CRM1 in a bipartite manner by means of an amino-terminal LR-NES and its nucleotide-binding domain. The LR-NES is a combined {alpha}-helical-extended structure that occupies a hydrophobic groove between two CRM1 outer helices. The LR-NES interface explains the consensus hydrophobic pattern, preference for intervening electronegative residues and inhibition by leptomycin B. The second nuclear export signal epitope is a basic surface on the snurportin 1 nucleotide-binding domain, which binds an acidic patch on CRM1 adjacent to the LR-NES site. Multipartite recognition of individually weak nuclear export signal epitopes may be common to CRM1 substrates, enhancing CRM1 binding beyond the generally low affinity LR-NES. Similar energetic construction is also used in multipartite nuclear localization signals to provide broad substrate specificity and rapid evolution in nuclear transport.

  14. Anti-leucine-rich glioma-inactivated 1 limbic encephalitis: A case report and literature review

    PubMed Central

    LIU, JINGYAO; LI, MIN; LI, GUIBO; ZHOU, CHUNKUI; ZHANG, RENSHENG

    2016-01-01

    This study describes the case of a 41-year-old woman admitted for anterograde memory loss, right facial grimacing and right arm posturing that had begun 1 month previously. Cranial magnetic resonance-diffusion weighted imaging and -fluid-attenuated inversion recovery imaging revealed a hyperintense signal in the left hippocampus and right basal ganglia, but no contrast enhancement. An electroencephalogram revealed rhythmic sharp and slow waves and rhythmic θ build-ups in the left temporal area. Single-photon emission computed tomography showed increased regional blood flow perfusion in the left cerebral frontal lobe and the right basal ganglia. The cerebrospinal fluid was normal, with the exception of the presence of leucine-rich glioma-inactivated 1 (LGI1) antibodies, and LGI1 antibodies were also found in the blood serum. The presence of the antibodies, the faciobrachial dystonic seizures (FBDSs) and the memory loss indicated limbic encephalitis. After 3 months of immunotherapy, the patient was free from epileptic seizures and had undergone a partial memory restoration. FBDSs alone justify the immediate initiation of immunotherapy, even prior to laboratory confirmation of the disease, as early treatment limits the duration of the illness. PMID:26889260

  15. Key roles for the small leucine-rich proteoglycans in renal and pulmonary pathophysiology

    PubMed Central

    Nastase, Madalina V.; Iozzo, Renato V.; Schaefer, Liliana

    2014-01-01

    Background Small leucine-rich proteoglycans (SLRPs) are molecules that have signaling roles in a multitude of biological processes. In this respect, SLRPs play key roles in the evolution of a variety of diseases throughout the human body. Scope of Review We will critically review current developments in the roles of SLRPs in several types of disease of the kidney and lungs. Particular emphasis will be given to the roles of decorin and biglycan, the best characterized members of the SLRP gene family. Major Conclusions In both renal and pulmonary disorders, SLRPs are essential elements that regulate several pathophysiological processes including fibrosis, inflammation and tumor progression. Decorin has remarkable antifibrotic and antitumorigenic properties and is considered a valuable potential treatment of these diseases. Biglycan can modulate inflammatory processes in lung and renal inflammation and is a potential target in the treatment of inflammatory conditions. General significance SLRPs can serve as either treatment targets or as potential treatment in renal or lung disease. PMID:24508120

  16. The estrous cycle modulates small leucine-rich proteoglycans expression in mouse uterine tissues.

    PubMed

    Salgado, Renato M; Favaro, Rodolfo R; Martin, Sebastian San; Zorn, Telma M T

    2009-01-01

    In the pregnant mouse uterus, small leucine-rich proteoglycans (SLRPs) are drastically remodeled within a few hours after fertilization, suggesting that ovarian hormone levels modulate their synthesis and degradation. In this study, we followed by immunoperoxidase approach, the presence of four members of the SLRP family (decorin, lumican, biglycan, and fibromodulin) in the uterine tissues along the estrous cycle of the mouse. All molecules except fibromodulin, which predominates in the myometrium, showed a striking modulation in their distribution in the endometrial stroma, following the rise in the level of estrogen. Moreover, notable differences in the distribution of SLRPs were observed between superficial and deep stroma, as well as between the internal and external layers of the myometrium. Only biglycan and fibromodulin were expressed in the luminal and glandular epithelia. All four SLRPs were found in cytoplasmic granules of mononucleated cells. The pattern of distribution of the immunoreaction for these molecules in the uterine tissues was found to be estrous cycle-stage dependent, suggesting that these molecules undergo ovarian hormonal control and probably participate in the preparation of the uterus for decidualization and embryo implantation. In addition, this and previous results from our laboratory suggest the existence of two subpopulations of endometrial fibroblasts that may be related to the centrifugal development of the decidua. Anat Rec, 2008. (c) 2008 Wiley-Liss, Inc. PMID:18951514

  17. The small leucine-rich proteoglycan BGN accumulates in CADASIL and binds to NOTCH3

    PubMed Central

    Zhang, Xiaojie; Lee, Soo Jung; Young, Marian F.; Wang, Michael M.

    2015-01-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited form of cerebral small vessel disease caused by mutations in conserved residues of NOTCH3. Affected arteries of CADASIL feature fibrosis and accumulation of NOTCH3. A variety of collagen subtypes (types I, III, IV, and VI) have been identified in fibrotic CADASIL vessels. Biglycan (BGN) and decorin (DCN), are Class I members of the small leucine-rich proteoglycan (SLRP) family that regulate collagen fibril size. Because DCN has been shown to deposit in arteries in cerebral small vessel disease, we tested whether BGN accumulates in arteries of CADASIL brains. BGN was strongly expressed in both small penetrating and leptomeningeal arteries of CADASIL brain. BGN protein was localized to all three layers of arteries (intima, media, and adventitia). Substantially more immunoreactivity was observed in CADASIL brains compared to controls. Immunoblotting of brain lysates showed a 4-fold increase in CADASIL brains (compared to controls). Messenger RNA encoding BGN was also increased in CADASIL and was localized by in situ hybridization to all three vascular layers in CADASIL. Human cerebrovascular smooth muscle cells exposed to purified NOTCH3 ectodomain upregulated BGN, DCN, and COL4A1 through mechanisms that are sensitive to rapamycin, a potent mTOR inhibitor. In addition, BGN protein interacted directly with NOTCH3 protein in cell culture and in direct protein interaction assays. In conclusion, BGN is a CADASIL-enriched protein that potentially accumulates in vessels by mTOR-mediated transcriptional activation and/or post-translational accumulation via protein interactions with NOTCH3 and collagen. PMID:25578324

  18. The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution.

    PubMed

    Tarasevich, Barbara J; Philo, John S; Maluf, Nasib Karl; Krueger, Susan; Buchko, Garry W; Lin, Genyao; Shaw, Wendy J

    2015-04-01

    Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in controlling growth and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, there is limited information on the quaternary structure of LRAP. Here, sedimentation velocity analytical ultracentrifugation (SV) and small angle neutron scattering (SANS) were used to study the tertiary and quaternary structure of LRAP at various pH values, ionic strengths, and concentrations. We found that the monomer is the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7-4.1, pH 4.5-8, 50 mmol/L(mM) to 200 mM NaCl, 0.065-2 mg/mL). The monomer is also the dominant species for unphosphorylated LRAP (LRAP(-P)) at pH 7.4 and for LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregates in a narrow pH range near the isoelectric point of pH 4.1. SV and SANS show that the LRAP monomer has a radius of ∼2.0 nm and an asymmetric structure, and solution NMR studies indicate that the monomer is largely unstructured. This work provides new insights into the secondary, tertiary, and quaternary structure of LRAP in solution and provides evidence that the monomeric species may be an important functional form of some amelogenins. PMID:25449314

  19. The Leucine-Rich Amelogenin Protein (LRAP) is Primarily Monomeric and Unstructured in Physiological Solution

    PubMed Central

    Tarasevich, Barbara J.; Philo, John S.; Maluf, Nasib Karl; Krueger, Susan; Buchko, Garry W.; Lin, Genyao; Shaw, Wendy J.

    2015-01-01

    Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in controlling growth and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, there is limited information on the quaternary structure of LRAP. Here, sedimentation velocity analytical ultracentrifugation (SV) and small angle neutron scattering (SANS) were used to study the tertiary and quaternary structure of LRAP at various pH values, ionic strengths, and concentrations. We found that the monomer is the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7 to 4.1, pH 4.5 to 8, 50 mmol/L(mM) to 200 mM NaCl, 0.065 to 2 mg/mL). The monomer is also the dominant species for unphosphorylated LRAP (LRAP(−P)) at pH 7.4 and for LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregates in a narrow pH range near the isoelectric point of pH 4.1. SV and SANS show that the LRAP monomer has a radius of ~2.0 nm and an asymmetric structure, and solution NMR studies indicate that the monomer is largely unstructured. This work provides new insights into the secondary, tertiary, and quaternary structure of LRAP in solution and provides evidence that the monomeric species may be an important functional form of some amelogenins. PMID:25449314

  20. Inhibitory Role of the Small Leucine-Rich Proteoglycan Biglycan in Bladder Cancer

    PubMed Central

    Niedworok, Christian; Röck, Katharina; Kretschmer, Inga; Freudenberger, Till; Nagy, Nadine; Szarvas, Tibor; vom Dorp, Frank; Reis, Henning; Rübben, Herbert; Fischer, Jens W.

    2013-01-01

    Background Urothelial bladder cancer is the ninth most common cancer. Despite surgical and chemotherapeutic treatment the prognosis is still poor once bladder cancer progresses to a muscle-invasive state. Discovery of new diagnostic markers and pathophysiologic effectors might help to contribute to novel diagnostic and therapeutic options. The extracellular matrix microenvironment shaped by the extracellular matrix critically affects tumor cell and stroma cell functions. Therefore, aim of the present study was to assess the possible implication of the small leucine-rich proteoglycan biglycan in progression of human urothelial bladder cancer. Methods and Results For this purpose tumor biopsies of 76 bladder cancer patients with different tumor stages (pTa, pT1-T4) were investigated with respect to biglycan expression and correlated with a long-term (10 years) clinical follow-up. Interestingly, higher biglycan mRNA expression was associated with higher tumor stages and muscle invasiveness. In vitro knock-down of endogenous biglycan in human urothelial carcinoma cells (J82 cells) increased proliferation, whereas addition of recombinant biglycan and overexpression of biglycan inhibited tumor cell proliferation. In line with this growth-inhibitory effect of biglycan, transplantation of J82 cells after knock-down of biglycan resulted in significantly increased growth of subcutaneous xenograft tumors in nude mice in vivo. Furthermore, treatment with two anti-proliferative, multi-receptor tyrosine kinase inhibitors—sunitinib and sorafenib—strongly upregulated biglycan expression. Collectively, the experimental data suggest that high biglycan expression is associated with reduced tumor cell proliferation. In accordance, Kaplan-Meier analysis revealed higher 10-year survival in patients with high biglycan mRNA expression in tumor biopsies. Conclusion In conclusion, the present data suggest that biglycan is an endogenous inhibitor of bladder cancer cell proliferation that

  1. The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution

    DOE PAGESBeta

    Tarasevich, Barbara J.; Philo, John S.; Maluf, Nasib Karl; Krueger, Susan; Buchko, Garry W.; Lin, Genyao; Shaw, Wendy J.

    2014-10-25

    Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in promoting nucleation, controlling growth, and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, the quaternary structure of LRAP is not as well studied. Here, analytical ultracentrifugation sedimentation velocity (SV) and small angle neutron scatteringmore » (SANS) were used to study the tertiary and quaternary structure of LRAP over a range of pH values, ionic strengths, and concentrations. SV has advantages over other techniques in accurately quantifying protein speciation in polydisperse solutions. We found that the monomer was the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7 to 4.1, pH 4.5 to 8, 50 mmol/L( mM) to 200 mM NaCl, 0.065 to 2 mg/mL). The monomer was also the dominant species for unphosphorylated LRAP (LRAP(-P)) at pH 7.4 and LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregated in a narrow pH range near the isoelectric point (pH 4.1). We conclude that LRAP does not form nanospheres under physiological solution conditions. Both SV and SANS showed that the LRAP monomer has a radius of ~2.0 nm and adopts an extended structure which solution NMR studies show is intrinsically disordered. This work provides new insights into the tertiary and quaternary structure of LRAP and further evidence that the monomeric species is an important functional form of amelogenins« less

  2. The leucine-rich amelogenin protein (LRAP) is primarily monomeric and unstructured in physiological solution

    SciTech Connect

    Tarasevich, Barbara J.; Philo, John S.; Maluf, Nasib Karl; Krueger, Susan; Buchko, Garry W.; Lin, Genyao; Shaw, Wendy J.

    2014-10-25

    Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in promoting nucleation, controlling growth, and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, the quaternary structure of LRAP is not as well studied. Here, analytical ultracentrifugation sedimentation velocity (SV) and small angle neutron scattering (SANS) were used to study the tertiary and quaternary structure of LRAP over a range of pH values, ionic strengths, and concentrations. SV has advantages over other techniques in accurately quantifying protein speciation in polydisperse solutions. We found that the monomer was the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7 to 4.1, pH 4.5 to 8, 50 mmol/L( mM) to 200 mM NaCl, 0.065 to 2 mg/mL). The monomer was also the dominant species for unphosphorylated LRAP (LRAP(-P)) at pH 7.4 and LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregated in a narrow pH range near the isoelectric point (pH 4.1). We conclude that LRAP does not form nanospheres under physiological solution conditions. Both SV and SANS showed that the LRAP monomer has a radius of ~2.0 nm and adopts an extended structure which solution NMR studies show is intrinsically disordered. This work provides new insights into the tertiary and quaternary structure of LRAP and further evidence that the monomeric species is an important functional form of amelogenins

  3. The Leucine-Rich Amelogenin Protein (LRAP) is primarily monomeric and unstructured in physiological solution

    SciTech Connect

    Tarasevich, Barbara J.; Philo, John S.; Maluf, Nasib K.; Krueger, Susan; Buchko, Garry W.; Lin, Genyao; Shaw, Wendy J.

    2015-04-01

    Amelogenin proteins are critical to the formation of enamel in teeth and may have roles in promoting nucleation, controlling growth, and regulating microstructures of the intricately woven hydroxyapatite (HAP). Leucine-rich amelogenin protein (LRAP) is a 59-residue splice variant of amelogenin and contains the N- and C-terminal charged regions of the full-length protein thought to control crystal growth. Although the quaternary structure of full-length amelogenin in solution has been well studied and can consist of self-assemblies of monomers called nanospheres, the quaternary structure of LRAP is not as well studied. Here, analytical ultracentrifugation sedimentation velocity (SV) and small angle neutron scattering (SANS) were used to study the tertiary and quaternary structure of LRAP over a range of pH values, ionic strengths, and concentrations. SV has advantages over other techniques in accurately quantifying protein speciation in polydisperse solutions. We found that the monomer was the dominant species of phosphorylated LRAP (LRAP(+P)) over a range of solution conditions (pH 2.7 to 3.9, pH 4.5 to 8, 50 mmol/L( mM) to 200 mM NaCl, 0.065 to 2 mg/mL). The monomer was also the dominant species for unphosphorylated LRAP (LRAP(-P)) at pH 7.4 and LRAP(+P) in the presence of 2.5 mM calcium at pH 7.4. LRAP aggregated in a narrow pH range near the isoelectric point (pH 4.1). We conclude that LRAP does not form nanospheres under physiological solution conditions. Both SV and SANS showed that the LRAP monomer has a radius of ~2.0 nm and adopts an extended structure which solution NMR studies show is intrinsically disordered. This work provides new insights into the tertiary and quaternary structure of LRAP and further evidence that the monomeric species is an important functional form of amelogenins

  4. Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes.

    PubMed

    Kurucz, Eva; Márkus, Róbert; Zsámboki, János; Folkl-Medzihradszky, Katalin; Darula, Zsuzsanna; Vilmos, Péter; Udvardy, Andor; Krausz, Ildikó; Lukacsovich, Tamás; Gateff, Elisabeth; Zettervall, Carl-Johan; Hultmark, Dan; Andó, István

    2007-04-01

    The hemocytes, the blood cells of Drosophila, participate in the humoral and cellular immune defense reactions against microbes and parasites [1-8]. The plasmatocytes, one class of hemocytes, are phagocytically active and play an important role in immunity and development by removing microorganisms as well as apoptotic cells. On the surface of circulating and sessile plasmatocytes, we have now identified a protein, Nimrod C1 (NimC1), which is involved in the phagocytosis of bacteria. Suppression of NimC1 expression in plasmatocytes inhibited the phagocytosis of Staphylococcus aureus. Conversely, overexpression of NimC1 in S2 cells stimulated the phagocytosis of both S. aureus and Escherichia coli. NimC1 is a 90-100 kDa single-pass transmembrane protein with ten characteristic EGF-like repeats (NIM repeats). The nimC1 gene is part of a cluster of ten related nimrod genes at 34E on chromosome 2, and similar clusters of nimrod-like genes are conserved in other insects such as Anopheles and Apis. The Nimrod proteins are related to other putative phagocytosis receptors such as Eater and Draper from D. melanogaster and CED-1 from C. elegans. Together, they form a superfamily that also includes proteins that are encoded in the human genome. PMID:17363253

  5. Role of Tropomodulin’s Leucine Rich Repeat Domain in the Formation of Neurite-like Processes

    PubMed Central

    2015-01-01

    Actin dynamics is fundamental for neurite development; monomer depolymerization from pointed ends is rate-limiting in actin treadmilling. Tropomodulins (Tmod) make up a family of actin pointed end-capping proteins. Of the four known isoforms, Tmod1–Tmod3 are expressed in brain cells. We investigated the role of Tmod’s C-terminal (LRR) domain in the formation of neurite-like processes by overexpressing Tmod1 and Tmod2 with deleted or mutated LRR domains in PC12 cells, a model system used to study neuritogenesis. Tmod1 overexpression results in a normal quantity and a normal length of processes, while Tmod2 overexpression reduces both measures. The Tmod2 overexpression phenotype is mimicked by overexpression of Tmod1 with the LRR domain removed or with three point mutations in the LRR domain that disrupt exposed clusters of conserved residues. Removal of Tmod2’s LRR domain does not significantly alter the outgrowth of neurite-like processes compared to that of Tmod2. Overexpression of chimeras with the N-terminal and C-terminal domains switched between Tmod1 and Tmod2 reinforces the idea that Tmod1’s LRR domain counteracts the reductive effect of the Tmod N-terminal domain upon formation of processes while Tmod2’s LRR domain does not. We suggest that the TM-dependent actin capping ability of both Tmods inhibits the formation of processes, but in Tmod1, this inhibition can be controlled via its LRR domain. Circular dichroism, limited proteolysis, and molecular dynamics demonstrate structural differences in the C-terminal region of the LRR domains of Tmod1, Tmod2, and the Tmod1 mutant. PMID:24746171

  6. The influence of Leucine-rich amelogenin peptide on MSC fate by inducing Wnt10b expression.

    PubMed

    Wen, Xin; Cawthorn, William P; MacDougald, Ormond A; Stupp, Samuel I; Snead, Malcolm L; Zhou, Yan

    2011-09-01

    Amelogenin is the most abundant protein of the enamel organic matrix and is a structural protein indispensable for enamel formation. One of the amelogenin splicing isoforms, Leucine-rich Amelogenin Peptide (LRAP) induces osteogenesis in various cell types. Previously, we demonstrated that LRAP activates the canonical Wnt signaling pathway to induce osteogenic differentiation of mouse ES cells through the concerted regulation of Wnt agonists and antagonists. There is a reciprocal relationship between osteogenic and adipogenic differentiation in bone marrow mesenchymal stem cells (BMMSCs). Wnt10b-mediated activation of canonical Wnt signaling has been shown to regulate mesenchymal stem cell fate. Using the bipotential bone marrow stromal cell line ST2, we have demonstrated that LRAP activates the canonical Wnt/β-catenin signaling pathway. A specific Wnt inhibitor sFRP-1 abolishes the effect of LRAP on the stimulation of osteogenesis and the inhibition of adipogenesis of ST2 cells. LRAP treatment elevates the Wnt10b expression level whereas Wnt10b knockdown by siRNA abrogates the effect of LRAP. We show here that LRAP promotes osteogenesis of mesenchymal stem cells at the expense of adipogenesis through upregulating Wnt10b expression to activate Wnt signaling. PMID:21663957

  7. A leucine-rich diet and exercise affect the biomechanical characteristics of the digital flexor tendon in rats after nutritional recovery.

