2,563 research outputs found

    Fast computational mutation-response scanning of proteins

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    Studying the effect of perturbations on protein structure is a basic approach in protein research. Important problems, such as predicting pathological mutations and understanding patterns of structural evolution, have been addressed by computational simulations that model mutations using forces and predict the resulting deformations. In single mutation-response scanning simulations, a sensitivity matrix is obtained by averaging deformations over point mutations. In double mutation-response scanning simulations, a compensation matrix is obtained by minimizing deformations over pairs of mutations. These very useful simulation-based methods may be too slow to deal with large proteins, protein complexes, or large protein databases. To address this issue, I derived analytical closed formulas to calculate the sensitivity and compensation matrices directly, without simulations. Here, I present these derivations and show that the resulting analytical methods are much faster than their simulation counterparts.Fil: Echave, Julián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias Físicas. - Universidad Nacional de San Martín. Instituto de Ciencias Físicas; Argentin

    Investigating the Role of Gene Duplication in Ribosomal Protein Evolution and Testing a Model of Duplicate Gene Retention in Mammals

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    Since Susumu Ohno’s seminal work in 1970, gene duplication has been widely recognized as the origin of multi-gene families and a major mechanism of evolutionary change. Understanding forces that govern the evolution of gene families through retention or loss of duplicated genes has been the subject of much inquiry and debate. The key challenge in this debate is accounting for retention of duplicate genes when, in the absence of some countervailing selective pressure leading to their retention, population genetics predicts that the overwhelming majority of duplicated genes should be lost. In an attempt to investigate the generation and retention of duplicate genes in mammals, the Nelson lab undertook annotation of duplication events in five mammalian genomes. We classified each event by duplication mechanism and duplicate gene fate. This led to two important and unexpected findings: First, half of all conserved duplicates are generated by RNA-based duplication (Retroduplication) events; second, ribosomal protein genes constitute one of the largest classes of conserved duplicated genes in mammals with majority of these duplicates being RNA-based. The work in this thesis begins with identifying and characterizing all gene duplicates of mammalian ribosomal protein gene (RPG) families. We found an unexpected large amount of intact retroduplicates (RTs) which cannot be readily explained by Ohno’s classic gene duplication trajectories. Hence, we propose a novel gene duplication model, Duplication Purification and Inactivation (DPI) that would be able to account for this phenomenon and ultimately serve in conjunction with other established models. Specifically, we hypothesize that dominant negative phenotypes prevent fixation of missense mutations in duplicated genes, thereby extending the survival of intact copies in the genome. Together, this thesis work provides a comprehensive history of ribosomal protein evolution in mammals, comprises a body of evidence that meets or exceeds that available for any other model of duplicate retention, and establishes the impact of forces that could influence the fate of every gene duplication event. Thus, the work described here has the potential to provide one of the most rigorously tested and widely applicable models of duplicate gene retention since Ohno first articulated the problem in the 1970’s

    Interaction of nutrition and genetics via DNMT3L-mediated DNA methylation determines cognitive decline

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    Low homocysteine levels and B vitamin treatment are reported to protect against declining cognitive health. Both B vitamins and homocysteine are involved in the production of S-adenosylmethionine, a universal methyl donor essential for the process of DNA methylation. We investigated the effect of a damaging coding variant within the DNA methyltransferase gene, DNMT3L (R278G, A/G) by examining B vitamin intake, homocysteine levels, cognitive performance, and brain atrophy in individuals in the VITACOG study of Mild Cognitive Impairment and the TwinsUK cohort. In the VITACOG study, individuals who received a two- year treatment of B vitamins and carried the G allele, showed better ‘visuospatial associative memory’ and slower rates of brain atrophy. In the TwinsUK study, improved ‘visuospatial associative memory’ was evident in individuals who reported regular vitamin intake and were A/A homozygotes. In silico modelling indicated that R278G disrupts protein interaction between DNMT3L and DNMT3A, affecting the DNMT3A-3L-H3 complex required for DNA methylation. These findings show that vitamin intake and genetic variation within DNMT3L interact to influence cognitive decline

    Fragment-based screening identifies molecules targeting the substrate-binding ankyrin repeat domains of tankyrase.

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    The PARP enzyme and scaffolding protein tankyrase (TNKS, TNKS2) uses its ankyrin repeat clusters (ARCs) to bind a wide range of proteins and thereby controls diverse cellular functions. A number of these are implicated in cancer-relevant processes, including Wnt/β-catenin signalling, Hippo signalling and telomere maintenance. The ARCs recognise a conserved tankyrase-binding peptide motif (TBM). All currently available tankyrase inhibitors target the catalytic domain and inhibit tankyrase's poly(ADP-ribosyl)ation function. However, there is emerging evidence that catalysis-independent "scaffolding" mechanisms contribute to tankyrase function. Here we report a fragment-based screening programme against tankyrase ARC domains, using a combination of biophysical assays, including differential scanning fluorimetry (DSF) and nuclear magnetic resonance (NMR) spectroscopy. We identify fragment molecules that will serve as starting points for the development of tankyrase substrate binding antagonists. Such compounds will enable probing the scaffolding functions of tankyrase, and may, in the future, provide potential alternative therapeutic approaches to inhibiting tankyrase activity in cancer and other conditions

    Structural model of the anion exchanger 1 (SLC4A1) and identification of transmembrane segments forming the transport site.