    PubMed

    Barbosa, Alexandre Wesley Carvalho; Benevides, Gustavo Pereira; Alferes, Leda Maria Totti; Salomão, Emilianne Miguel; Gomes-Marcondes, Maria Cristina Cintra; Gomes, Laurecir

    2012-01-01

    An increase in the capacity of athletic performance depends on adequate nutrition, which ensures optimal function of the musculoskeletal system, including tendon stability. However, little is known about the status of tendons and extracellular matrix modifications during malnutrition and nutritional recovery when leucine is used in response to exercise conditioning. The purpose of this study was to evaluate the collagen content and biomechanical aspects of the deep digital flexor tendon (DDFT) in malnourished rats submitted to nutritional recovery (control diet or leucine-rich diet) and aerobic physical activity. After 60 days of undernourishment (6% protein diet), the malnourished rats were subsequently nutritionally recovered with a control diet or leucine-rich diet and trained or not (swimming, without overload) for 5 weeks. The biomechanical analysis and quantification of hydroxyproline were assessed in the DDFT in all experimental groups. The leucine-rich diet increased hydroxyproline content in the tension region, independently of the training. In the compression region, hydroxyproline content was higher in the malnourished and leucine-trained groups. Biomechanical analysis showed a lower load in the malnourished and all-trained groups. The lowest stress was observed with control-trained animals. The nutritional-recovered groups showed higher strain values corresponding to control group, while the lowest values were observed in malnourished and trained groups. The results suggest that a leucine-rich diet stimulates collagen synthesis of the DDFT, especially when in combination with physical exercise, and seems to determine the increase of resistance and the biomechanical characteristics of tendons. PMID:21107621

  8. Proline-, glutamic acid-, and leucine-rich protein 1 mediates estrogen rapid signaling and neuroprotection in the brain

    PubMed Central

    Sareddy, Gangadhara R.; Zhang, Quanguang; Wang, Ruimin; Scott, Erin; Zou, Yi; O'Connor, Jason C.; Chen, Yidong; Dong, Yan; Vadlamudi, Ratna K.; Brann, Darrell

    2015-01-01

    17-β estradiol (E2) has been implicated as neuroprotective in a variety of neurodegenerative disorders. However, the underlying mechanism remains unknown. Here, we provide genetic evidence, using forebrain-specific knockout (FBKO) mice, that proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), an estrogen receptor coregulator protein, is essential for the extranuclear signaling and neuroprotective actions of E2 in the hippocampal CA1 region after global cerebral ischemia (GCI). E2-mediated extranuclear signaling (including activation of extracellular signal-regulated kinase and Akt) and antiapoptotic effects [such as attenuation of JNK signaling and increase in phosphorylation of glycogen synthase kinase-3β (GSK3β)] after GCI were compromised in PELP1 FBKO mice. Mechanistic studies revealed that PELP1 interacts with GSK3β, E2 modulates interaction of PELP1 with GSK3β, and PELP1 is a novel substrate for GSK3β. RNA-seq analysis of control and PELP1 FBKO mice after ischemia demonstrated alterations in several genes related to inflammation, metabolism, and survival in PELP1 FBKO mice, as well as a significant reduction in the activation of the Wnt/β-catenin signaling pathway. In addition, PELP1 FBKO studies revealed that PELP1 is required for E2-mediated neuroprotection and for E2-mediated preservation of cognitive function after GCI. Collectively, our data provide the first direct in vivo evidence, to our knowledge, of an essential role for PELP1 in E2-mediated rapid extranuclear signaling, neuroprotection, and cognitive function in the brain. PMID:26627258

  9. Over-Expression of Cysteine Leucine Rich Protein Is Related to SAG Resistance in Clinical Isolates of Leishmania donovani

    PubMed Central

    Das, Sanchita; Shah, Priyanka; Tandon, Rati; Yadav, Narendra Kumar; Sahasrabuddhe, Amogh A.; Sundar, Shyam; Siddiqi, Mohammad Imran; Dube, Anuradha

    2015-01-01

    Background Resistance emergence against antileishmanial drugs, particularly Sodium Antimony Gluconate (SAG) has severely hampered the therapeutic strategy against visceral leishmaniasis, the mechanism of resistance being indistinguishable. Cysteine leucine rich protein (CLrP), was recognized as one of the overexpressed proteins in resistant isolates, as observed in differential proteomics between sensitive and resistant isolates of L. donovani. The present study deals with the characterization of CLrP and for its possible connection with SAG resistance. Methodology and Principal Findings In pursuance of deciphering the role of CLrP in SAG resistance, gene was cloned, over-expressed in E. coli system and thereafter antibody was raised. The expression profile of CLrP and was found to be over-expressed in SAG resistant clinical isolates of L. donovani as compared to SAG sensitive ones when investigated by real-time PCR and western blotting. CLrP has been characterized through bioinformatics, immunoblotting and immunolocalization analysis, which reveals its post-translational modification along with its dual existence in the nucleus as well as in the membrane of the parasite. Further investigation using a ChIP assay confirmed its DNA binding potential. Over-expression of CLrP in sensitive isolate of L. donovani significantly decreased its responsiveness to SAG (SbV and SbIII) and a shift towards the resistant mode was observed. Further, a significant increase in its infectivity in murine macrophages has been observed. Conclusion/Significance The study reports the differential expression of CLrP in SAG sensitive and resistant isolates of L. donovani. Functional intricacy of CLrP increases with dual localization, glycosylation and DNA binding potential of the protein. Further over-expressing CLrP in sensitive isolate of L. donovani shows significantly decreased sensitivity towards SAG and increased infectivity as well, thus assisting the parasite in securing a safe niche

  10. Clinical analysis of leucine-rich glioma inactivated-1 protein antibody associated with limbic encephalitis onset with seizures

    PubMed Central

    Li, Zhimei; Cui, Tao; Shi, Weixiong; Wang, Qun

    2016-01-01

    Abstract We summarized the clinical characteristics of patients presenting with seizures and limbic encephalitis (LE) associated with leucine-rich glioma inactivated-1 protein antibody (LGI1) in order help recognize and treat this condition at its onset. We analyzed clinical, video electroencephalogram (VEEG), magnetic resonance imaging (MRI), and laboratory data of 10 patients who presented with LGI1-LE and followed up their outcomes from 2 to 16 (9.4 ± 4.2) months. All patients presented with seizures onset, including faciobrachial dystonic seizure (FBDS), partial seizure (PS), and generalized tonic-clonic seizure (GTCS). Four patients (Cases 3, 5, 7, and 8) had mild cognitive deficits. Interictal VEEG showed normal patterns, focal slowing, or sharp waves in the temporal or frontotemporal lobes. Ictal VEEG of Cases 4, 5, and 7 showed diffuse voltage depression preceding FBDS, a left frontal/temporal origin, and a bilateral temporal origin, respectively. Ictal foci could not be localized in other cases. MRI scan revealed T2/fluid-attenuated inversion recovery (FLAIR) hyperintensity and evidence of edema in the right medial temporal lobe in Case 3, left hippocampal atrophy in Case 5, hyperintensities in the bilateral medial temporal lobes in Case 7, and hyperintensities in the basal ganglia and frontal cortex in Case 10. All 10 serum samples were positive for LGI1 antibody, but it was only detected in the cerebrospinal fluid (CSF) of 7 patients. Five patients (Cases 2, 4, 6, 7, and 8) presented with hyponatremia. One patient (Case 2) was diagnosed with small cell lung cancer. While responses to antiepileptic drugs (AEDs) were poor, most patients (except Case 2) responded favorably to immunotherapy. LGI1-LE may initially manifest with various types of seizures, particularly FBDS and complex partial seizures (CPS) of mesial temporal origin, and slowly progressive cognitive involvement. Clinical follow-up, VEEG monitoring, and MRI scan are helpful in early

  11. A case of autoimmune epilepsy associated with anti-leucine-rich glioma inactivated subunit 1 antibodies manifesting electrical shock-like sensations and transparent sadness

    PubMed Central

    Murata, Yoshiko; Watanabe, Osamu; Taniguchi, Go; Sone, Daichi; Fujioka, Mao; Okazaki, Mitsutoshi; Nakagawa, Eiji; Watanabe, Yutaka; Watanabe, Masako

    2015-01-01

    Autoimmune epilepsy is an isolated phenotype of autoimmune encephalitis, which may be suspected in patients with unexplained adult-onset seizure disorders or resistance to antiepileptic drugs (AEDs). Antibodies against leucine-rich glioma inactivated subunit 1 of the voltage-gated potassium channel (VGKC) complex, recently termed anti-LGI-1 antibodies, are one of the causes of autoimmune epilepsies. Bizarre symptoms with extremely short duration and high frequency are clues to the possible presence of autoimmune epilepsy with anti-LGI-1 antibodies. Precise diagnosis is important because autoimmune epilepsy is treatable and the prognosis can be predicted. PMID:26543815

  12. Subcellular localization of CrmA: identification of a novel leucine-rich nuclear export signal conserved in anti-apoptotic serpins.

    PubMed Central

    Rodriguez, Jose A; Span, Simone W; Kruyt, Frank A E; Giaccone, Giuseppe

    2003-01-01

    The cowpox virus-encoded anti-apoptotic protein cytokine response modifier A (CrmA) is a member of the serpin family that specifically inhibits the cellular proteins caspase 1, caspase 8 and granzyme B. In this study, we have used Flag- and yellow fluorescent protein (YFP)-tagged versions of CrmA to investigate the mechanisms that regulate its subcellular localization. We show that CrmA can actively enter and exit the nucleus and we demonstrate the role of the nuclear export receptor CRM1 in this shuttling process. CrmA contains a novel leucine-rich nuclear export signal (NES) that is functionally conserved in the anti-apoptotic cellular serpin PI-9. Besides this leucine-rich export signal, additional sequences mapping to a 103-amino-acid region flanking the NES contribute to the CRM1-dependent nuclear export of CrmA. Although YFP-tagged CrmA is primarily located in the cytoplasm, shifting its localization to be predominantly nuclear by fusion of a heterologous nuclear localization signal did not impair its ability to prevent Fas-induced apoptosis. We propose that nucleocytoplasmic shuttling would allow CrmA to efficiently target cellular pro-apoptotic proteins not only in the cytoplasm, but also in the nucleus, and thus to carry out its anti-apoptotic function in both compartments. PMID:12667137

  13. Nuclear export of cutaneous HPV8 E7 oncoprotein is mediated by a leucine-rich nuclear export signal via a CRM1 pathway

    SciTech Connect

    Onder, Zeynep; Chang, Vivian; Moroianu, Junona

    2015-01-01

    We recently determined that the nuclear import of cutaneous beta genus HPV8 E7 oncoprotein it is mediated by its zinc-binding domain via direct hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Onder and Moroianu, 2014). Here we investigated the nuclear export of HPV8 E7 oncoprotein using confocal microscopy after transfections of HeLa cells with EGFP–8cE7 and mutant plasmids and treatment with Ratjadone A nuclear export inhibitor. We determined that HPV8 E7 contains a leucine-rich nuclear export signal (NES), {sub 76}IRTFQELLF{sub 84}, within its zinc-binding domain that mediates its nuclear export via a CRM1 pathway. We found that HPV8 E7 interacts with CRM1 and that the hydrophobic amino acid residues I76, F79 and L82 of the NES are essential for this interaction and for nuclear export of HPV8 E7 oncoprotein. - Highlights: • HPV8 E7 has a leucine-rich NES within its zinc-binding domain that mediates its nuclear export. • CRM1 nuclear export receptor interacts with HPV8 E7 and mediates its export. • Identification of the critical hydrophobic amino acids of the NES of HPV8 E7.

  14. [Isolation and characterization of an unknown, leucine-rich 3.1-S-alpha2-glycoprotein from human serum (author's transl)].

    PubMed

    Haupt, H; Baudner, S

    1977-06-01

    This article describes the isolation and characterization of a previously unknown, leucine-rich 3.1S-alpha2-glycoprotein from human serum. The starting material was Supernatant II, which is a byproduct in the large-scale preparation of albumin and gamma-globulin by the ethacridine lactate/ammonium sulfate procedure. The purified protein is homogenous both in carrier-free and molecular-sieve electrophoresis. Its electrophoretic mobility indicates that it belongs to the alpha2-globulins. Isoelectric focussing splits it into 4 bands with isoelectric points between 3.8 and 4.1. In the ultracentrifuge it sediments in a single band at 3.1S. The molecular weight determined by equilibrium sedimentation is 49 600 +/- 4 000. Subunits were not detected. Chemical analysis reveals it to be a glycoprotein with a carbohydrate content of 23%. The amino acid content is unusual in that the leucine content is almost 17%, i.e. about every fifth amino acid is a leucine. The average concentration of the leucine-rich 3.1S-alpha2-glycoprotein in human serum was determined by a quantitative immunological method as 2.1 mg per 100 ml. The protein is not related to any of the previously known well characterized serum proteins. PMID:69600

  15. Abundant Intergenic TAACTGA Direct Repeats and Putative Alternate RNA Polymerase β′ Subunits in Marine Beggiatoaceae Genomes: Possible Regulatory Roles and Origins

    PubMed Central

    MacGregor, Barbara J.

    2015-01-01

    The genome sequences of several giant marine sulfur-oxidizing bacteria present evidence of a possible post-transcriptional regulatory network that may have been transmitted to or from two distantly related bacteria lineages. The draft genome of a Cand. “Maribeggiatoa” filament from the Guaymas Basin (Gulf of California, Mexico) seafloor contains 169 sets of TAACTGA direct repeats and one indirect repeat, with two to six copies per set. Related heptamers are rarely or never found as direct repeats. TAACTGA direct repeats are also found in some other Beggiatoaceae, Thiocystis violascens, a range of Cyanobacteria, and five Bacteroidetes. This phylogenetic distribution suggests they may have been transmitted horizontally, but no mechanism is evident. There is no correlation between total TAACTGA occurrences and repeats per genome. In most species the repeat units are relatively short, but longer arrays of up to 43 copies are found in several Bacteroidetes and Cyanobacteria. The majority of TAACTGA repeats in the Cand. “Maribeggiatoa” Orange Guaymas (BOGUAY) genome are within several nucleotides upstream of a putative start codon, suggesting they may be binding sites for a post-transcriptional regulator. Candidates include members of the ribosomal protein S1, Csp (cold shock protein), and Csr (carbon storage regulator) families. No pattern was evident in the predicted functions of the open reading frames (ORFs) downstream of repeats, but some encode presumably essential products such as ribosomal proteins. Among these is an ORF encoding a possible alternate or modified RNA polymerase beta prime subunit, predicted to have the expected subunit interaction domains but lacking most catalytic residues. A similar ORF was found in the Thioploca ingrica draft genome, but in no others. In both species they are immediately upstream of putative sensor kinase genes with nearly identical domain structures. In the marine Beggiatoaceae, a role for the TAACTGA repeats in

  16. Abundant Intergenic TAACTGA Direct Repeats and Putative Alternate RNA Polymerase β' Subunits in Marine Beggiatoaceae Genomes: Possible Regulatory Roles and Origins.

    PubMed

    MacGregor, Barbara J

    2015-01-01

    The genome sequences of several giant marine sulfur-oxidizing bacteria present evidence of a possible post-transcriptional regulatory network that may have been transmitted to or from two distantly related bacteria lineages. The draft genome of a Cand. "Maribeggiatoa" filament from the Guaymas Basin (Gulf of California, Mexico) seafloor contains 169 sets of TAACTGA direct repeats and one indirect repeat, with two to six copies per set. Related heptamers are rarely or never found as direct repeats. TAACTGA direct repeats are also found in some other Beggiatoaceae, Thiocystis violascens, a range of Cyanobacteria, and five Bacteroidetes. This phylogenetic distribution suggests they may have been transmitted horizontally, but no mechanism is evident. There is no correlation between total TAACTGA occurrences and repeats per genome. In most species the repeat units are relatively short, but longer arrays of up to 43 copies are found in several Bacteroidetes and Cyanobacteria. The majority of TAACTGA repeats in the Cand. "Maribeggiatoa" Orange Guaymas (BOGUAY) genome are within several nucleotides upstream of a putative start codon, suggesting they may be binding sites for a post-transcriptional regulator. Candidates include members of the ribosomal protein S1, Csp (cold shock protein), and Csr (carbon storage regulator) families. No pattern was evident in the predicted functions of the open reading frames (ORFs) downstream of repeats, but some encode presumably essential products such as ribosomal proteins. Among these is an ORF encoding a possible alternate or modified RNA polymerase beta prime subunit, predicted to have the expected subunit interaction domains but lacking most catalytic residues. A similar ORF was found in the Thioploca ingrica draft genome, but in no others. In both species they are immediately upstream of putative sensor kinase genes with nearly identical domain structures. In the marine Beggiatoaceae, a role for the TAACTGA repeats in translational

  17. Structural and Molecular Characterization of Iron-sensing Hemerythrin-like Domain within F-box and Leucine-rich Repeat Protein 5 (FBXL5)♦

    PubMed Central

    Thompson, Joel W.; Salahudeen, Ameen A.; Chollangi, Srinivas; Ruiz, Julio C.; Brautigam, Chad A.; Makris, Thomas M.; Lipscomb, John D.; Tomchick, Diana R.; Bruick, Richard K.

    2012-01-01

    Mammalian cells maintain iron homeostasis by sensing changes in bioavailable iron levels and promoting adaptive responses. FBXL5 is a subunit of an E3 ubiquitin ligase complex that mediates the stability of iron regulatory protein 2, an important posttranscriptional regulator of several genes involved in iron metabolism. The stability of FBXL5 is regulated in an iron- and oxygen-responsive manner, contingent upon the presence of its N-terminal domain. Here we present the atomic structure of the FBXL5 N terminus, a hemerythrin-like α-helical bundle fold not previously observed in mammalian proteins. The core of this domain employs an unusual assortment of amino acids necessary for the assembly and sensing properties of its diiron center. These regulatory features govern the accessibility of a mapped sequence required for proteasomal degradation of FBXL5. Detailed molecular and structural characterization of the ligand-responsive hemerythrin domain provides insights into the mechanisms by which FBXL5 serves as a unique mammalian metabolic sensor. PMID:22253436

  18. Diverged Alleles of the Anopheles gambiae Leucine-Rich Repeat Gene APL1A Display Distinct Protective Profiles against Plasmodium falciparum

    PubMed Central

    Mitri, Christian; Riehle, Michelle M.; Bischoff, Emmanuel; Brito-Fravallo, Emma; Takashima, Eizo; Thiery, Isabelle; Zettor, Agnes; Petres, Stephane; Bourgouin, Catherine; Vernick, Kenneth D.; Eiglmeier, Karin

    2012-01-01

    Functional studies have demonstrated a role for the Anopheles gambiae APL1A gene in resistance against the human malaria parasite, Plasmodium falciparum. Here, we exhaustively characterize the structure of the APL1 locus and show that three structurally different APL1A alleles segregate in the Ngousso colony. Genetic association combined with RNAi-mediated gene silencing revealed that APL1A alleles display distinct protective profiles against P. falciparum. One APL1A allele is sufficient to explain the protective phenotype of APL1A observed in silencing experiments. Epitope-tagged APL1A isoforms expressed in an in vitro hemocyte-like cell system showed that under assay conditions, the most protective APL1A isoform (APL1A2) localizes within large cytoplasmic vesicles, is not constitutively secreted, and forms only one protein complex, while a less protective isoform (APL1A1) is constitutively secreted in at least two protein complexes. The tested alleles are identical to natural variants in the wild A. gambiae population, suggesting that APL1A genetic variation could be a factor underlying natural heterogeneity of vector susceptibility to P. falciparum. PMID:23285147

  19. Hemerythrin-like Domain within F-box and Leucine-rich Repeat Protein 5 (FBXL5) Communicates Cellular Iron and Oxygen Availability by Distinct Mechanisms

    PubMed Central

    Chollangi, Srinivas; Thompson, Joel W.; Ruiz, Julio C.; Gardner, Kevin H.; Bruick, Richard K.

    2012-01-01

    Iron regulatory proteins play a principal role in maintaining cellular iron homeostasis by post-transcriptionally regulating factors responsible for iron uptake, utilization, and storage. An E3 ubiquitin ligase complex containing FBXL5 targets IRP2 for proteasomal degradation under iron- and oxygen-replete conditions, whereas FBXL5 itself is degraded when iron and oxygen availability decreases. FBXL5 contains a hemerythrin-like (Hr) domain at its N terminus that mediates its own differential stability. Here, we investigated the iron- and oxygen-dependent conformational changes within FBXL5-Hr that underlie its role as a cellular sensor. As predicted, FBXL5-Hr undergoes substantive structural changes when iron becomes limiting, accounting for its switch-like behavior. However, these same changes are not observed in response to oxygen depletion, indicating that this domain accommodates two distinct sensing mechanisms. Moreover, FBXL5-Hr does not behave as a dynamic sensor that continuously samples the cellular environment, assuming conformations in equilibrium with ever-changing cellular iron levels. Instead, the isolated domain appears competent to incorporate iron only at or near the time of its own synthesis. These observations have important implications for mechanisms by which these metabolites are sensed within mammalian cells. PMID:22648410

  20. Structural and molecular characterization of iron-sensing hemerythrin-like domain within F-box and leucine-rich repeat protein 5 (FBXL5).

    PubMed

    Thompson, Joel W; Salahudeen, Ameen A; Chollangi, Srinivas; Ruiz, Julio C; Brautigam, Chad A; Makris, Thomas M; Lipscomb, John D; Tomchick, Diana R; Bruick, Richard K

    2012-03-01

    Mammalian cells maintain iron homeostasis by sensing changes in bioavailable iron levels and promoting adaptive responses. FBXL5 is a subunit of an E3 ubiquitin ligase complex that mediates the stability of iron regulatory protein 2, an important posttranscriptional regulator of several genes involved in iron metabolism. The stability of FBXL5 is regulated in an iron- and oxygen-responsive manner, contingent upon the presence of its N-terminal domain. Here we present the atomic structure of the FBXL5 N terminus, a hemerythrin-like α-helical bundle fold not previously observed in mammalian proteins. The core of this domain employs an unusual assortment of amino acids necessary for the assembly and sensing properties of its diiron center. These regulatory features govern the accessibility of a mapped sequence required for proteasomal degradation of FBXL5. Detailed molecular and structural characterization of the ligand-responsive hemerythrin domain provides insights into the mechanisms by which FBXL5 serves as a unique mammalian metabolic sensor. PMID:22253436

  1. Hemerythrin-like domain within F-box and leucine-rich repeat protein 5 (FBXL5) communicates cellular iron and oxygen availability by distinct mechanisms.

    PubMed

    Chollangi, Srinivas; Thompson, Joel W; Ruiz, Julio C; Gardner, Kevin H; Bruick, Richard K

    2012-07-01

    Iron regulatory proteins play a principal role in maintaining cellular iron homeostasis by post-transcriptionally regulating factors responsible for iron uptake, utilization, and storage. An E3 ubiquitin ligase complex containing FBXL5 targets IRP2 for proteasomal degradation under iron- and oxygen-replete conditions, whereas FBXL5 itself is degraded when iron and oxygen availability decreases. FBXL5 contains a hemerythrin-like (Hr) domain at its N terminus that mediates its own differential stability. Here, we investigated the iron- and oxygen-dependent conformational changes within FBXL5-Hr that underlie its role as a cellular sensor. As predicted, FBXL5-Hr undergoes substantive structural changes when iron becomes limiting, accounting for its switch-like behavior. However, these same changes are not observed in response to oxygen depletion, indicating that this domain accommodates two distinct sensing mechanisms. Moreover, FBXL5-Hr does not behave as a dynamic sensor that continuously samples the cellular environment, assuming conformations in equilibrium with ever-changing cellular iron levels. Instead, the isolated domain appears competent to incorporate iron only at or near the time of its own synthesis. These observations have important implications for mechanisms by which these metabolites are sensed within mammalian cells. PMID:22648410

  2. Recombination within a nucleotide-binding-site/leucine-rich-repeat gene cluster produces new variants conditioning resistance to soybean mosaic virus in soybeans.

    PubMed Central

    Hayes, A J; Jeong, S C; Gore, M A; Yu, Y G; Buss, G R; Tolin, S A; Maroof, M A Saghai

    2004-01-01

    The soybean Rsv1 gene for resistance to soybean mosaic virus (SMV; Potyvirus) has previously been described as a single-locus multi-allelic gene mapping to molecular linkage group (MLG) F. Various Rsv1 alleles condition different responses to the seven (G1-G7) described strains of SMV, including extreme resistance, localized and systemic necrosis, and mosaic symptoms. We describe the cloning of a cluster of NBS-LRR resistance gene candidates from MLG F of the virus-resistant soybean line PI96983 and demonstrate that multiple genes within this cluster interact to condition unique responses to SMV strains. In addition to cloning 3gG2, a strong candidate for the major Rsv1 resistance gene from PI96983, we describe various unique resistant and necrotic reactions coincident with the presence or absence of other members of this gene cluster. Responses of recombinant lines from a high-resolution mapping population of PI96983 (resistant) x Lee 68 (susceptible) demonstrate that more than one gene in this region of the PI96983 chromosome conditions resistance and/or necrosis to SMV. In addition, the soybean cultivars Marshall and Ogden, which carry other previously described Rsv1 alleles, are shown to possess the 3gG2 gene in a NBS-LRR gene cluster background distinct from PI96983. These observations suggest that two or more related non-TIR-NBS-LRR gene products are likely involved in the allelic response of several Rsv1-containing lines to SMV. PMID:15020438

  3. Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen.

    PubMed

    Veena, Mariswamy; Melvin, Prasad; Prabhu, Sreedhara Ashok; Shailasree, Sekhar; Shetty, Hunthrike Shekar; Kini, Kukkundoor Ramachandra

    2016-03-01

    Downy mildew caused by Sclerospora graminicola is a devastating disease of pearl millet. Based on candidate gene approach, a set of 22 resistance gene analogues were identified. The clone RGPM 301 (AY117410) containing a partial sequence shared 83% similarity to rice R-proteins. A full-length R-gene RGA RGPM 301 of 3552 bp with 2979 bp open reading frame encoding 992 amino acids was isolated by the degenerate primers and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) approach. It had a molecular mass of 113.96 kDa and isoelectric point (pI) of 8.71. The sequence alignment and phylogenetic analysis grouped it to a non-TIR NBS LRR group. The quantitative real-time PCR (qRT-PCR) analysis revealed higher accumulation of the transcripts following inoculation with S. graminicola in the resistant cultivar (IP18296) compared to susceptible cultivar (7042S). Further, significant induction in the transcript levels were observed when treated with abiotic elicitor β-aminobutyric acid (BABA) and biotic elicitor Pseudomonas fluorescens. Exogenous application of phytohormones jasmonic acid or salicylic acid also up-regulated the expression levels of RGA RGPM 301. The treatment of cultivar IP18296 with mitogen-activated protein kinase (MPK) inhibitors (PD98059 and U0126) suppressed the levels of RGA RGPM 301. A 3.5 kb RGA RGPM 301 which is a non-TIR NBS-LRR protein was isolated from pearl millet and its up-regulation during downy mildew interaction was demonstrated by qRT-PCR. These studies indicate a role for this RGA in pearl millet downy mildew interaction. PMID:26842722

  4. Effects of hyperbaric oxygen therapy on NACHT domain-leucine-rich-repeat- and pyrin domain-containing protein 3 inflammasome expression in rats following spinal cord injury.

    PubMed

    Liang, Fang; Li, Chunsheng; Gao, Chunjin; Li, Zhuo; Yang, Jing; Liu, Xuehua; Wang, Yong

    2015-06-01

    The clinical application of hyperbaric oxygen therapy (HBOT) in spinal cord injury (SCI) has been reported, however the mechanism underlying its therapeutic effects remains to be elucidated. In the present study, SCI was modeled in male Sprague‑Dawley rats. A total of 120 rats were randomly divided into four groups: Sham‑operated group (SH); sham‑operated and hyperbaric oxygen group (SH+HBO); spinal cord injury group (SCI) and spinal cord injury and hyperbaric oxygen treatment group (SCI+HBO). The rats in each group were randomly divided into five smaller groups (12 h, 1, 3, 7 and 14 days after surgery). The mRNA and protein expression levels of NACHT domain‑, leucine‑rich‑repeat‑ and pyrin domain‑containing protein 3 (NALP3) inflammasome, including NALP3, adaptor molecule apoptosis‑associated speck‑like protein (ASC) and caspase‑1 were determined at several time points following injury. The results of the present study demonstrated that HBOT compromised the mRNA and protein expression levels of NALP3, ASC and caspase‑1 in the SCI model rats and HBOT mitigated SCI‑induced interleukin 1β release in the injured spinal cord tissue. It was concluded that HBOT is an effective approach, which can prevent against spinal cord injury, likely by inactivating NALP3 inflammasome. PMID:25672366

  5. Mechanisms of haplotype divergence at the RGA08 nucleotide-binding leucine-rich repeat gene locus in wild banana (Musa balbisiana)

    PubMed Central

    2010-01-01

    Background Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW). Results Sequence comparison revealed two regions of contrasting features. The first is a highly colinear gene-rich region where the two haplotypes diverge only by single nucleotide polymorphisms and two repetitive element insertions. The second corresponds to a large cluster of RGA08 genes, with 13 and 18 predicted RGA genes and pseudogenes spread over 131 and 152 kb respectively on each haplotype. The RGA08 cluster is enriched in repetitive element insertions, in duplicated non-coding intergenic sequences including low complexity regions and shows structural variations between haplotypes. Although some allelic relationships are retained, a large diversity of RGA08 genes occurs in this single M. balbisiana genotype, with several RGA08 paralogs specific to each haplotype. The RGA08 gene family has evolved by mechanisms of unequal recombination, intragenic sequence exchange and diversifying selection. An unequal recombination event taking place between duplicated non-coding intergenic sequences resulted in a different RGA08 gene content between haplotypes pointing out the role of such duplicated regions in the evolution of RGA clusters. Based on the synonymous substitution rate in coding sequences, we estimated a 1 million year divergence time for these M. balbisiana haplotypes. Conclusions A large RGA08 gene cluster identified in wild banana corresponds to a highly variable genomic region between haplotypes surrounded by conserved flanking regions. High level of sequence identity (70 to 99%) of the genic and intergenic regions suggests a recent and rapid evolution of this cluster in M. balbisiana. PMID:20637079

  6. Deletion of the PH-domain and Leucine-rich Repeat Protein Phosphatase 1 (Phlpp1) Increases Fibroblast Growth Factor (Fgf) 18 Expression and Promotes Chondrocyte Proliferation*

    PubMed Central

    Bradley, Elizabeth W.; Carpio, Lomeli R.; Newton, Alexandra C.; Westendorf, Jennifer J.

    2015-01-01

    Endochondral ossification orchestrates formation of the vertebrate skeleton and is often induced during disease and repair processes of the musculoskeletal system. Here we show that the protein phosphatase Phlpp1 regulates endochondral ossification. Phlpp1 null mice exhibit decreased bone mass and notable changes in the growth plate, including increased BrdU incorporation and matrix production. Phosphorylation of known Phlpp1 substrates, Akt2, PKC, and p70 S6 kinase, were enhanced in ex vivo cultured Phlpp1−/− chondrocytes. Furthermore, Phlpp1 deficiency diminished FoxO1 levels leading to increased expression of Fgf18, Mek/Erk activity, and chondrocyte metabolic activity. Phlpp inhibitors also increased matrix content, Fgf18 production and Erk1/2 phosphorylation. Chemical inhibition of Fgfr-signaling abrogated elevated Erk1/2 phosphorylation and metabolic activity in Phlpp1-null cultures. These results demonstrate that Phlpp1 controls chondrogenesis via multiple mechanisms and that Phlpp1 inhibition could be a strategy to promote cartilage regeneration and repair. PMID:25953896

  7. Chaperone complex BAG2-HSC70 regulates localization of Caenorhabditis elegans leucine-rich repeat kinase LRK-1 to the Golgi.

    PubMed

    Fukuzono, Takashi; Pastuhov, Strahil Iv; Fukushima, Okinobu; Li, Chun; Hattori, Ayuna; Iemura, Shun-Ichiro; Natsume, Tohru; Shibuya, Hiroshi; Hanafusa, Hiroshi; Matsumoto, Kunihiro; Hisamoto, Naoki

    2016-04-01

    Mutations in LRRK2 are linked to autosomal dominant forms of Parkinson's disease. We identified two human proteins that bind to LRRK2: BAG2 and HSC70, which are known to form a chaperone complex. We characterized the role of their Caenorhabditis elegans homologues, UNC-23 and HSP-1, in the regulation of LRK-1, the sole homologue of human LRRK2. In C. elegans, LRK-1 determines the polarized sorting of synaptic vesicle (SV) proteins to the axons by excluding SV proteins from the dendrite-specific transport machinery in the Golgi. In unc-23 mutants, SV proteins are localized to both presynaptic and dendritic endings in neurons, a phenotype also observed in lrk-1 deletion mutants. Furthermore, we isolated mutations in the hsp-1 gene that can suppress the unc-23, but not the lrk-1 defect. We show that UNC-23 determines LRK-1 localization to the Golgi apparatus in cooperation with HSP-1. These results describe a chaperone-dependent mechanism through which LRK-1 localization is regulated. PMID:26853528

  8. Crystal structure of the leucine-rich repeat domain of the NOD-like receptor NLRP1: implications for binding of muramyl dipeptide.

    PubMed

    Reubold, Thomas F; Hahne, Gernot; Wohlgemuth, Sabine; Eschenburg, Susanne

    2014-09-17

    The NOD-like receptor NLRP1 (NLR family, pyrin domain containing 1) senses the presence of the bacterial cell wall component l-muramyl dipeptide (MDP) inside the cell. We determined the crystal structure of the LRR domain of human NLRP1 in the absence of MDP to a resolution of 1.65Å. The fold of the structure can be assigned to the ribonuclease inhibitor-like class of LRR proteins. We compared our structure with X-ray models of the LRR domains of NLRX1 and NLRC4 and a homology model of the LRR domain of NOD2. We conclude that the MDP binding site of NLRP1 is not located in the LRR domain. PMID:25064844

  9. Acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) contributes to retinoic acid-induced differentiation of leukemic cells

    SciTech Connect

    Yu, Yun; Shen, Shao-Ming; Zhang, Fei-Fei; Wu, Zhao-Xia; Han, Bin; Wang, Li-Shun

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer ANP32B was down-regulated during ATRA-induced leukemic cell differentiation. Black-Right-Pointing-Pointer Knockdown of ANP32B enhanced ATRA-induced leukemic cell differentiation. Black-Right-Pointing-Pointer Ectopic expression of ANP32B inhibited ATRA-induced leukemic cell differentiation. Black-Right-Pointing-Pointer ANP32B inhibited ATRA activated transcriptional activity of RAR{alpha}. -- Abstract: The acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) is a member of a conserved superfamily of nuclear proteins whose functions are largely unknown. In our previous work, ANP32B was identified as a novel direct substrate for caspase-3 and acted as a negative regulator for leukemic cell apoptosis. In this work, we provided the first demonstration that ANP32B expression was down-regulated during differentiation induction of leukemic cells by all-trans retinoic acid (ATRA). Knockdown of ANP32B expression by specific shRNA enhanced ATRA-induced leukemic cell differentiation, while ectopic expression of ANP32B attenuated it, indicating an inhibitory role of ANP32B against leukemic cell differentiation. Furthermore, luciferase reporter assay revealed that ANP32B might exert this role through inhibiting the ATRA dependent transcriptional activity of retinoic acid receptor (RAR{alpha}). These data will shed new insights into understanding the biological functions of ANP32B protein.

  10. [A case of smoldering anti-leucine-rich glioma-inactivated 1 (LGI1) antibody-associated limbic encephalitis with faciobrachial dystonic seizure].

    PubMed

    Nakaoku, Yuriko; Maki, Takakuni; Kanazawa, Kyoko; Matsumoto, Riki; Fukuyama, Hidenao; Takahashi, Ryosuke; Ikeda, Akio

    2013-01-01

    We report a 59-year-old right-handed woman with smoldering leucine-rich glioma-inactivated 1 (LGI1) antibody-associated limbic encephalitis (LE) following faciobrachial dystonic seizures. During 8 months before her admission, she developed partial seizures manifesting very brief and very frequent dystonia in her right hand sometimes with oral automatism and loss of awareness. In addition, she showed psychiatric disturbances such as emotionally labile condition and personality changes. On admission, neuropsychological examination revealed short-term memory impairment. During electroencephalography (EEG) monitoring, ictal EEG showed rhythmic delta waves and interictal EEG showed intermittent irregular slow waves at the bilateral frontotemporal area. Brain MRI demonstrated high T2/FLAIR signal changes in the left amygdala expanding into the left hippocampus. FDG-PET showed hypermetabolism in the left amygdala, hippocampus and the bilateral basal ganglia. Cerebrospinal fluid analysis was unremarkable. There were no signs of malignant tumor detected on systemic examination. LGI1 antibody was positive in the serum and the cerebrospinal fluid and the clinical diagnosis of LGI1 antibody-associated LE was confirmed. Her symptoms and the abnormalities in the brain MRI/FDG-PET showed immediate improvement after anti-epileptic and steroid therapy. PMID:24097318

  11. Molecular cloning and expression of the gene for a major leucine-rich protein from human hepatoblastoma cells (HepG2).