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    International audienceThe anion exchanger 1 (AE1), member of bicarbonate transporter family SLC4, mediates an electroneutral chloride/bicarbonate exchange in physiological conditions. However, some point mutations in AE1 membrane spanning domain convert the electroneutral anion exchanger into a Na+ and K+ conductance or induce a cation leak in a still functional anion exchanger. The molecular determinants that govern ion movement through this transporter are still unknown. The present study was intended to identify the ion translocation pathway within AE1. In the absence of resolutive 3D structure of AE1 membrane spanning domain, in silico modeling combined with site directed mutagenesis experiments have been done. A structural model of AE1 membrane spanning domain is proposed and this model is based on the structure of Uracil-proton symporter. This model was used to design cysteine-scanning mutagenesis on transmembrane segments (TM) 3 and 5. By measuring AE1 anion exchange activity or cation leak it is proposed that there is a unique transport site comprising TM3-5 and 8 that should function as an anion exchanger and a cation leak

    STRAIN-SPECIFIC PROTEIN INTERACTION AND LOCALIZATION OF TWO STRAINS OF POTATO YELLOW DWARF VIRUS AND FUNCTIONAL DOMAINS OF THEIR MATRIX PROTEIN

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    Potato yellow dwarf virus (PYDV) is the type species of the genus nucleorhabdovirus which is typified by its nucleotropic characters of the members. The virus accomplishes its replication and morphogenesis in the nuclei of infected cells. Two strains, Constricta strain (CYDV) and Sanguinolenta strain (SYDV) have been described at the level of vector-specificity. CYDV is vectored by Agallia constricta and SYDV is transmitted by Aceratagllia sanguinolenta. The full-length genome of CYDV was sequenced. The 12,792 nt antisense genome encodes seven open reading frames in the order of, nucleocapsid protein (N), unknown protein (X), phosphoprotein (P), movement protein (Y), matrix protein (M), glycoprotein (G), and large polymerase protein (L). The features of each protein including a nuclear localization signal, isoelectric point, and transmembrane domain, were determined by predictive algorithms. The gene coding region was flanked by leader and trailer, and each ORF was separated by a conserved intergenic junction. In the intergenic junctions, the highly conserved cis-regulatory elements, polyadenylation signal, gene spacer, and transcription start site, were identified. The similarities of amino acid sequences between each cognate protein of SYDV and CYDV were higher than 80% except for X and P proteins. The protein localization and interaction assays of each CYDV protein identified strain-specific associations in comparison with those of SYDV and generated unique protein interaction and localization map compared to SYDV. Phylogenetic analysis using L protein identified that CYDV forms a clade with other leafhopper-transmitted rhabdoviruses. Protein sequence comparisons revealed that CYDV X has greater similarity to the cognate protein of Eggplant mottle disease virus than to SYDV X. The localization patterns of CYDV-N and -Y were different compared the cognate proteins of SYDV. The functional nuclear export domain of SYDV M was identified using c-terminal fragments of the Mwt(aa 211-243), MLL223AA(aa 211-243), and MKR225AA(aa 211-243). Based on the data, the functional domains M mediating membrane association, nuclear import and export were mapped for both strains and suggested a model whereby M mediates intra- and intercellular movement of PYDV nucleocapsid

    Production of oilseed rape with increased seed shattering resistance

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    Rapeseed (Brassica napus) production is limited by the crop’s natural propagation mechanism which involves growing siliques that dry out upon maturity and break easily. The resulting pre-harvest yield loss makes shatter resistance an important breeding goal. Studies on Arabidopsis thaliana revealed a set of transcription factors controlling dehiscence zone establishment. INDEHISCENT (IND) and ALCATRAZ (ALC) are major regulators of tissue differentiation in the critical parts of the silique. While ALC is required for the development of a partially degraded separation layer, IND also regulates the essential lignification of neighboring cells, probably through induction of NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1 and 3 (NST1/3) expression. This study aimed at producing rapeseed lines with robust siliques through the use of Bnalc, Bnind, and Bnnst1 mutations. CRISPR/Cas9-mediated gene editing of the two BnALC homoeologs of cultivar ‘Haydn’ efficiently yielded four mutant alleles in a single transgenic T1 plant which were stably inherited. A tensile force test suggested the increased shatter resistance of T2 double mutants. However, the effect was masked by the innate silique robustness of ‘Haydn’. The Bnalc phenotype was therefore confirmed with EMS-induced mutant alleles in the shatter-prone cultivar ‘Express’. Bnind mutations derived from the same ‘Express’ mutant population were utilized for detailed analyses of shatter mechanics. Three phenotyping tests consistently identified a double mutant with especially robust siliques. No lignification defects were observed. Instead, shatter resistance was accounted to a broader replum-valve joint area in combination with smaller cells therein. CRISPR/Cas9-induced mutagenesis of four BnNST1 homoeologs yielded a chimeric T1 plant with multiple mutant alleles per gene copy which now have to be fixed in the progeny. Altogether, the developed mutant material provides novel variation for shatter resistance breeding
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