    PubMed

    Hou, J; Wang, F; McKeehan, W L

    1994-02-01

    The human hepatoblastoma cell line, HepG2, exhibits an array of stable properties in culture that have made it a popular cell culture model for studies on regulation of liver-specific gene expression and properties of hepatoma cells. In contrast to other hepatoma cell lines, HepG2 cells overexpress a characteristic detergent-extractable, wheat germ lectin-binding protein with apparent molecular mass of 130 kDa. Using an antibody to screen a phage expression library of HepG2 complementary DNA (cDNA), we identified and cloned a 4734 base pair cDNA which codes for a 130-kDa leucine-rich protein (lrp 130) when expressed in transfected cells. The deduced sequence of lrp130 exhibits sequences weakly homologous to the consensus sequence for the ATP binding site in ATP-dependent kinases and the protein kinase C phosphorylation site of the epidermal growth factor receptor. Consistent with the higher levels of expression of lrp130 antigen, Northern hybridization analysis indicated that HepG2 cells express high levels of the major 4.8 kilobase lrp130 mRNA relative to other hepatoma cells. Although currently of unknown function, lrp130 may be of utility as a marker for liver cell lineages represented by the HepG2 cell line. PMID:8012652

  12. Acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) association with lymph node metastasis predicts poor survival in oral squamous cell carcinoma patients

    PubMed Central

    Lee, Chien-Hung; Lin, Shu-Hui; Chin, Mei-Chung; Chiang, Shang-Lun; Wang, Zhi-Hong; Hua, Chun-Hung; Tsai, Ming-Hsui; Chang, Jan-Gowth; Ko, Ying-Chin

    2016-01-01

    Acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) is a multifunctional protein aberrantly expressed in various types of cancers. However, its expression pattern and clinical significance in oral squamous cell carcinoma (OSCC) remains unclear. In this study, we immunohistochemically investigated the expression pattern of ANP32A in 259 OSCC patients and the results were correlated with clinicopathological factors using Allred, Klein and Immunoreactive scoring (IRS) system. Our data indicated that high expression of ANP32A was significantly associated with N stage and tumor differentiation status in OSCC patients. High ANP32A expression with N2/N3 stage had an increased mortality risk than low ANP32A expressing OSCC patients with N0/N1 stage. Functional studies revealed that knockdown of ANP32A significantly decreased the migration and invasion ability thereby concomitantly increasing E-cadherin and decreasing Slug, Claudin-1 and Vimentin expression in vitro. These results suggest that ANP32A is commonly increased in oral squamous cell carcinoma and ANP32A protein could act as a potential biomarker for prognosis assessment of oral cancer patients with lymph node metastasis. PMID:26918356

  13. Leptoglycin: a new Glycine/Leucine-rich antimicrobial peptide isolated from the skin secretion of the South American frog Leptodactylus pentadactylus (Leptodactylidae).

    PubMed

    Sousa, Juliana C; Berto, Raquel F; Gois, Elicélia A; Fontenele-Cardi, Nauíla C; Honório, José E R; Konno, Katsuhiro; Richardson, Michael; Rocha, Marcos F G; Camargo, Antônio A C M; Pimenta, Daniel C; Cardi, Bruno A; Carvalho, Krishnamurti M

    2009-07-01

    Antimicrobial peptides are components of innate immunity that is the first-line defense against invading pathogens for a wide range of organisms. Here, we describe the isolation, biological characterization and amino acid sequencing of a novel neutral Glycine/Leucine-rich antimicrobial peptide from skin secretion of Leptodactylus pentadactylus named leptoglycin. The amino acid sequence of the peptide purified by RP-HPLC (C(18) column) was deduced by mass spectrometric de novo sequencing and confirmed by Edman degradation: GLLGGLLGPLLGGGGGGGGGLL. Leptoglycin was able to inhibit the growth of Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Citrobacter freundii with minimal inhibitory concentrations (MICs) of 8 microM, 50 microM, and 75 microM respectively, but it did not show antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Enterococcus faecalis), yeasts (Candida albicans and Candida tropicalis) and dermatophytes fungi (Microsporum canis and Trichophyton rubrum). No hemolytic activity was observed at the 2-200 microM range concentration. The amino acid sequence of leptoglycin with high level of glycine (59.1%) and leucine (36.4%) containing an unusual central proline suggests the existence of a new class of Gly/Leu-rich antimicrobial peptides. Taken together, these results suggest that this natural antimicrobial peptide could be a tool to develop new antibiotics. PMID:19298834

  14. Potential of mean force analysis of the self-association of leucine-rich transmembrane α-helices: Difference between atomistic and coarse-grained simulations

    NASA Astrophysics Data System (ADS)

    Nishizawa, Manami; Nishizawa, Kazuhisa

    2014-08-01

    Interaction of transmembrane (TM) proteins is important in many biological processes. Large-scale computational studies using coarse-grained (CG) simulations are becoming popular. However, most CG model parameters have not fully been calibrated with respect to lateral interactions of TM peptide segments. Here, we compare the potential of mean forces (PMFs) of dimerization of TM helices obtained using a MARTINI CG model and an atomistic (AT) Berger lipids-OPLS/AA model (ATOPLS). For helical, tryptophan-flanked, leucine-rich peptides (WL15 and WALP15) embedded in a parallel configuration in an octane slab, the ATOPLS PMF profiles showed a shallow minimum (with a depth of approximately 3 kJ/mol; i.e., a weak tendency to dimerize). A similar analysis using the CHARMM36 all-atom model (ATCHARMM) showed comparable results. In contrast, the CG analysis generally showed steep PMF curves with depths of approximately 16-22 kJ/mol, suggesting a stronger tendency to dimerize compared to the AT model. This CG > AT discrepancy in the propensity for dimerization was also seen for dilauroylphosphatidylcholine (DLPC)-embedded peptides. For a WL15 (and WALP15)/DLPC bilayer system, ATOPLS PMF showed a repulsive mean force for a wide range of interhelical distances, in contrast to the attractive forces observed in the octane system. The change from the octane slab to the DLPC bilayer also mitigated the dimerization propensity in the CG system. The dimerization energies of CG (AALALAA)3 peptides in DLPC and dioleoylphosphatidylcholine bilayers were in good agreement with previous experimental data. The lipid headgroup, but not the length of the lipid tails, was a key causative factor contributing to the differences between octane and DLPC. Furthermore, the CG model, but not the AT model, showed high sensitivity to changes in amino acid residues located near the lipid-water interface and hydrophobic mismatch between the peptides and membrane. These findings may help interpret CG and AT

  15. Leucine-rich amelogenin peptide (LRAP) as a surface primer for biomimetic remineralization of superficial enamel defects: An in vitro study.

    PubMed

    Shafiei, Farhad; Hossein, Bagheri G; Farajollahi, Mohammad M; Fathollah, Moztarzadeh; Marjan, Behroozibakhsh; Tahereh, Jafarzadeh Kashi

    2015-01-01

    This study was carried out to obtain more information about the assembly of hydroxyapatite bundles formed in the presence of Leucine-Rich Amelogenin Peptide (LRAP) and to evaluate its effect on the remineralization of enamel defects through a biomimetic approach. One or 2 mg/mL LRAP solutions containing 2.5 mM of Ca(+2) and 1.5 mM phosphate were prepared (pH = 7.2) and stored at 37 °C for 24 h. The products of the reaction were studied using atomic force microscopy (AFM), transmission electron microscopy (TEM), and selected area electron diffraction (SAED). Vickers surface microhardness recovery (SMR%) of acid-etched bovine enamel, with or without LRAP surface treatment, were calculated to evaluate the influence of peptide on the lesion remineralization. Distilled water and 1 or 2 mg/mL LRAP solution (pH = 7.2) were applied on the lesions and the specimens were incubated in mineralization solution (2.5mM Ca(+2) , 1.5mM PO4 (-3) , pH = 7.2) for 24 h. One-way ANOVA and Tukey's multi-comparison tests were used for statistical analysis. The pattern of enamel surface repair was studied using FE-SEM. AFM showed the formation of highly organized hierarchical structures, composed of hydroxyapatite (HA) crystals, similar to the dental enamel microstructure. ANOVA procedure showed significant effect of peptide treatment on the calculated SMR% (p < 0.001). Tukey's test revealed that peptide treated groups had significantly higher values of SMR%. In conclusion, LRAP is able to regulate the formation of HA and enhances the remineralization of acid-etched enamel as a surface treatment agent. PMID:25676352

  16. ICP27 mediates HSV RNA export by shuttling through a leucine-rich nuclear export signal and binding viral intronless RNAs through an RGG motif

    PubMed Central

    Sandri-Goldin, Rozanne M.

    1998-01-01

    Infection of metazoan cells with some viruses alters the balance of cellular mRNA export to favor viral RNA export and to retain cellular transcripts in the nucleus. Here, evidence is presented to show that the herpes simplex virus 1 (HSV-1) essential regulatory protein ICP27, which inhibits host cell-splicing, resulting in the accumulation of unspliced transcripts in the nucleus, mediates RNA export of viral intronless mRNAs. ICP27 was shown to shuttle between the nucleus and cytoplasm through a leucine-rich nuclear export signal, which alone was able to direct the export of the heterologous green fluorescent protein. In vivo UV irradiation studies demonstrated that ICP27 could be crosslinked to poly(A)+ RNA in the nucleus and the cytoplasm, supporting a role in export. Furthermore, the amount of hnRNP A1, which has been implicated in the export of cellular spliced mRNAs, that was bound to poly(A)+ RNA in HSV-1-infected cells was reduced compared with uninfected cells. In addition, it was demonstrated that ICP27 bound seven intronless HSV-1 transcripts in both the nucleus and the cytoplasm, and export of these transcripts was diminished substantially during infection with an ICP27 null mutant virus. In contrast, ICP27 did not bind to two HSV-1 mRNAs that undergo splicing. Finally, binding of ICP27 to RNA in vivo required an arginine-glycine region that resembles an RGG box. These results indicate that ICP27 is an important viral export factor that promotes the transport of HSV-1 intronless RNAs. PMID:9512520

  17. Potential of mean force analysis of the self-association of leucine-rich transmembrane α-helices: Difference between atomistic and coarse-grained simulations

    SciTech Connect

    Nishizawa, Manami; Nishizawa, Kazuhisa

    2014-08-21

    Interaction of transmembrane (TM) proteins is important in many biological processes. Large-scale computational studies using coarse-grained (CG) simulations are becoming popular. However, most CG model parameters have not fully been calibrated with respect to lateral interactions of TM peptide segments. Here, we compare the potential of mean forces (PMFs) of dimerization of TM helices obtained using a MARTINI CG model and an atomistic (AT) Berger lipids-OPLS/AA model (AT{sup OPLS}). For helical, tryptophan-flanked, leucine-rich peptides (WL15 and WALP15) embedded in a parallel configuration in an octane slab, the AT{sup OPLS} PMF profiles showed a shallow minimum (with a depth of approximately 3 kJ/mol; i.e., a weak tendency to dimerize). A similar analysis using the CHARMM36 all-atom model (AT{sup CHARMM}) showed comparable results. In contrast, the CG analysis generally showed steep PMF curves with depths of approximately 16–22 kJ/mol, suggesting a stronger tendency to dimerize compared to the AT model. This CG > AT discrepancy in the propensity for dimerization was also seen for dilauroylphosphatidylcholine (DLPC)-embedded peptides. For a WL15 (and WALP15)/DLPC bilayer system, AT{sup OPLS} PMF showed a repulsive mean force for a wide range of interhelical distances, in contrast to the attractive forces observed in the octane system. The change from the octane slab to the DLPC bilayer also mitigated the dimerization propensity in the CG system. The dimerization energies of CG (AALALAA){sub 3} peptides in DLPC and dioleoylphosphatidylcholine bilayers were in good agreement with previous experimental data. The lipid headgroup, but not the length of the lipid tails, was a key causative factor contributing to the differences between octane and DLPC. Furthermore, the CG model, but not the AT model, showed high sensitivity to changes in amino acid residues located near the lipid-water interface and hydrophobic mismatch between the peptides and membrane. These

  18. Identification of a putative nuclear export signal motif in human NANOG homeobox domain

    SciTech Connect

    Park, Sung-Won; Do, Hyun-Jin; Huh, Sun-Hyung; Sung, Boreum; Uhm, Sang-Jun; Song, Hyuk; Kim, Nam-Hyung; Kim, Jae-Hwan

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer We found the putative nuclear export signal motif within human NANOG homeodomain. Black-Right-Pointing-Pointer Leucine-rich residues are important for human NANOG homeodomain nuclear export. Black-Right-Pointing-Pointer CRM1-specific inhibitor LMB blocked the potent human NANOG NES-mediated nuclear export. -- Abstract: NANOG is a homeobox-containing transcription factor that plays an important role in pluripotent stem cells and tumorigenic cells. To understand how nuclear localization of human NANOG is regulated, the NANOG sequence was examined and a leucine-rich nuclear export signal (NES) motif ({sup 125}MQELSNILNL{sup 134}) was found in the homeodomain (HD). To functionally validate the putative NES motif, deletion and site-directed mutants were fused to an EGFP expression vector and transfected into COS-7 cells, and the localization of the proteins was examined. While hNANOG HD exclusively localized to the nucleus, a mutant with both NLSs deleted and only the putative NES motif contained (hNANOG HD-{Delta}NLSs) was predominantly cytoplasmic, as observed by nucleo/cytoplasmic fractionation and Western blot analysis as well as confocal microscopy. Furthermore, site-directed mutagenesis of the putative NES motif in a partial hNANOG HD only containing either one of the two NLS motifs led to localization in the nucleus, suggesting that the NES motif may play a functional role in nuclear export. Furthermore, CRM1-specific nuclear export inhibitor LMB blocked the hNANOG potent NES-mediated export, suggesting that the leucine-rich motif may function in CRM1-mediated nuclear export of hNANOG. Collectively, a NES motif is present in the hNANOG HD and may be functionally involved in CRM1-mediated nuclear export pathway.

  19. Activation of Autophagy and Nucleotide-Binding Domain Leucine-Rich Repeat–Containing-Like Receptor Family, Pyrin Domain–Containing 3 Inflammasome during Leishmania infantum–Associated Glomerulonephritis

    PubMed Central

    Esch, Kevin J.; Schaut, Robert G.; Lamb, Ian M.; Clay, Gwendolyn; Morais Lima, Ádila L.; do Nascimento, Paulo R.P.; Whitley, Elizabeth M.; Jeronimo, Selma M.B.; Sutterwala, Fayyaz S.; Haynes, Joseph S.; Petersen, Christine A.

    2016-01-01

    Chronic kidney disease is a major contributor to human and companion animal morbidity and mortality. Renal complications are sequelae of canine and human visceral leishmaniasis (VL). Despite the high incidence of infection-mediated glomerulonephritis, little is known about pathogenesis of VL-associated renal disease. Leishmania infantum–infected dogs are a naturally occurring model of VL-associated glomerulonephritis. Membranoproliferative glomerulonephritis type I [24 of 25 (96%)], with interstitial lymphoplasmacytic nephritis [23 of 25 (92%)], and glomerular and interstitial fibrosis [12 of 25 (48%)] were predominant lesions. An ultrastructural evaluation of glomeruli from animals with VL identified mesangial cell proliferation and interposition. Immunohistochemistry demonstrated significant Leishmania antigen, IgG, and C3b deposition in VL dog glomeruli. Asymptomatic and symptomatic dogs had increased glomerular nucleotide-binding domain leucine-rich repeat–containing-like receptor family, pyrin domain containing 3 and autophagosome-associated microtubule-associated protein 1 light chain 3 associated with glomerular lesion severity. Transcriptional analyses from symptomatic dogs confirmed induction of autophagy and inflammasome genes within glomeruli and tubules. On the basis of temporal VL staging, glomerulonephritis was initiated by IgG and complement deposition. This deposition preceded presence of nucleotide-binding domain leucine-rich repeat–containing-like receptor family, pyrin domain containing 3–associated inflammasomes and increased light chain 3 puncta indicative of autophagosomes in glomeruli from dogs with clinical VL and renal failure. These findings indicate potential roles for inflammasome complexes in glomerular damage during VL and autophagy in ensuing cellular responses. PMID:26079813

  20. Essential role of PH domain and leucine-rich repeat protein phosphatase 2 in Nrf2 suppression via modulation of Akt/GSK3β/Fyn kinase axis during oxidative hepatocellular toxicity.

    PubMed

    Rizvi, F; Shukla, S; Kakkar, P

    2014-01-01

    Instances of sustained oxidative activity have been shown to involve dysregulation of Nrf2-mediated transcriptional induction; however, mechanisms warranting Nrf2-repression remain unclear. In this study, using primary rat hepatocytes, we have attempted to identify factors that may negatively influence Nrf2 survival pathway. Though studies indicate a conspicuous association between Akt and Nrf2, a confirmatory link between the two is unaddressed. On inhibiting PI3K/Akt pathway, we observed compromised activities of antioxidant and detoxification enzymes culminating in oxidative cytotoxicity. This was accompanied by reduced nuclear retention of Nrf2 and its ARE binding affinity, increased Nrf2 ubiquitination and concurrent decline in its downstream targets. Moreover, Akt inhibition enhanced nuclear translocation as well as phosphorylation of Fyn kinase, an enzyme linked to Nrf2 degradation, by relieving GSK3β from phosphorylation-mediated repression. The involvement of Akt and Fyn kinase in influencing Nrf2 signaling was further confirmed in oxidatively stressed hepatocytes by using tert-butyl hydroperoxide (tBHP). tBHP-induced decrease in Nrf2 levels was associated with enhanced Fyn kinase phosphorylation, Fyn kinase nuclear translocation and decreased levels of phosphorylated GSK3β(Ser9) in a time-dependent manner. Interestingly, tBHP induced site-specific deactivation of Akt as only Akt(Ser473) phosphorylation was observed to be affected. Further, protein expression as well as nuclear localization of PHLPP2, a phosphatase specific for Akt(Ser473), was found to be significantly enhanced in tBHP-stressed hepatocytes. Silencing of PHLPP2 not only resulted in considerable restoration of Nrf2 signaling, enhanced Nrf2-ARE binding and reduced Nrf2 ubiquitination but also significantly suppressed tBHP-induced ROS generation and alterations in mitochondrial permeability. We infer that cellular PHLPP2 levels may aggravate oxidative toxicity by suppressing Nrf2/ARE transcriptional regulation via Akt(Se473)/GSK3β/Fyn kinase axis. The study indicates that PHLPP2 could serve as a new target for developing strategies to manage pathological conditions exacerbated due to oxidative stress. PMID:24675471

  1. The Viral Restriction Factor Tetherin Prevents Leucine-rich Pentatricopeptide Repeat-containing Protein (LRPPRC) from Association with Beclin 1 and B-cell CLL/lymphoma 2 (Bcl-2) and Enhances Autophagy and Mitophagy*

    PubMed Central

    Zou, Jing; Li, Wenjiao; Misra, Anisha; Yue, Fei; Song, Kun; Chen, Qi; Guo, Guanghua; Yi, Jinglin; Kimata, Jason T.; Liu, Leyuan

    2015-01-01

    Tetherin has been characterized as a key factor that restricts viral particles such as HIV and hepatitis C virus on plasma membranes, acts as a ligand of the immunoglobulin-like transcript 7 (ILT7) receptor in tumor cells, and suppresses antiviral innate immune responses mediated by human plasmacytoid dendritic cells. However, the normal cellular function of Tetherin without viral infection is unknown. Here we show that Tetherin not only serves as a substrate of autophagy but itself regulates the initiation of autophagy. Tetherin interacts with the autophagy/mitophagy suppressor LRPPRC and prevents LRPPRC from forming a ternary complex with Beclin 1 and Bcl-2 so that Beclin 1 is released to bind with PI3KCIII (class III PI3K) to activate the initiation of autophagy. Suppression of Tetherin leads to impairment of autophagy, whereas overexpression of Tetherin causes activation of autophagy. Under mitophagic stress, Tetherin is concentrated on mitochondria engulfed in autophagosomes. Tetherin plays a general role in the degradation of autophagosomes containing not only the symbiotic mitochondria but also, possibly, the infected virus. Therefore, Tetherin may enhance autophagy and mitophagy to suppress tumorigenesis, enhance innate immune responses, or prevent T cell apoptosis or pyroptosis. PMID:25631043

  2. The flexible structure of the K24S28 region of Leucine-Rich Amelogenin Protein (LRAP) bound to apatites as a function of surface type, calcium, mutation, and ionic strength

    SciTech Connect

    Lu, Junxia; Burton, Sarah D.; Xu, Yimin; Buchko, Garry W.; Shaw, Wendy J.

    2014-07-11

    Leucine-Rich Amelogenin Protein (LRAP) is a member of the amelogenin family of biomineralization proteins, proteins which play a critical role in enamel formation. Recent studies have revealed the structure and orientation of the N- and C-terminus of LRAP bound to hydroxyapatite (HAP), a surface used as an analog of enamel. The structure of one region, K24 to S28, was found to be sensitive to phosphorylation of S16, the only naturally observed site of serine phosphorylation in LRAP, suggesting that the residues from K24 to S28 may sit at a key region of structural flexibility and play a role in the protein’s function. In this work, we investigated the sensitivity of the structure and orientation of this region when bound to HAP as a function of several factors which may vary during enamel formation to influence structure: the ionic strength (0.05 M, 0.15 M, 0.2 M), the calcium concentration (0.07 mM and 0.4 mM), and the surface to which it is binding (HAP and carbonated apatite (CAP), a more direct mimic of enamel). A naturally occurring mutation found in amelogenin (T21I), was also investigated. The structure in the K24S28 region of the protein was found to be sensitive to these conditions, with the CAP surface and excess Ca2+ (8:1 [Ca2+]:[LRAP-K24S28(+P)]) resulting in a much tighter helix, while low ionic strength relaxed the helical structure. Higher ionic strength and the point mutation did not result in any structural change in this region. The distance of the backbone of K24 from the surface was most sensitive to excess Ca2+ and in the T21I-mutation. Collectively, these data suggest that the protein is able to accommodate structural changes while maintaining its interaction with the surface, and provides further evidence of the structural sensitivity of the K24 to S28 region, a sensitivity that may contribute to function in biomineralization. This research was supported by NIH-NIDCR Grant DE-015347. The research was performed at the Pacific Northwest

  3. The flexible structure of the K24S28 region of Leucine-Rich Amelogenin Protein (LRAP) bound to apatites as a function of surface type, calcium, mutation, and ionic strength

    PubMed Central

    Lu, Jun-xia; Burton, Sarah D.; Xu, Yimin S.; Buchko, Garry W.; Shaw, Wendy J.

    2014-01-01

    Leucine-Rich Amelogenin Protein (LRAP) is a member of the amelogenin family of biomineralization proteins, proteins which play a critical role in enamel formation. Recent studies have revealed the structure and orientation of the N- and C-terminus of LRAP bound to hydroxyapatite (HAP), a surface used as an analog of enamel. The structure of one region, K24 to S28, was found to be sensitive to phosphorylation of S16, the only naturally observed site of serine phosphorylation in LRAP, suggesting that K24S28 may sit at a key region of structural flexibility and play a role in the protein's function. In this work, we investigated the sensitivity of the structure and orientation of this region when bound to HAP as a function of several factors which may vary during enamel formation to influence structure: the ionic strength (0.05, 0.15, 0.2 M), the calcium concentration (0.07 and 0.4 mM), and the surface to which it is binding [HAP and carbonated apatite (CAP), a more direct mimic of enamel]. A naturally occurring mutation found in amelogenin (T21I) was also investigated. The structure in the K24S28 region of the protein was found to be sensitive to these conditions, with the CAP surface and excess Ca2+ (8:1 [Ca2+]:[LRAP-K24S28(+P)]) resulting in a tighter helix, while low ionic strength relaxed the helical structure. Higher ionic strength and the point mutation did not result in any structural change in this region. The distance of the backbone of K24 from the surface was most sensitive to excess Ca2+ and in the T21I-mutation. Collectively, these data suggest that phosphorylated LRAP is able to accommodate structural changes while maintaining its interaction with the surface, and provides further evidence of the structural sensitivity of the K24S28 region, a sensitivity that may contribute to function in biomineralization. PMID:25071599

  4. Transcription of TP0126, Treponema pallidum putative OmpW homolog, is regulated by the length of a homopolymeric guanosine repeat.

    PubMed

    Giacani, Lorenzo; Brandt, Stephanie L; Ke, Wujian; Reid, Tara B; Molini, Barbara J; Iverson-Cabral, Stefanie; Ciccarese, Giulia; Drago, Francesco; Lukehart, Sheila A; Centurion-Lara, Arturo

    2015-06-01

    An effective mechanism for introduction of phenotypic diversity within a bacterial population exploits changes in the length of repetitive DNA elements located within gene promoters. This phenomenon, known as phase variation, causes rapid activation or silencing of gene expression and fosters bacterial adaptation to new or changing environments. Phase variation often occurs in surface-exposed proteins, and in Treponema pallidum subsp. pallidum, the syphilis agent, it was reported to affect transcription of three putative outer membrane protein (OMP)-encoding genes. When the T. pallidum subsp. pallidum Nichols strain genome was initially annotated, the TP0126 open reading frame was predicted to include a poly(G) tract and did not appear to have a predicted signal sequence that might suggest the possibility of its being an OMP. Here we show that the initial annotation was incorrect, that this poly(G) is instead located within the TP0126 promoter, and that it varies in length in vivo during experimental syphilis. Additionally, we show that TP0126 transcription is affected by changes in the poly(G) length consistent with regulation by phase variation. In silico analysis of the TP0126 open reading frame based on the experimentally identified transcriptional start site shortens this hypothetical protein by 69 amino acids, reveals a predicted cleavable signal peptide, and suggests structural homology with the OmpW family of porins. Circular dichroism of recombinant TP0126 supports structural homology to OmpW. Together with the evidence that TP0126 is fully conserved among T. pallidum subspecies and strains, these data suggest an important role for TP0126 in T. pallidum biology and syphilis pathogenesis. PMID:25802057

  5. Transcription of TP0126, Treponema pallidum Putative OmpW Homolog, Is Regulated by the Length of a Homopolymeric Guanosine Repeat

    PubMed Central

    Brandt, Stephanie L.; Ke, Wujian; Reid, Tara B.; Molini, Barbara J.; Iverson-Cabral, Stefanie; Ciccarese, Giulia; Drago, Francesco; Lukehart, Sheila A.; Centurion-Lara, Arturo

    2015-01-01

    An effective mechanism for introduction of phenotypic diversity within a bacterial population exploits changes in the length of repetitive DNA elements located within gene promoters. This phenomenon, known as phase variation, causes rapid activation or silencing of gene expression and fosters bacterial adaptation to new or changing environments. Phase variation often occurs in surface-exposed proteins, and in Treponema pallidum subsp. pallidum, the syphilis agent, it was reported to affect transcription of three putative outer membrane protein (OMP)-encoding genes. When the T. pallidum subsp. pallidum Nichols strain genome was initially annotated, the TP0126 open reading frame was predicted to include a poly(G) tract and did not appear to have a predicted signal sequence that might suggest the possibility of its being an OMP. Here we show that the initial annotation was incorrect, that this poly(G) is instead located within the TP0126 promoter, and that it varies in length in vivo during experimental syphilis. Additionally, we show that TP0126 transcription is affected by changes in the poly(G) length consistent with regulation by phase variation. In silico analysis of the TP0126 open reading frame based on the experimentally identified transcriptional start site shortens this hypothetical protein by 69 amino acids, reveals a predicted cleavable signal peptide, and suggests structural homology with the OmpW family of porins. Circular dichroism of recombinant TP0126 supports structural homology to OmpW. Together with the evidence that TP0126 is fully conserved among T. pallidum subspecies and strains, these data suggest an important role for TP0126 in T. pallidum biology and syphilis pathogenesis. PMID:25802057

  6. Control of repeat protein curvature by computational protein design

    PubMed Central

    Park, Keunwan; Shen, Betty W.; Parmeggiani, Fabio; Huang, Po-Ssu; Stoddard, Barry L.; Baker, David

    2014-01-01

    Shape complementarity is an important component of molecular recognition, and the ability to precisely adjust the shape of a binding scaffold to match a target of interest would greatly facilitate the creation of high affinity protein reagents and therapeutics. Here we describe a general approach to control the shape of the binding surface on repeat protein scaffolds, and apply it to leucine rich repeat proteins. First, a set of self-compatible building block modules are designed that when polymerized each generate surfaces with unique but constant curvatures. Second, a set of junction modules that connect the different building blocks are designed. Finally, new proteins with custom designed shapes are generated by appropriately combining building block and junction modules. Crystal structures of the designs illustrate the power of the approach in controlling repeat protein curvature. PMID:25580576

  7. Understanding and identifying amino acid repeats.

    PubMed

    Luo, Hong; Nijveen, Harm

    2014-07-01

    Amino acid repeats (AARs) are abundant in protein sequences. They have particular roles in protein function and evolution. Simple repeat patterns generated by DNA slippage tend to introduce length variations and point mutations in repeat regions. Loss of normal and gain of abnormal function owing to their variable length are potential risks leading to diseases. Repeats with complex patterns mostly refer to the functional domain repeats, such as the well-known leucine-rich repeat and WD repeat, which are frequently involved in protein–protein interaction. They are mainly derived from internal gene duplication events and stabilized by ‘gate-keeper’ residues, which play crucial roles in preventing inter-domain aggregation. AARs are widely distributed in different proteomes across a variety of taxonomic ranges, and especially abundant in eukaryotic proteins. However, their specific evolutionary and functional scenarios are still poorly understood. Identifying AARs in protein sequences is the first step for the further investigation of their biological function and evolutionary mechanism. In principle, this is an NP-hard problem, as most of the repeat fragments are shaped by a series of sophisticated evolutionary events and become latent periodical patterns. It is not possible to define a uniform criterion for detecting and verifying various repeat patterns. Instead, different algorithms based on different strategies have been developed to cope with different repeat patterns. In this review, we attempt to describe the amino acid repeat-detection algorithms currently available and compare their strategies based on an in-depth analysis of the biological significance of protein repeats. PMID:23418055

  8. Expansion and Function of Repeat Domain Proteins During Stress and Development in Plants.

    PubMed

    Sharma, Manisha; Pandey, Girdhar K

    2015-01-01

    The recurrent repeats having conserved stretches of amino acids exists across all domains of life. Subsequent repetition of single sequence motif and the number and length of the minimal repeating motifs are essential characteristics innate to these proteins. The proteins with tandem peptide repeats are essential for providing surface to mediate protein-protein interactions for fundamental biological functions. Plants are enriched in tandem repeat containing proteins typically distributed into various families. This has been assumed that the occurrence of multigene repeats families in plants enable them to cope up with adverse environmental conditions and allow them to rapidly acclimatize to these conditions. The evolution, structure, and function of repeat proteins have been studied in all kingdoms of life. The presence of repeat proteins is particularly profuse in multicellular organisms in comparison to prokaryotes. The precipitous expansion of repeat proteins in plants is presumed to be through internal tandem duplications. Several repeat protein gene families have been identified in plants. Such as Armadillo (ARM), Ankyrin (ANK), HEAT, Kelch-like repeats, Tetratricopeptide (TPR), Leucine rich repeats (LRR), WD40, and Pentatricopeptide repeats (PPR). The structure and functions of these repeat proteins have been extensively studied in plants suggesting a critical role of these repeating peptides in plant cell physiology, stress and development. In this review, we illustrate the structural, functional, and evolutionary prospects of prolific repeat proteins in plants. PMID:26793205

  9. Expansion and Function of Repeat Domain Proteins During Stress and Development in Plants

    PubMed Central

    Sharma, Manisha; Pandey, Girdhar K.

    2016-01-01

    The recurrent repeats having conserved stretches of amino acids exists across all domains of life. Subsequent repetition of single sequence motif and the number and length of the minimal repeating motifs are essential characteristics innate to these proteins. The proteins with tandem peptide repeats are essential for providing surface to mediate protein–protein interactions for fundamental biological functions. Plants are enriched in tandem repeat containing proteins typically distributed into various families. This has been assumed that the occurrence of multigene repeats families in plants enable them to cope up with adverse environmental conditions and allow them to rapidly acclimatize to these conditions. The evolution, structure, and function of repeat proteins have been studied in all kingdoms of life. The presence of repeat proteins is particularly profuse in multicellular organisms in comparison to prokaryotes. The precipitous expansion of repeat proteins in plants is presumed to be through internal tandem duplications. Several repeat protein gene families have been identified in plants. Such as Armadillo (ARM), Ankyrin (ANK), HEAT, Kelch-like repeats, Tetratricopeptide (TPR), Leucine rich repeats (LRR), WD40, and Pentatricopeptide repeats (PPR). The structure and functions of these repeat proteins have been extensively studied in plants suggesting a critical role of these repeating peptides in plant cell physiology, stress and development. In this review, we illustrate the structural, functional, and evolutionary prospects of prolific repeat proteins in plants. PMID:26793205

  10. Molecular cloning and characterization of a novel repeat-containing Leishmania major gene, ppg1, that encodes a membrane-associated form of proteophosphoglycan with a putative glycosylphosphatidylinositol anchor.

    PubMed

    Ilg, T; Montgomery, J; Stierhof, Y D; Handman, E

    1999-10-29

    Leishmania parasites secrete a variety of proteins that are modified by phosphoglycan chains structurally similar to those of the cell surface glycolipid lipophosphoglycan. These proteins are collectively called proteophosphoglycans. We report here the cloning and sequencing of a novel Leishmania major proteophosphoglycan gene, ppg1. It encodes a large polypeptide of approximately 2300 amino acids. The N-terminal domain of approximately 70 kDa exhibits 11 imperfect amino acid repeats that show some homology to promastigote surface glycoproteins of the psa2/gp46 complex. The large central domain apparently consists exclusively of approximately 100 repetitive peptides of the sequence APSASSSSA(P/S)SSSSS(+/-S). Gene fusion experiments demonstrate that these peptide repeats are the targets of phosphoglycosylation in Leishmania and that they form extended filamentous structures reminiscent of mammalian mucins. The C-terminal domain contains a functional glycosylphosphatidylinositol anchor addition signal sequence, which confers cell surface localization to a normally secreted Leishmania acid phosphatase, when fused to its C terminus. Antibody binding studies show that the ppg1 gene product is phosphoglycosylated by phosphoglycan repeats and cap oligosaccharides. In contrast to previously characterized proteophosphoglycans, the ppg1 gene product is predominantly membrane-associated and it is expressed on the promastigote cell surface. Therefore this membrane-bound proteophosphoglycan may be important for direct host-parasite interactions. PMID:10531342

  11. A WD-repeat protein stabilizes ORC binding to chromatin.

    PubMed

    Shen, Zhen; Sathyan, Kizhakke M; Geng, Yijie; Zheng, Ruiping; Chakraborty, Arindam; Freeman, Brian; Wang, Fei; Prasanth, Kannanganattu V; Prasanth, Supriya G

    2010-10-01

    Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells. PMID:20932478

  12. The evolution and function of protein tandem repeats in plants.

    PubMed

    Schaper, Elke; Anisimova, Maria

    2015-04-01

    Sequence tandem repeats (TRs) are abundant in proteomes across all domains of life. For plants, little is known about their distribution or contribution to protein function. We exhaustively annotated TRs and studied the evolution of TR unit variations for all Ensembl plants. Using phylogenetic patterns of TR units, we detected conserved TRs with unit number and order preserved during evolution, and those TRs that have diverged via recent TR unit gains/losses. We correlated the mode of evolution of TRs to protein function. TR number was strongly correlated with proteome size, with about one-half of all TRs recognized as common protein domains. The majority of TRs have been highly conserved over long evolutionary distances, some since the separation of red algae and green plants c. 1.6 billion yr ago. Conversely, recurrent recent TR unit mutations were rare. Our results suggest that the first TRs by far predate the first plants, and that TR appearance is an ongoing process with similar rates across the plant kingdom. Interestingly, the few detected highly mutable TRs might provide a source of variation for rapid adaptation. In particular, such TRs are enriched in leucine-rich repeats (LRRs) commonly found in R genes, where TR unit gain/loss may facilitate resistance to emerging pathogens. PMID:25420631

  13. l-Ala-γ-d-Glu-meso-diaminopimelic Acid (DAP) Interacts Directly with Leucine-rich Region Domain of Nucleotide-binding Oligomerization Domain 1, Increasing Phosphorylation Activity of Receptor-interacting Serine/Threonine-protein Kinase 2 and Its Interaction with Nucleotide-binding Oligomerization Domain 1*

    PubMed Central

    Laroui, Hamed; Yan, Yutao; Narui, Yoshie; Ingersoll, Sarah A.; Ayyadurai, Saravanan; Charania, Moiz A.; Zhou, Feimeng; Wang, Binghe; Salaita, Khalid; Sitaraman, Shanthi V.; Merlin, Didier

    2011-01-01

    The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-γ-d-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-κB activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a Kd value of 34.5 μm. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a Kd value of 4.13 μm. However, NOD1/RICK binding was of higher affinity (Kd of 3.26 μm) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity. PMID:21757725

  14. PPR (pentatricopeptide repeat) proteins in mammals: important aids to mitochondrial gene expression.

    PubMed

    Lightowlers, Robert N; Chrzanowska-Lightowlers, Zofia M A

    2008-11-15

    Genes encoding PPR (pentatricopeptide repeat)-containing proteins constitute one of the largest gene families in plants. The majority of these proteins are predicted to target organelles and to bind to RNA. Strikingly, there is a dearth of these proteins in mammals, although genomic searches reveal six candidates, all of which are also predicted to target the mitochondrion. Two of these proteins, POLRMT (the mitochondrial RNA polymerase) and MRPS27, a mitoribosomal protein, are involved in transcription and translation respectively. PTCD1 (pentatricopeptide repeat domain protein 1) and PTCD3 are predicted to be involved in the assembly of respiratory chain complexes, whereas mutations in one other protein, LRPPRC (leucine-rich pentatricopeptide repeat cassette), have been shown to cause defects in the levels of cytochrome c oxidase, the terminal member of the respiratory chain. In this issue of the Biochemical Journal, Xu et al. turn their attention to the remaining candidate, PTCD2. Depletion in a mouse model led to deficiencies of the third complex of the respiratory chain that caused profound ultrastructural changes in the heart. The exact molecular function of PTCD2 remains unclear, but depletion leads to an apparent lack of processing of the mitochondrial transcript encoding apocytochrome b, a critical member of complex III. These data are consistent with PTCD2 playing an important role in the post-transcriptional expression of the mitochondrial genome. PMID:18939947

  15. Repeating thermocouple

    SciTech Connect

    Falk, R. A.

    1985-06-04

    Disclosed herein is a repeating use thermocouple assembly and method of making the same in which a cavity adjacent the tip of the thermocouple is filled with a thermosetting foundry sand and baked in place to provide support for the thermocouple tube without causing stresses during use which could cause breakage of the thermocouple tube.

  16. The Candidate Phylum Poribacteria by Single-Cell Genomics: New Insights into Phylogeny, Cell-Compartmentation, Eukaryote-Like Repeat Proteins, and Other Genomic Features

    PubMed Central

    Kamke, Janine; Rinke, Christian; Schwientek, Patrick; Mavromatis, Kostas; Ivanova, Natalia; Sczyrba, Alexander; Woyke, Tanja; Hentschel, Ute

    2014-01-01

    The candidate phylum Poribacteria is one of the most dominant and widespread members of the microbial communities residing within marine sponges. Cell compartmentalization had been postulated along with their discovery about a decade ago and their phylogenetic association to the Planctomycetes, Verrucomicrobia, Chlamydiae superphylum was proposed soon thereafter. In the present study we revised these features based on genomic data obtained from six poribacterial single cells. We propose that Poribacteria form a distinct monophyletic phylum contiguous to the PVC superphylum together with other candidate phyla. Our genomic analyses supported the possibility of cell compartmentalization in form of bacterial microcompartments. Further analyses of eukaryote-like protein domains stressed the importance of such proteins with features including tetratricopeptide repeats, leucin rich repeats as well as low density lipoproteins receptor repeats, the latter of which are reported here for the first time from a sponge symbiont. Finally, examining the most abundant protein domain family on poribacterial genomes revealed diverse phyH family proteins, some of which may be related to dissolved organic posphorus uptake. PMID:24498082

  17. The leucine rich amelogenin protein (LRAP) adsorbs as monomers or dimers onto surfaces

    SciTech Connect

    Tarasevich, Barbara J.; Lea, Alan S.; Shaw, Wendy J.

    2010-03-15

    Amelogenin and amelogenin splice variants are believed to be involved in controlling the formation of the highly anisotropic and ordered hydroxyapatite crystallites that form enamel. The adsorption behavior of amelogenin proteins onto substrates is very important because protein-surface interactions are critical to it’s function. We have studied the adsorption of LRAP, a splice variant of amelogenin which may also contribute to enamel function, onto model self-assembled monolayers on gold containing of COOH, CH3, and NH2 end groups. Dynamic light scattering (DLS) experiments indicated that LRAP in phosphate buffered saline (PBS) and solutions at saturation with calcium phosphate contained aggregates of nanospheres. Null ellipsometry and atomic force microscopy (AFM) were used to study protein adsorption amounts and structures. Relatively high amounts of adsorption occurred onto the CH3 and NH2 surfaces from both calcium phosphate and PBS solutions. Adsorption was also promoted onto COOH surfaces when calcium was present in the solutions suggesting an interaction that involves calcium bridging with the negatively charged C-terminus. The ellipsometry and AFM studies suggested that the protein adsorbed onto all surfaces as LRAP monomers. We propose that the monomers adsorb onto the surfaces by disassembling or “shedding” from the nanospheres that are present in solution. This work reveals the importance of small subnanosphere-sized structures of LRAP at interfaces, structures that may be important in the biomineralization of tooth enamel.

  18. Leucine-rich protein 130 contributes to apoptosis resistance of human hepatocarcinoma cells.

    PubMed

    Michaud, Mickaël; Barakat, Stéphane; Magnard, Sandrine; Rigal, Dominique; Baggetto, Loris G

    2011-01-01

    LRP130 is a ubiquitous protein involved in cellular homeostasis, microtubule alteration, and transactivation of a few multidrug resistance genes. Its role in resistance to apoptosis in HepG2 and HUH7 hepatocarcinoma cells was investigated. Using shRNA-producing lentiviruses to down-regulate the LRP130 gene, we showed that i) LRP130 did not affect the capacity of hepatocarcinoma cells to extrude drugs since LRP130 down-regulation was insufficient to significantly reduce P-glycoprotein production in these cells, and ii) the expression of 11 apoptosis-related genes measured by PCR-array was significantly reduced. Interestingly, six of these genes encode extrinsic pathway proapoptotic proteins whose expression was higher in LRP130-non producing than in LRP130-producing HepG2 cells. Fluorescence microscopy confirmed this new anti-apoptotic role of LRP130, which is strengthened by a significantly reduced cytochrome c oxidase activity in LRP130-down-regulated hepatocarcinoma cells. PMID:21109938

  19. Structural identification of putative USPs in Catharanthus roseus.

    PubMed

    Bahieldin, Ahmed; Atef, Ahmed; Shokry, Ahmed M; Al-Karim, Saleh; Al Attas, Sanaa G; Gadallah, Nour O; Edris, Sherif; Al-Kordy, Magdy A; Omer, Abdulkader M Shaikh; Sabir, Jamal S M; Ramadan, Ahmed M; Al-Hajar, Abdulrahman S M; Makki, Rania M; Hassan, Sabah M; El-Domyati, Fotouh M

    2015-10-01

    Nucleotide sequences of the C. roseus SRA database were assembled and translated in order to detect putative universal stress proteins (USPs). Based on the known conserved USPA domain, 24 Pfam putative USPA proteins in C. roseus were detected and arranged in six architectures. The USPA-like domain was detected in all architectures, while the protein kinase-like (or PK-like), (tyr)PK-like and/or U-box domains are shown downstream it. Three other domains were also shown to coexist with the USPA domain in C. roseus putative USPA sequences. These domains are tetratricopeptide repeat (or TPR), apolipophorin III (or apoLp-III) and Hsp90 co-chaperone Cdc37. Subsequent analysis divided USPA-like domains based on the ability to bind ATP. The multiple sequence alignment indicated the occurrence of eight C. roseus residues of known features of the bacterial 1MJH secondary structure. The data of the phylogenetic tree indicated several distinct groups of USPA-like domains confirming the presence of high level of sequence conservation between the plant and bacterial USPA-like sequences. PMID:26318047

  20. Transcript Abundance of Putative Lipid Phosphate Phosphatases During Development of Trypanosoma brucei in the Tsetse Fly.

    PubMed

    Alves e Silva, Thiago Luiz; Savage, Amy F; Aksoy, Serap

    2016-04-01

    African trypanosomes (Trypanosoma brucei spp.) cause devastating diseases in sub-Saharan Africa. Trypanosomes differentiate repeatedly during development in tsetse flies before gaining mammalian infectivity in fly salivary glands. Lipid phosphate phosphatases (LPPs) are involved in diverse biological processes, such as cell differentiation and cell migration. Gene sequences encoding two putative T. brucei LPP proteins were used to search the T. brucei genome, revealing two additional putative family members. Putative structural features and transcript abundance during parasite development in tsetse fly were characterized. Three of the four LPP proteins are predicted to have six transmembrane domains, while the fourth shows only one. Semiquantitative gene expression revealed differential regulation of LPPs during parasite development. Transcript abundance for three of the four putative LPP genes was elevated in parasites infecting salivary glands, but not mammalian-infective metacyclic cells in fly saliva, indicating a potential role of this family in parasite establishment in tsetse salivary glands. PMID:26856918

  1. Repeated Course Enrollments.

    ERIC Educational Resources Information Center

    Windham, Patricia

    This report resents tables of repeated course enrollment data in Florida community colleges for the fall 1993 cohort. Overall, the percent of repeats in college preparatory courses was greater than that of college credit courses. Within ICS codes, the highest percentage of credit repeat enrollments was in mathematics; the second highest was in…

  2. Structures of designed armadillo-repeat proteins show propagation of inter-repeat interface effects

    PubMed Central

    Reichen, Christian; Madhurantakam, Chaithanya; Hansen, Simon; Grütter, Markus G.; Plückthun, Andreas; Mittl, Peer R. E.

    2016-01-01

    The armadillo repeat serves as a scaffold for the development of modular peptide-recognition modules. In order to develop such a system, three crystal structures of designed armadillo-repeat proteins with third-generation N-caps (YIII-type), four or five internal repeats (M-type) and second-generation C-caps (AII-type) were determined at 1.8 Å (His-YIIIM4AII), 2.0 Å (His-YIIIM5AII) and 1.95 Å (YIIIM5AII) resolution and compared with those of variants with third-generation C-caps. All constructs are full consensus designs in which the internal repeats have exactly the same sequence, and hence identical conformations of the internal repeats are expected. The N-cap and internal repeats M1 to M3 are indeed extremely similar, but the comparison reveals structural differences in internal repeats M4 and M5 and the C-cap. These differences are caused by long-range effects of the C-cap, contacting molecules in the crystal, and the intrinsic design of the repeat. Unfortunately, the rigid-body movement of the C-terminal part impairs the regular arrangement of internal repeats that forms the putative peptide-binding site. The second-generation C-cap improves the packing of buried residues and thereby the stability of the protein. These considerations are useful for future improvements of an armadillo-repeat-based peptide-recognition system. PMID:26894544

  3. Structures of designed armadillo-repeat proteins show propagation of inter-repeat interface effects.

    PubMed

    Reichen, Christian; Madhurantakam, Chaithanya; Hansen, Simon; Grütter, Markus G; Plückthun, Andreas; Mittl, Peer R E

    2016-01-01

    The armadillo repeat serves as a scaffold for the development of modular peptide-recognition modules. In order to develop such a system, three crystal structures of designed armadillo-repeat proteins with third-generation N-caps (YIII-type), four or five internal repeats (M-type) and second-generation C-caps (AII-type) were determined at 1.8 Å (His-YIIIM4AII), 2.0 Å (His-YIIIM5AII) and 1.95 Å (YIIIM5AII) resolution and compared with those of variants with third-generation C-caps. All constructs are full consensus designs in which the internal repeats have exactly the same sequence, and hence identical conformations of the internal repeats are expected. The N-cap and internal repeats M1 to M3 are indeed extremely similar, but the comparison reveals structural differences in internal repeats M4 and M5 and the C-cap. These differences are caused by long-range effects of the C-cap, contacting molecules in the crystal, and the intrinsic design of the repeat. Unfortunately, the rigid-body movement of the C-terminal part impairs the regular arrangement of internal repeats that forms the putative peptide-binding site. The second-generation C-cap improves the packing of buried residues and thereby the stability of the protein. These considerations are useful for future improvements of an armadillo-repeat-based peptide-recognition system. PMID:26894544

  4. Reconfigurable multiport EPON repeater

    NASA Astrophysics Data System (ADS)

    Oishi, Masayuki; Inohara, Ryo; Agata, Akira; Horiuchi, Yukio

    2009-11-01

    An extended reach EPON repeater is one of the solutions to effectively expand FTTH service areas. In this paper, we propose a reconfigurable multi-port EPON repeater for effective accommodation of multiple ODNs with a single OLT line card. The proposed repeater, which has multi-ports in both OLT and ODN sides, consists of TRs, BTRs with the CDR function and a reconfigurable electrical matrix switch, can accommodate multiple ODNs to a single OLT line card by controlling the connection of the matrix switch. Although conventional EPON repeaters require full OLT line cards to accommodate subscribers from the initial installation stage, the proposed repeater can dramatically reduce the number of required line cards especially when the number of subscribers is less than a half of the maximum registerable users per OLT. Numerical calculation results show that the extended reach EPON system with the proposed EPON repeater can save 17.5% of the initial installation cost compared with a conventional repeater, and can be less expensive than conventional systems up to the maximum subscribers especially when the percentage of ODNs in lightly-populated areas is higher.

  5. Revisiting the TALE repeat.

    PubMed

    Deng, Dong; Yan, Chuangye; Wu, Jianping; Pan, Xiaojing; Yan, Nieng

    2014-04-01

    Transcription activator-like (TAL) effectors specifically bind to double stranded (ds) DNA through a central domain of tandem repeats. Each TAL effector (TALE) repeat comprises 33-35 amino acids and recognizes one specific DNA base through a highly variable residue at a fixed position in the repeat. Structural studies have revealed the molecular basis of DNA recognition by TALE repeats. Examination of the overall structure reveals that the basic building block of TALE protein, namely a helical hairpin, is one-helix shifted from the previously defined TALE motif. Here we wish to suggest a structure-based re-demarcation of the TALE repeat which starts with the residues that bind to the DNA backbone phosphate and concludes with the base-recognition hyper-variable residue. This new numbering system is consistent with the α-solenoid superfamily to which TALE belongs, and reflects the structural integrity of TAL effectors. In addition, it confers integral number of TALE repeats that matches the number of bound DNA bases. We then present fifteen crystal structures of engineered dHax3 variants in complex with target DNA molecules, which elucidate the structural basis for the recognition of bases adenine (A) and guanine (G) by reported or uncharacterized TALE codes. Finally, we analyzed the sequence-structure correlation of the amino acid residues within a TALE repeat. The structural analyses reported here may advance the mechanistic understanding of TALE proteins and facilitate the design of TALEN with improved affinity and specificity. PMID:24622844

  6. Quantum repeated games revisited

    NASA Astrophysics Data System (ADS)

    Frąckiewicz, Piotr

    2012-03-01

    We present a scheme for playing quantum repeated 2 × 2 games based on Marinatto and Weber’s approach to quantum games. As a potential application, we study the twice repeated Prisoner’s Dilemma game. We show that results not available in the classical game can be obtained when the game is played in the quantum way. Before we present our idea, we comment on the previous scheme of playing quantum repeated games proposed by Iqbal and Toor. We point out the drawbacks that make their results unacceptable.

  7. The Putative Son's Attractiveness Alters the Perceived Attractiveness of the Putative Father.

    PubMed

    Prokop, Pavol

    2015-08-01

    A body of literature has investigated female mate choice in the pre-mating context (pre-mating sexual selection). Humans, however, are long-living mammals forming pair-bonds which sequentially produce offspring. Post-mating evaluations of a partner's attractiveness may thus significantly influence the reproductive success of men and women. I tested herein the theory that the attractiveness of putative sons provides extra information about the genetic quality of fathers, thereby influencing fathers' attractiveness across three studies. As predicted, facially attractive boys were more frequently attributed to attractive putative fathers and vice versa (Study 1). Furthermore, priming with an attractive putative son increased the attractiveness of the putative father with the reverse being true for unattractive putative sons. When putative fathers were presented as stepfathers, the effect of the boy's attractiveness on the stepfather's attractiveness was lower and less consistent (Study 2). This suggests that the presence of an attractive boy has the strongest effect on the perceived attractiveness of putative fathers rather than on non-fathers. The generalized effect of priming with beautiful non-human objects also exists, but its effect is much weaker compared with the effects of putative biological sons (Study 3). Overall, this study highlighted the importance of post-mating sexual selection in humans and suggests that the heritable attractive traits of men are also evaluated by females after mating and/or may be used by females in mate poaching. PMID:25731909

  8. Toddlers' Duration of Attention toward Putative Threat

    ERIC Educational Resources Information Center

    Kiel, Elizabeth J.; Buss, Kristin A.

    2011-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk of developing anxious behavior, toddlers' attention toward a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined…

  9. The Pentapeptide Repeat Proteins

    SciTech Connect

    Vetting,M.; Hegde, S.; Fajardo, J.; Fiser, A.; Roderick, S.; Takiff, H.; Blanchard, J.

    2006-01-01

    The Pentapeptide Repeat Protein (PRP) family has over 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S, T,A, V][D, N][L, F]-[S, T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral {beta}-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable size, shape and electrostatic similarity to DNA.

  10. Honesty through repeated interactions.

    PubMed

    Rich, Patricia; Zollman, Kevin J S

    2016-04-21

    In the study of signaling, it is well known that the cost of deception is an essential element for stable honest signaling in nature. In this paper, we show how costs for deception can arise endogenously from repeated interactions between individuals. Utilizing the Sir Philip Sidney game as an illustrative case, we show that repeated interactions can sustain honesty with no observable signal costs, even when deception cannot be directly observed. We provide a number of potential experimental tests for this theory which distinguish it from the available alternatives. PMID:26869213

  11. Bidirectional Manchester repeater

    NASA Technical Reports Server (NTRS)

    Ferguson, J.

    1980-01-01

    Bidirectional Manchester repeater is inserted at periodic intervals along single bidirectional twisted pair transmission line to detect, amplify, and transmit bidirectional Manchester 11 code signals. Requiring only 18 TTL 7400 series IC's, some line receivers and drivers, and handful of passive components, circuit is simple and relatively inexpensive to build.

  12. Triggering of repeated earthquakes

    NASA Astrophysics Data System (ADS)

    Sobolev, G. A.; Zakrzhevskaya, N. A.; Sobolev, D. G.

    2016-03-01

    Based on the analysis of the world's earthquakes with magnitudes M ≥ 6.5 for 1960-2013, it is shown that they cause global-scale coherent seismic oscillations which most distinctly manifest themselves in the period interval of 4-6 min during 1-3 days after the event. After these earthquakes, a repeated shock has an increased probability to occur in different seismically active regions located as far away as a few thousand km from the previous event, i.e., a remote interaction of seismic events takes place. The number of the repeated shocks N( t) decreases with time, which characterizes the memory of the lithosphere about the impact that has occurred. The time decay N( t) can be approximated by the linear, exponential, and powerlaw dependences. No distinct correlation between the spatial locations of the initial and repeated earthquakes is revealed. The probable triggering mechanisms of the remote interaction between the earthquakes are discussed. Surface seismic waves traveling several times around the Earth's, coherent oscillations, and global source are the most preferable candidates. This may lead to the accumulation and coalescence of ruptures in the highly stressed or weakened domains of a seismically active region, which increases the probability of a repeated earthquake.

  13. Repeated Causal Decision Making

    ERIC Educational Resources Information Center

    Hagmayer, York; Meder, Bjorn

    2013-01-01

    Many of our decisions refer to actions that have a causal impact on the external environment. Such actions may not only allow for the mere learning of expected values or utilities but also for acquiring knowledge about the causal structure of our world. We used a repeated decision-making paradigm to examine what kind of knowledge people acquire in…

  14. Magnetism and the putative early Martian life

    NASA Astrophysics Data System (ADS)

    Rochette, P.

    2001-08-01

    A short critical review is provided on three questions linking magnetism and the putative early Mars life. Was there a large internal Martian magnetic field, during which period, and is it a requisite for life? What is the origin of the paleomagnetic signal of Martian meteorites, including ALH84001? What is the present credibility of the case for fossil bacterial magnetite grains in ALH84001?

  15. Accumulate repeat accumulate codes

    NASA Technical Reports Server (NTRS)

    Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

    2004-01-01

    In this paper we propose an innovative channel coding scheme called 'Accumulate Repeat Accumulate codes' (ARA). This class of codes can be viewed as serial turbo-like codes, or as a subclass of Low Density Parity Check (LDPC) codes, thus belief propagation can be used for iterative decoding of ARA codes on a graph. The structure of encoder for this class can be viewed as precoded Repeat Accumulate (RA) code or as precoded Irregular Repeat Accumulate (IRA) code, where simply an accumulator is chosen as a precoder. Thus ARA codes have simple, and very fast encoder structure when they representing LDPC codes. Based on density evolution for LDPC codes through some examples for ARA codes, we show that for maximum variable node degree 5 a minimum bit SNR as low as 0.08 dB from channel capacity for rate 1/2 can be achieved as the block size goes to infinity. Thus based on fixed low maximum variable node degree, its threshold outperforms not only the RA and IRA codes but also the best known LDPC codes with the dame maximum node degree. Furthermore by puncturing the accumulators any desired high rate codes close to code rate 1 can be obtained with thresholds that stay close to the channel capacity thresholds uniformly. Iterative decoding simulation results are provided. The ARA codes also have projected graph or protograph representation that allows for high speed decoder implementation.

  16. Duct Leakage Repeatability Testing

    SciTech Connect

    Walker, Iain; Sherman, Max

    2014-01-01

    Duct leakage often needs to be measured to demonstrate compliance with requirements or to determine energy or Indoor Air Quality (IAQ) impacts. Testing is often done using standards such as ASTM E1554 (ASTM 2013) or California Title 24 (California Energy Commission 2013 & 2013b), but there are several choices of methods available within the accepted standards. Determining which method to use or not use requires an evaluation of those methods in the context of the particular needs. Three factors that are important considerations are the cost of the measurement, the accuracy of the measurement and the repeatability of the measurement. The purpose of this report is to evaluate the repeatability of the three most significant measurement techniques using data from the literature and recently obtained field data. We will also briefly discuss the first two factors. The main question to be answered by this study is to determine if differences in the repeatability of these tests methods is sufficient to indicate that any of these methods is so poor that it should be excluded from consideration as an allowed procedure in codes and standards.

  17. Putative Excitatory and Putative Inhibitory Inputs Localize to Different Dendritic Domains in a Drosophila Flight Motoneuron

    PubMed Central

    Kuehn, Claudia; Duch, Carsten

    2012-01-01

    Input-output computations of individual neurons may be affected by the three-dimensional structure of their dendrites and by the targeting of input synapses to specific parts of their dendrites. However, only few examples exist where dendritic architecture can be related to behaviorally relevant computations of a neuron. By combining genetic, immunohistochemical, and confocal laser scanning methods this study estimates the location of the spike initiating zone and the dendritic distribution patterns of putative synaptic inputs on an individually identified Drosophila flight motorneuron, MN5. MN5 is a monopolar neuron with more than 4000 dendritic branches. The site of spike initiation was estimated by mapping sodium channel immunolabel onto geometric reconstructions of MN5. Maps of putative excitatory cholinergic and of putative inhibitory GABAergic inputs on MN5 dendrites were created by charting tagged Dα7 nicotinic acetylcholine receptors and Rdl GABAA receptors onto MN5 dendritic surface reconstructions. Although these methods provided only an estimate of putative input synapse distributions, the data indicated that inhibitory and excitatory synapses were targeted preferentially to different dendritic domains of MN5, and thus, computed mostly separately. Most putative inhibitory inputs were close to spike initiation, which was consistent with sharp inhibition, as predicted previously based on recordings of motoneuron firing patterns during flight. By contrast, highest densities of putative excitatory inputs at more distant dendritic regions were consistent with the prediction that in response to different power demands during flight, tonic excitatory drive to flight motoneuron dendrites must be smoothly translated into different tonic firing frequencies. PMID:23279094

  18. Duct Leakage Repeatability Testing

    SciTech Connect

    Walker, Iain; Sherman, Max

    2014-08-01

    The purpose of this report is to evaluate the repeatability of the three most significant measurement techniques for duct leakage using data from the literature and recently obtained field data. We will also briefly discuss the first two factors. The main question to be answered by this study is to determine if differences in the repeatability of these tests methods is sufficient to indicate that any of these methods is so poor that it should be excluded from consideration as an allowed procedure in codes and standards. The three duct leak measurement methods assessed in this report are the two duct pressurization methods that are commonly used by many practitioners and the DeltaQ technique. These are methods B, C and A, respectively of the ASTM E1554 standard. Although it would be useful to evaluate other duct leak test methods, this study focused on those test methods that are commonly used and are required in various test standards, such as BPI (2010), RESNET (2014), ASHRAE 62.2 (2013), California Title 24 (CEC 2012), DOE Weatherization and many other energy efficiency programs.

  19. Repeated measures with zeros.

    PubMed

    Berk, K N; Lachenbruch, P A

    2002-08-01

    Consider repeated measures data with many zeros. For the case with one grouping factor and one repeated measure, we examine several models, assuming that the nonzero data are roughly lognormal. One of the simplest approaches is to model the zeros as left-censored observations from the lognormal distribution. A random effect is assumed for subjects. The censored model makes a strong assumption about the relationship between the zeros and the nonzero values. To check on this, you can instead assume that some of the zeros are 'true' zeros and model them as Bernoulli. Then the other values are modeled with a censored lognormal. A logistic model is used for the Bernoulli p, the probability of a true nonzero. The fit of the pure left-censored lognormal can be assessed by testing the hypothesis that p is 1, as described by Moulton and Halsey. The model can also be simplified by omitting the censoring, leaving a logistic model for the zeros and a lognormal model for the nonzero values. This is approximately equivalent to modeling the zero and nonzero values separately, a two-part model. In contrast to the censored model, this model assumes only a slight relationship (a covariance component) between the occurrence of zeros and the size of the nonzero values. The models are compared in terms of an example with data from children's private speech. PMID:12197298

  20. Ten Putative Contributors to the Obesity Epidemic

    PubMed Central

    McAllister, Emily J.; Dhurandhar, Nikhil V.; Keith, Scott W.; Aronne, Louis J.; Barger, Jamie; Baskin, Monica; Benca, Ruth M.; Biggio, Joseph; Boggiano, Mary M.; Eisenmann, Joe C.; Elobeid, Mai; Fontaine, Kevin R.; Gluckman, Peter; Hanlon, Erin C.; Katzmarzyk, Peter; Pietrobelli, Angelo; Redden, David T.; Ruden, Douglas M.; Wang, Chenxi; Waterland, Robert A.; Wright, Suzanne M.; Allison, David B.

    2010-01-01

    The obesity epidemic is a global issue and shows no signs of abating, while the cause of this epidemic remains unclear. Marketing practices of energy-dense foods and institutionally-driven declines in physical activity are the alleged perpetrators for the epidemic, despite a lack of solid evidence to demonstrate their causal role. While both may contribute to obesity, we call attention to their unquestioned dominance in program funding and public efforts to reduce obesity, and propose several alternative putative contributors that would benefit from equal consideration and attention. Evidence for microorganisms, epigenetics, increasing maternal age, greater fecundity among people with higher adiposity, assortative mating, sleep debt, endocrine disruptors, pharmaceutical iatrogenesis, reduction in variability of ambient temperatures, and intrauterine and intergenerational effects, as contributing factors to the obesity epidemic are reviewed herein. While the evidence is strong for some contributors such as pharmaceutical-induced weight gain, it is still emerging for other reviewed factors. Considering the role of such putative etiological factors of obesity may lead to comprehensive, cause specific, and effective strategies for prevention and treatment of this global epidemic. PMID:19960394

  1. Ising Model Reprogramming of a Repeat Protein's Equilibrium Unfolding Pathway.

    PubMed

    Millership, C; Phillips, J J; Main, E R G

    2016-05-01

    Repeat proteins are formed from units of 20-40 aa that stack together into quasi one-dimensional non-globular structures. This modular repetitive construction means that, unlike globular proteins, a repeat protein's equilibrium folding and thus thermodynamic stability can be analysed using linear Ising models. Typically, homozipper Ising models have been used. These treat the repeat protein as a series of identical interacting subunits (the repeated motifs) that couple together to form the folded protein. However, they cannot describe subunits of differing stabilities. Here we show that a more sophisticated heteropolymer Ising model can be constructed and fitted to two new helix deletion series of consensus tetratricopeptide repeat proteins (CTPRs). This analysis, showing an asymmetric spread of stability between helices within CTPR ensembles, coupled with the Ising model's predictive qualities was then used to guide reprogramming of the unfolding pathway of a variant CTPR protein. The designed behaviour was engineered by introducing destabilising mutations that increased the thermodynamic asymmetry within a CTPR ensemble. The asymmetry caused the terminal α-helix to thermodynamically uncouple from the rest of the protein and preferentially unfold. This produced a specific, highly populated stable intermediate with a putative dimerisation interface. As such it is the first step in designing repeat proteins with function regulated by a conformational switch. PMID:26947150

  2. Repeat Customer Success in Extension

    ERIC Educational Resources Information Center

    Bess, Melissa M.; Traub, Sarah M.

    2013-01-01

    Four multi-session research-based programs were offered by two Extension specialist in one rural Missouri county. Eleven participants who came to multiple Extension programs could be called "repeat customers." Based on the total number of participants for all four programs, 25% could be deemed as repeat customers. Repeat customers had…

  3. Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins.

    PubMed

    Mori, Kohji; Arzberger, Thomas; Grässer, Friedrich A; Gijselinck, Ilse; May, Stephanie; Rentzsch, Kristin; Weng, Shih-Ming; Schludi, Martin H; van der Zee, Julie; Cruts, Marc; Van Broeckhoven, Christine; Kremmer, Elisabeth; Kretzschmar, Hans A; Haass, Christian; Edbauer, Dieter

    2013-12-01

    Massive GGGGCC repeat expansion in the first intron of the gene C9orf72 is the most common known cause of familial frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Despite its intronic localization and lack of an ATG start codon, the repeat region is translated in all three reading frames into aggregating dipeptide-repeat (DPR) proteins, poly-(Gly-Ala), poly-(Gly-Pro) and poly-(Gly-Arg). We took an antibody-based approach to further validate the translation of DPR proteins. To test whether the antisense repeat RNA transcript is also translated, we raised antibodies against the predicted products, poly-(Ala-Pro) and poly-(Pro-Arg). Both antibodies stained p62-positive neuronal cytoplasmic inclusions throughout the cerebellum and hippocampus indicating that not only sense but also antisense strand repeats are translated into DPR proteins in the absence of ATG start codons. Protein products of both strands co-aggregate suggesting concurrent translation of both strands. Moreover, an antibody targeting the putative carboxyl terminus of DPR proteins can detect inclusion pathology in C9orf72 repeat expansion carriers suggesting that the non-ATG translation continues through the entire repeat and beyond. A highly sensitive monoclonal antibody against poly-(Gly-Arg), visualized abundant inclusion pathology in all cortical regions and some inclusions also in motoneurons. Together, our data show that the GGGGCC repeat is bidirectionally translated into five distinct DPR proteins that co-aggregate in the characteristic p62-positive TDP-43 negative inclusions found in FTLD/ALS cases with C9orf72 repeat expansion. Novel monoclonal antibodies against poly-(Gly-Arg) will facilitate pathological diagnosis of C9orf72 FTLD/ALS. PMID:24132570

  4. FUNCTIONAL ANALYSIS OF A RING DOMAIN ANKYRIN REPEAT PROTEIN THAT IS HIGHLY EXPRESSED DURING FLOWER SENESCENCE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A gene encoding a RING zinc finger ankyrin repeat protein (MjXB3), a putative E3 ubiquitin ligase, is highly expressed in petals of senescing four o'clock (Mirabilis jalapa) flowers, increasing >40 000-fold during the onset of visible senescence. The gene has homologues in many other species, and t...

  5. RepeatsDB: a database of tandem repeat protein structures

    PubMed Central

    Di Domenico, Tomás; Potenza, Emilio; Walsh, Ian; Gonzalo Parra, R.; Giollo, Manuel; Minervini, Giovanni; Piovesan, Damiano; Ihsan, Awais; Ferrari, Carlo; Kajava, Andrey V.; Tosatto, Silvio C.E.

    2014-01-01

    RepeatsDB (http://repeatsdb.bio.unipd.it/) is a database of annotated tandem repeat protein structures. Tandem repeats pose a difficult problem for the analysis of protein structures, as the underlying sequence can be highly degenerate. Several repeat types haven been studied over the years, but their annotation was done in a case-by-case basis, thus making large-scale analysis difficult. We developed RepeatsDB to fill this gap. Using state-of-the-art repeat detection methods and manual curation, we systematically annotated the Protein Data Bank, predicting 10 745 repeat structures. In all, 2797 structures were classified according to a recently proposed classification schema, which was expanded to accommodate new findings. In addition, detailed annotations were performed in a subset of 321 proteins. These annotations feature information on start and end positions for the repeat regions and units. RepeatsDB is an ongoing effort to systematically classify and annotate structural protein repeats in a consistent way. It provides users with the possibility to access and download high-quality datasets either interactively or programmatically through web services. PMID:24311564

  6. Major Quantitative Trait Loci and Putative Candidate Genes for Powdery Mildew Resistance and Fruit-Related Traits Revealed by an Intraspecific Genetic Map for Watermelon (Citrullus lanatus var. lanatus)

    PubMed Central

    Kim, Kwang-Hwan; Hwang, Ji-Hyun; Han, Dong-Yeup; Park, Minkyu; Kim, Seungill; Choi, Doil; Kim, Yongjae; Lee, Gung Pyo; Kim, Sun-Tae; Park, Young-Hoon

    2015-01-01

    An intraspecific genetic map for watermelon was constructed using an F2 population derived from ‘Arka Manik’ × ‘TS34’ and transcript sequence variants and quantitative trait loci (QTL) for resistance to powdery mildew (PMR), seed size (SS), and fruit shape (FS) were analyzed. The map consists of 14 linkage groups (LGs) defined by 174 cleaved amplified polymorphic sequences (CAPS), 2 derived-cleaved amplified polymorphic sequence markers, 20 sequence-characterized amplified regions, and 8 expressed sequence tag-simple sequence repeat markers spanning 1,404.3 cM, with a mean marker interval of 6.9 cM and an average of 14.6 markers per LG. Genetic inheritance and QTL analyses indicated that each of the PMR, SS, and FS traits is controlled by an incompletely dominant effect of major QTLs designated as pmr2.1, ss2.1, and fsi3.1, respectively. The pmr2.1, detected on chromosome 2 (Chr02), explained 80.0% of the phenotypic variation (LOD = 30.76). This QTL was flanked by two CAPS markers, wsb2-24 (4.00 cM) and wsb2-39 (13.97 cM). The ss2.1, located close to pmr2.1 and CAPS marker wsb2-13 (1.00 cM) on Chr02, explained 92.3% of the phenotypic variation (LOD = 68.78). The fsi3.1, detected on Chr03, explained 79.7% of the phenotypic variation (LOD = 31.37) and was flanked by two CAPS, wsb3-24 (1.91 cM) and wsb3-9 (7.00 cM). Candidate gene-based CAPS markers were developed from the disease resistance and fruit shape gene homologs located on Chr.02 and Chr03 and were mapped on the intraspecific map. Colocalization of these markers with the major QTLs indicated that watermelon orthologs of a nucleotide-binding site-leucine-rich repeat class gene containing an RPW8 domain and a member of SUN containing the IQ67 domain are candidate genes for pmr2.1 and fsi3.1, respectively. The results presented herein provide useful information for marker-assisted breeding and gene cloning for PMR and fruit-related traits. PMID:26700647

  7. Major Quantitative Trait Loci and Putative Candidate Genes for Powdery Mildew Resistance and Fruit-Related Traits Revealed by an Intraspecific Genetic Map for Watermelon (Citrullus lanatus var. lanatus).

    PubMed

    Kim, Kwang-Hwan; Hwang, Ji-Hyun; Han, Dong-Yeup; Park, Minkyu; Kim, Seungill; Choi, Doil; Kim, Yongjae; Lee, Gung Pyo; Kim, Sun-Tae; Park, Young-Hoon

    2015-01-01

    An intraspecific genetic map for watermelon was constructed using an F2 population derived from 'Arka Manik' × 'TS34' and transcript sequence variants and quantitative trait loci (QTL) for resistance to powdery mildew (PMR), seed size (SS), and fruit shape (FS) were analyzed. The map consists of 14 linkage groups (LGs) defined by 174 cleaved amplified polymorphic sequences (CAPS), 2 derived-cleaved amplified polymorphic sequence markers, 20 sequence-characterized amplified regions, and 8 expressed sequence tag-simple sequence repeat markers spanning 1,404.3 cM, with a mean marker interval of 6.9 cM and an average of 14.6 markers per LG. Genetic inheritance and QTL analyses indicated that each of the PMR, SS, and FS traits is controlled by an incompletely dominant effect of major QTLs designated as pmr2.1, ss2.1, and fsi3.1, respectively. The pmr2.1, detected on chromosome 2 (Chr02), explained 80.0% of the phenotypic variation (LOD = 30.76). This QTL was flanked by two CAPS markers, wsb2-24 (4.00 cM) and wsb2-39 (13.97 cM). The ss2.1, located close to pmr2.1 and CAPS marker wsb2-13 (1.00 cM) on Chr02, explained 92.3% of the phenotypic variation (LOD = 68.78). The fsi3.1, detected on Chr03, explained 79.7% of the phenotypic variation (LOD = 31.37) and was flanked by two CAPS, wsb3-24 (1.91 cM) and wsb3-9 (7.00 cM). Candidate gene-based CAPS markers were developed from the disease resistance and fruit shape gene homologs located on Chr.02 and Chr03 and were mapped on the intraspecific map. Colocalization of these markers with the major QTLs indicated that watermelon orthologs of a nucleotide-binding site-leucine-rich repeat class gene containing an RPW8 domain and a member of SUN containing the IQ67 domain are candidate genes for pmr2.1 and fsi3.1, respectively. The results presented herein provide useful information for marker-assisted breeding and gene cloning for PMR and fruit-related traits. PMID:26700647

  8. The structure and chromosome location of the human chondroadherin gene (CHAD)

    SciTech Connect

    Grover, J.; Roughley, P.J. |; Chen, Xiao-Ning; Korenberg, J.R.

    1997-10-15

    The cDNA sequence of the human chondroadherin gene was cloned using PCR-based techniques. The gene encodes a protein of 359 amino acids, of which the first 21 amino acids represent a putative signal peptide sequence and which possesses 11 leucine-rich repeats flanked by cysteine-rich regions. The cDNA possesses a 5{prime} untranslated region of 149 bp, a coding region of 1080 hp including the stop codon, and a 3{prime} untranslated region of 561 bp terminating in a poly(A) tail. The cDNA hybridizes with a single messenger RNA of 1.9 kb, which is present in chondrocytes at all ages. Analysis of genomic DNA revealed that the chondroadherin gene possesses two introns, both of which reside within the coding region. The first intron has a length of about 2.3 kb and separates the codons for lysine(258) and phenylalanine(259). The second intron has a length of about 0.5 kb and splits the codon for tryptophan(314). This genomic organization results in exon 1 encoding the signal peptide, the amino-terminal cysteine-rich region, and the first 9 leucine-rich repeats; exon 2 encoding the last 2 leucine-rich repeats and part of the carboxy-terminal cysteine-rich region; and exon 3 encoding the remainder of the carboxy-terminal cysteine-rich region. The gene does not possess a TATA box prior to its transcription start site. Isolation of a cosmid clone spanning the chondroadherin gene enabled its chromosome location to be established. The gene was shown to reside at chromosome 17q21.33. 32 refs., 5 figs.

  9. Saturation of repeated quantum measurements

    NASA Astrophysics Data System (ADS)

    Haapasalo, Erkka; Heinosaari, Teiko; Kuramochi, Yui

    2016-08-01

    We study sequential measurement scenarios where the system is repeatedly subjected to the same measurement process. We first provide examples of such repeated measurements where further repetitions of the measurement do not increase our knowledge on the system after some finite number of measurement steps. We also prove, however, that repeating the Lüders measurement of an unsharp two-outcome observable never saturates in this sense, and we characterize the observable measured in the limit of infinitely many repetitions. Our result implies that a repeated measurement can be used to correct the inherent noise of an unsharp observable.

  10. The Biogeography of Putative Microbial Antibiotic Production.

    PubMed

    Morlon, Hélène; O'Connor, Timothy K; Bryant, Jessica A; Charkoudian, Louise K; Docherty, Kathryn M; Jones, Evan; Kembel, Steven W; Green, Jessica L; Bohannan, Brendan J M

    2015-01-01

    Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics. PMID:26102275

  11. The Biogeography of Putative Microbial Antibiotic Production

    PubMed Central

    Bryant, Jessica A.; Charkoudian, Louise K.; Docherty, Kathryn M.; Jones, Evan; Kembel, Steven W.; Green, Jessica L.; Bohannan, Brendan J. M.

    2015-01-01

    Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics. PMID:26102275

  12. Magnetic Pulse Affects a Putative Magnetoreceptor Mechanism

    PubMed Central

    Davila, Alfonso F.; Winklhofer, Michael; Shcherbakov, Valera P.; Petersen, Nikolai

    2005-01-01

    Clusters of superparamagnetic (SP) magnetite crystals have recently been identified in free nerve endings in the upper-beak skin of homing pigeons and are interpreted as being part of a putative magnetoreceptor system. Motivated by these findings, we developed a physical model that accurately predicts the dynamics of interacting SP clusters in a magnetic field. The main predictions are: 1), under a magnetic field, a group of SP clusters self-assembles into a chain-like structure that behaves like a compass needle under slowly rotating fields; 2), in a frequently changing field as encountered by a moving bird, a stacked chain is a structurally more stable configuration than a single chain; 3), chain-like structures of SP clusters disrupt under strong fields applied at oblique angles; and 4), reassemble on a timescale of hours to days (assuming a viscosity of the cell plasma η ∼ 1 P). Our results offer a novel mechanism for magnetic field perception and are in agreement with the response of birds observed after magnetic-pulse treatments, which have been conducted in the past to specifically test if ferrimagnetic material is involved in magnetoreception, but which have defied explanation so far. Our theoretical results are supported by experiments on a technical SP model system using a high-speed camera. We also offer new predictions that can be tested experimentally. PMID:15863473

  13. Toddlers’ Duration of Attention towards Putative Threat

    PubMed Central

    Kiel, Elizabeth J.; Buss, Kristin A.

    2010-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk for developing anxious behavior, toddlers’ attention towards a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined how attention towards an angry-looking gorilla mask in a room with alternative opportunities for play in 24-month-old toddlers predicted social inhibition when children entered kindergarten. Analyses examined attention to threat above and beyond and in interaction with both proximity to the mask and fear of novelty observed in other situations. Attention to threat interacted with proximity to the mask to predict social inhibition, such that attention to threat most strongly predicted social inhibition when toddlers stayed furthest from the mask. This relation occurred above and beyond the predictive relation between fear of novelty and social inhibition. Results are discussed within the broader literature of anxiety development and attentional processes in young children. PMID:21373365

  14. Biogenic Origin for Earth's Oldest Putative Microfossils

    SciTech Connect

    De Gregorio, B.; Sharp, T; Flynn, G; Wirick, S; Hervig, R

    2009-01-01

    Carbonaceous microbe-like features preserved within a local chert unit of the 3.5 Ga old Apex Basalt in Western Australia may represent some of the oldest evidence of life on Earth. However, the biogenicity of these putative microfossils has been called into question, primarily because the sample collection locality is a black, carbon-rich, brecciated chert dike representing an Archean submarine hydrothermal spring, suggesting a formation via an abiotic organic synthesis mechanism. Here we describe the macromolecular hydrocarbon structure, carbon bonding, functional group chemistry, and biotic element abundance of carbonaceous matter associated with these filamentous features. These characteristics are similar to those of biogenic kerogen from the ca. 1.9 Ga old Gunflint Formation. Although an abiotic origin cannot be entirely ruled out, it is unlikely that known abiotic synthesis mechanisms could recreate both the structural and compositional complexity of this ancient carbonaceous matter. Thus, we find that a biogenic origin for this material is more likely, implying that the Apex microbe-like features represent authentic biogenic organic matter.

  15. Differential Modulation of Nods Signaling Pathways by Fatty Acids in Human Colonic Epithelial HCT116 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nucleotide-binding oligomerization domain containing proteins (Nods) are intracellular pattern recognition receptors (PRRs) recognizing conserved moieties of bacterial peptidoglycan through their leucine-rich repeats (LRR) domain. The agonists for Nods activate proinflammtory signaling pathways incl...

  16. All-photonic quantum repeaters

    NASA Astrophysics Data System (ADS)

    Azuma, Koji; Tamaki, Kiyoshi; Lo, Hoi-Kwong

    2015-04-01

    Quantum communication holds promise for unconditionally secure transmission of secret messages and faithful transfer of unknown quantum states. Photons appear to be the medium of choice for quantum communication. Owing to photon losses, robust quantum communication over long lossy channels requires quantum repeaters. It is widely believed that a necessary and highly demanding requirement for quantum repeaters is the existence of matter quantum memories. Here we show that such a requirement is, in fact, unnecessary by introducing the concept of all-photonic quantum repeaters based on flying qubits. In particular, we present a protocol based on photonic cluster-state machine guns and a loss-tolerant measurement equipped with local high-speed active feedforwards. We show that, with such all-photonic quantum repeaters, the communication efficiency scales polynomially with the channel distance. Our result paves a new route towards quantum repeaters with efficient single-photon sources rather than matter quantum memories.

  17. All-photonic quantum repeaters.

    PubMed

    Azuma, Koji; Tamaki, Kiyoshi; Lo, Hoi-Kwong

    2015-01-01

    Quantum communication holds promise for unconditionally secure transmission of secret messages and faithful transfer of unknown quantum states. Photons appear to be the medium of choice for quantum communication. Owing to photon losses, robust quantum communication over long lossy channels requires quantum repeaters. It is widely believed that a necessary and highly demanding requirement for quantum repeaters is the existence of matter quantum memories. Here we show that such a requirement is, in fact, unnecessary by introducing the concept of all-photonic quantum repeaters based on flying qubits. In particular, we present a protocol based on photonic cluster-state machine guns and a loss-tolerant measurement equipped with local high-speed active feedforwards. We show that, with such all-photonic quantum repeaters, the communication efficiency scales polynomially with the channel distance. Our result paves a new route towards quantum repeaters with efficient single-photon sources rather than matter quantum memories. PMID:25873153

  18. All-photonic quantum repeaters

    PubMed Central

    Azuma, Koji; Tamaki, Kiyoshi; Lo, Hoi-Kwong

    2015-01-01

    Quantum communication holds promise for unconditionally secure transmission of secret messages and faithful transfer of unknown quantum states. Photons appear to be the medium of choice for quantum communication. Owing to photon losses, robust quantum communication over long lossy channels requires quantum repeaters. It is widely believed that a necessary and highly demanding requirement for quantum repeaters is the existence of matter quantum memories. Here we show that such a requirement is, in fact, unnecessary by introducing the concept of all-photonic quantum repeaters based on flying qubits. In particular, we present a protocol based on photonic cluster-state machine guns and a loss-tolerant measurement equipped with local high-speed active feedforwards. We show that, with such all-photonic quantum repeaters, the communication efficiency scales polynomially with the channel distance. Our result paves a new route towards quantum repeaters with efficient single-photon sources rather than matter quantum memories. PMID:25873153

  19. Sequence repeats and protein structure

    NASA Astrophysics Data System (ADS)

    Hoang, Trinh X.; Trovato, Antonio; Seno, Flavio; Banavar, Jayanth R.; Maritan, Amos

    2012-11-01

    Repeats are frequently found in known protein sequences. The level of sequence conservation in tandem repeats correlates with their propensities to be intrinsically disordered. We employ a coarse-grained model of a protein with a two-letter amino acid alphabet, hydrophobic (H) and polar (P), to examine the sequence-structure relationship in the realm of repeated sequences. A fraction of repeated sequences comprises a distinct class of bad folders, whose folding temperatures are much lower than those of random sequences. Imperfection in sequence repetition improves the folding properties of the bad folders while deteriorating those of the good folders. Our results may explain why nature has utilized repeated sequences for their versatility and especially to design functional proteins that are intrinsically unstructured at physiological temperatures.

  20. Estimating repeatability of egg size

    USGS Publications Warehouse

    Flint, P.L.; Rockwell, R.F.; Sedinger, J.S.

    2001-01-01

    Measures of repeatability have long been used to assess patterns of variation in egg size within and among females. We compared different analytical approaches for estimating repeatability of egg size of Black Brant. Separate estimates of repeatability for eggs of each clutch size and laying sequence number varied from 0.49 to 0.64. We suggest that using the averaging egg size within clutches results in underestimation of variation within females and thereby overestimates repeatability. We recommend a nested design that partitions egg-size variation within clutches, among clutches within females, and among females. We demonstrate little variation in estimates of repeatability resulting from a nested model controlling for egg laying sequence and a nested model in which we assumed laying sequence was unknown.

  1. Putative Lineage of Novel African Usutu Virus, Central Europe

    PubMed Central

    Cadar, Daniel; Bosch, Stefan; Jöst, Hanna; Börstler, Jessica; Garigliany, Mutien-Marie; Becker, Norbert

    2015-01-01

    We characterized the complete genome of a putative novel Usutu virus (USUV) strain (Usutu-BONN) detected in a dead blackbird from Germany. Genomic analysis revealed several unique amino acid substitutions among the polyprotein gene. Phylogenetic analyses demonstrated that Usutu-BONN constitutes a putative novel African USUV lineage, which was probably recently introduced to central Europe. PMID:26291923

  2. Identification of Putative Fallopian Tube Stem Cells

    PubMed Central

    Snegovskikh, Victoria; Mutlu, Levent; Massasa, Effi

    2014-01-01

    Stem cells are used to repair and regenerate multiple tissues in the adult. We have previously shown that stem cells play a significant role in mediating endometrial repair and tissue regeneration. We hypothesized that the oviduct may possess a similar population of stem cells that contribute to the maintenance of this tissue. Here we identify label-retaining cells (LRCs) in the murine oviduct which indicate the presence of a stem/progenitor cell population in this tissue as well. Two-day-old CD-1 mice were injected intraperitoneally with 5-bromo-2-deoxyuridine (BrdU) or vehicle control. Female animals (n = 36 for each group) were killed at 6 weeks post injection. Reproductive tracts were removed, specimens were embedded in paraffin, and 5-µ sections were prepared. Oviduct was identified by hematoxylin and eosin staining and morphology. Immunofluorescence studies were performed on serial sections tissues (n = 12 per animal) using antibodies against BrdU. Confocal microscopy was used to identify 4′,6-diamidino-2-phenylindole (DAPI)- and BrdU-stained nuclei. In the group of mice exposed to BrdU, we identified a population of LRCs in all specimens and not in controls. The putative stem cells are located at the base of each villi, suggesting the location of the stem cell niche. The number of DAPI-stained nuclei divided by the number of LRCs; LRCs constituted 0.5% of all nucleated cells. The oviduct contains a population of progenitor cells, likely used in the repair and regeneration of fallopian tube. Defective or insufficient stem cell reserve may underlie common tubal diseases, including hydrosalpinx and ectopic pregnancy. PMID:25305130

  3. Formation of the Arabidopsis Pentatricopeptide Repeat Family1[W

    PubMed Central

    Rivals, Eric; Bruyère, Clémence; Toffano-Nioche, Claire; Lecharny, Alain

    2006-01-01

    In Arabidopsis (Arabidopsis thaliana) the 466 pentatricopeptide repeat (PPR) proteins are putative RNA-binding proteins with essential roles in organelles. Roughly half of the PPR proteins form the plant combinatorial and modular protein (PCMP) subfamily, which is land-plant specific. PCMPs exhibit a large and variable tandem repeat of a standard pattern of three PPR variant motifs. The association or not of this repeat with three non-PPR motifs at their C terminus defines four distinct classes of PCMPs. The highly structured arrangement of these motifs and the similar repartition of these arrangements in the four classes suggest precise relationships between motif organization and substrate specificity. This study is an attempt to reconstruct an evolutionary scenario of the PCMP family. We developed an innovative approach based on comparisons of the proteins at two levels: namely the succession of motifs along the protein and the amino acid sequence of the motifs. It enabled us to infer evolutionary relationships between proteins as well as between the inter- and intraprotein repeats. First, we observed a polarized elongation of the repeat from the C terminus toward the N-terminal region, suggesting local recombinations of motifs. Second, the most N-terminal PPR triple motif proved to evolve under different constraints than the remaining repeat. Altogether, the evidence indicates different evolution for the PPR region and the C-terminal one in PCMPs, which points to distinct functions for these regions. Moreover, local sequence homogeneity observed across PCMP classes may be due to interclass shuffling of motifs, or to deletions/insertions of non-PPR motifs at the C terminus. PMID:16825340

  4. Protein Repeats from First Principles.

    PubMed

    Turjanski, Pablo; Parra, R Gonzalo; Espada, Rocío; Becher, Verónica; Ferreiro, Diego U

    2016-01-01

    Some natural proteins display recurrent structural patterns. Despite being highly similar at the tertiary structure level, repeating patterns within a single repeat protein can be extremely variable at the sequence level. We use a mathematical definition of a repetition and investigate the occurrences of these in sequences of different protein families. We found that long stretches of perfect repetitions are infrequent in individual natural proteins, even for those which are known to fold into structures of recurrent structural motifs. We found that natural repeat proteins are indeed repetitive in their families, exhibiting abundant stretches of 6 amino acids or longer that are perfect repetitions in the reference family. We provide a systematic quantification for this repetitiveness. We show that this form of repetitiveness is not exclusive of repeat proteins, but also occurs in globular domains. A by-product of this work is a fast quantification of the likelihood of a protein to belong to a family. PMID:27044676

  5. Protein Repeats from First Principles

    PubMed Central

    Turjanski, Pablo; Parra, R. Gonzalo; Espada, Rocío; Becher, Verónica; Ferreiro, Diego U.

    2016-01-01

    Some natural proteins display recurrent structural patterns. Despite being highly similar at the tertiary structure level, repeating patterns within a single repeat protein can be extremely variable at the sequence level. We use a mathematical definition of a repetition and investigate the occurrences of these in sequences of different protein families. We found that long stretches of perfect repetitions are infrequent in individual natural proteins, even for those which are known to fold into structures of recurrent structural motifs. We found that natural repeat proteins are indeed repetitive in their families, exhibiting abundant stretches of 6 amino acids or longer that are perfect repetitions in the reference family. We provide a systematic quantification for this repetitiveness. We show that this form of repetitiveness is not exclusive of repeat proteins, but also occurs in globular domains. A by-product of this work is a fast quantification of the likelihood of a protein to belong to a family. PMID:27044676

  6. Protein Repeats from First Principles

    NASA Astrophysics Data System (ADS)

    Turjanski, Pablo; Parra, R. Gonzalo; Espada, Rocío; Becher, Verónica; Ferreiro, Diego U.

    2016-04-01

    Some natural proteins display recurrent structural patterns. Despite being highly similar at the tertiary structure level, repeating patterns within a single repeat protein can be extremely variable at the sequence level. We use a mathematical definition of a repetition and investigate the occurrences of these in sequences of different protein families. We found that long stretches of perfect repetitions are infrequent in individual natural proteins, even for those which are known to fold into structures of recurrent structural motifs. We found that natural repeat proteins are indeed repetitive in their families, exhibiting abundant stretches of 6 amino acids or longer that are perfect repetitions in the reference family. We provide a systematic quantification for this repetitiveness. We show that this form of repetitiveness is not exclusive of repeat proteins, but also occurs in globular domains. A by-product of this work is a fast quantification of the likelihood of a protein to belong to a family.

  7. Integration of evolutionary and desolvation energy analysis identifies functional sites in a plant immunity protein

    PubMed Central

    Casasoli, Manuela; Federici, Luca; Spinelli, Francesco; Di Matteo, Adele; Vella, Nicoletta; Scaloni, Flavio; Fernandez-Recio, Juan; Cervone, Felice; De Lorenzo, Giulia

    2009-01-01

    Plant immune responses often depend on leucine-rich repeat receptors that recognize microbe-associated molecular patterns or pathogen-specific virulence proteins, either directly or indirectly. When the recognition is direct, a molecular arms race takes place where plant receptors continually and rapidly evolve in response to virulence factor evolution. A useful model system to study ligand-receptor coevolution dynamics at the protein level is represented by the interaction between pathogen-derived polygalacturonases (PGs) and plant polygalacturonase-inhibiting proteins (PGIPs). We have applied codon substitution models to PGIP sequences of different eudicotyledonous families to identify putative positively selected sites and then compared these sites with the propensity of protein surface residues to interact with protein partners, based on desolvation energy calculations. The 2 approaches remarkably correlated in pinpointing several residues in the concave face of the leucine-rich repeat domain. These residues were mutated into alanine and their effect on the recognition of several PGs was tested, leading to the identification of unique hotspots for the PGIP-PG interaction. The combined approach used in this work can be of general utility in cases where structural information about a pattern-recognition receptor or resistance-gene product is available. PMID:19372373

  8. Investigation of a Quadruplex-Forming Repeat Sequence Highly Enriched in Xanthomonas and Nostoc sp.

    PubMed

    Rehm, Charlotte; Wurmthaler, Lena A; Li, Yuanhao; Frickey, Tancred; Hartig, Jörg S

    2015-01-01

    In prokaryotes simple sequence repeats (SSRs) with unit sizes of 1-5 nucleotides (nt) are causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noticed in bacteria, reports about SSRs of 6-9 nt are rare. In particular G-rich repeat sequences with the propensity to fold into G-quadruplex (G4) structures have received little attention. In silico analysis of prokaryotic genomes show putative G4 forming sequences to be abundant. This report focuses on a surprisingly enriched G-rich repeat of the type GGGNATC in Xanthomonas and cyanobacteria such as Nostoc. We studied in detail the genomes of Xanthomonas campestris pv. campestris ATCC 33913 (Xcc), Xanthomonas axonopodis pv. citri str. 306 (Xac), and Nostoc sp. strain PCC7120 (Ana). In all three organisms repeats are spread all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed with repeat numbers ranging from two up to 26 units. However a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for G4 formation. Evidence for G4 formation of the consensus repeat sequences was found in biophysical studies utilizing CD spectroscopy. The G-rich repeats are preferably located between aligned open reading frames (ORFs) and are under-represented in coding regions or between divergent ORFs. The G-rich repeats are preferentially located within a distance of 50 bp upstream of an ORF on the anti-sense strand or within 50 bp from the stop codon on the sense strand. Analysis of whole transcriptome sequence data showed that the majority of repeat sequences are transcribed. The genetic loci in the vicinity of repeat regions show increased genomic stability. In conclusion, we introduce and characterize a special class of highly abundant and wide-spread quadruplex-forming repeat sequences in bacteria. PMID:26695179

  9. Investigation of a Quadruplex-Forming Repeat Sequence Highly Enriched in Xanthomonas and Nostoc sp.

    PubMed Central

    Rehm, Charlotte; Wurmthaler, Lena A.; Li, Yuanhao; Frickey, Tancred; Hartig, Jörg S.

    2015-01-01

    In prokaryotes simple sequence repeats (SSRs) with unit sizes of 1–5 nucleotides (nt) are causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noticed in bacteria, reports about SSRs of 6–9 nt are rare. In particular G-rich repeat sequences with the propensity to fold into G-quadruplex (G4) structures have received little attention. In silico analysis of prokaryotic genomes show putative G4 forming sequences to be abundant. This report focuses on a surprisingly enriched G-rich repeat of the type GGGNATC in Xanthomonas and cyanobacteria such as Nostoc. We studied in detail the genomes of Xanthomonas campestris pv. campestris ATCC 33913 (Xcc), Xanthomonas axonopodis pv. citri str. 306 (Xac), and Nostoc sp. strain PCC7120 (Ana). In all three organisms repeats are spread all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed with repeat numbers ranging from two up to 26 units. However a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for G4 formation. Evidence for G4 formation of the consensus repeat sequences was found in biophysical studies utilizing CD spectroscopy. The G-rich repeats are preferably located between aligned open reading frames (ORFs) and are under-represented in coding regions or between divergent ORFs. The G-rich repeats are preferentially located within a distance of 50 bp upstream of an ORF on the anti-sense strand or within 50 bp from the stop codon on the sense strand. Analysis of whole transcriptome sequence data showed that the majority of repeat sequences are transcribed. The genetic loci in the vicinity of repeat regions show increased genomic stability. In conclusion, we introduce and characterize a special class of highly abundant and wide-spread quadruplex-forming repeat sequences in bacteria. PMID:26695179

  10. Limitations on quantum key repeaters.

    PubMed

    Bäuml, Stefan; Christandl, Matthias; Horodecki, Karol; Winter, Andreas

    2015-01-01

    A major application of quantum communication is the distribution of entangled particles for use in quantum key distribution. Owing to noise in the communication line, quantum key distribution is, in practice, limited to a distance of a few hundred kilometres, and can only be extended to longer distances by use of a quantum repeater, a device that performs entanglement distillation and quantum teleportation. The existence of noisy entangled states that are undistillable but nevertheless useful for quantum key distribution raises the question of the feasibility of a quantum key repeater, which would work beyond the limits of entanglement distillation, hence possibly tolerating higher noise levels than existing protocols. Here we exhibit fundamental limits on such a device in the form of bounds on the rate at which it may extract secure key. As a consequence, we give examples of states suitable for quantum key distribution but unsuitable for the most general quantum key repeater protocol. PMID:25903096