RESUMO
CpG dinucleotides are efficiently methylated in vertebrate genomes except in the CpG islands having a high C+G content. Methylated CpGs are the single most mutated dinucleotide. Sequences surrounding disease causing CpG mutation sites were analyzed from locus-specific mutation databases. Both tetra- and heptanucleotide analyses indicated clear overall sequence preference for having pyrimidines 5' and purines 3' to the mutated 5-methylcytosine. The most mutated tetranucleotides are TCGA and TCGG, the former being also a frequent restriction and modification site. The results will help in elucidating the still controversial mutation mechanism of CpG doublets.
Assuntos
Fosfatos de Dinucleosídeos/genética , Mutação , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/genética , Sequência de Bases , Metilação de DNA , Fator VIII/genética , Genes p53 , Ligação Genética , Hemofilia A/genética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/genética , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/genética , Mutação Puntual , Proteínas Tirosina Quinases/genética , Cromossomo XRESUMO
The metal ion requirement of a Bacillus subtilis phytase has been studied. Removal of metal ions from the enzyme by EDTA resulted in complete inactivation. Circular dichroism spectroscopy was used to study the effect of metal ion removal on the protein conformation. The loss of enzymatic activity is most likely due to a conformational change, as the circular dichroism spectra of holoenzyme and metal-depleted enzyme were different. Metal-depleted enzyme was partially able to restore the active conformation when incubated in the presence of calcium. Only minor reactivation was detected with other divalent metal ions and their combinations. Based on the data we conclude that B. subtilis phytase requires calcium for active conformation. Calcium has also a strong stabilizing effect on the enzyme against thermal denaturation. However, the conformational change resulted by calcium depletion does not affect the protease susceptibility.
Assuntos
6-Fitase/metabolismo , Bacillus subtilis/enzimologia , Cálcio/metabolismo , Metais/metabolismo , Bacillus subtilis/metabolismo , Dicroísmo Circular , Endopeptidases/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Conformação ProteicaRESUMO
Immunodeficiencies form a distinct group of human hereditary diseases with several rare disorders. During recent years, information has been collected concerning immunodeficiency patients and mutations causing disorders. The large European (ESID) registry contains clinical data for some 7,000 patients. At present, international mutation databases have information for > 1,000 immunodeficiency patients, including X-linked chronic granulomatous disease (XCGD), Wiskott-Aldrich syndrome (WAS), and X-linked thrombocytopenia (XLT), X-linked hyper-IgM syndrome (XHIM), X-linked agammaglobulinemia (XLA), and X-linked severe combined immunodeficiency (XSCID). The databases are available on Internet. The mutation spectra of patients in these registries were compared. Mutational hotspots were found in CpG dinucleotides with a preference for selected flanking bases.
Assuntos
Síndromes de Imunodeficiência/epidemiologia , Síndromes de Imunodeficiência/genética , Mutação , Sistema de Registros/estatística & dados numéricos , Alergia e Imunologia , Bases de Dados como Assunto/estatística & dados numéricos , Humanos , Terminologia como AssuntoRESUMO
X-linked lymphoproliferative disease (XLP) is a rare and severe immune deficiency, characterized by abnormal immune responses to the Epstein-Barr virus. Recently, the gene responsible for XLP, SH2D1A, has been identified and shown to code for a small cytoplasmic protein with an SH2 domain that interacts with SLAM and 2B4, two receptorial molecules involved in signal transduction in T and NK cells, respectively. A variety of SH2D1A gene mutations have been reported thus far in XLP males. Here we describe a single-strand conformation polymorphism assay for mutation analysis in XLP. Four novel patients with SH2D1A mutations are described. These mutants, and the others previously reported in the literature, have been included in a Registry (SH2D1Abase) that is fully accessible on the World Wide Web. A three-dimensional model of the SH2 domain of the SH2D1A protein has been developed, based on homology with other SH2 domains. The structural consequences of disease-causing SH2D1A mutations are discussed.
Assuntos
Proteínas de Transporte/genética , Ligação Genética , Peptídeos e Proteínas de Sinalização Intracelular , Transtornos Linfoproliferativos/genética , Mutação , Cromossomo X/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Transporte/química , Sequência Conservada , Primers do DNA/genética , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Polimorfismo Conformacional de Fita Simples , Conformação Proteica , Homologia de Sequência de Aminoácidos , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Domínios de Homologia de src/genéticaRESUMO
Src homology 2 (SH2) domains recognize phosphotyrosine (pY)-containing sequences and thereby mediate their association to ligands. Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase, in which mutations cause a hereditary immunodeficiency disease, X-linked agammaglobulinemia (XLA). Mutations have been found in all Btk domains, including SH2. We have analyzed the structural and functional effects of six disease-related amino acid substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q. Also, we present a novel Btk SH2 missense mutation, H362R, leading to classical XLA. Based on circular dichroism analysis, the conformation of five of the XLA mutants studied differs from the native Btk SH2 domain, while mutant R307G is structurally identical. The binding of XLA mutation-containing SH2 domains to pY-Sepharose was reduced, varying between 1 and 13% of that for the native SH2 domain. The solubility of all the mutated proteins was remarkably reduced. SH2 domain mutations were divided into three categories: 1) Functional mutations, which affect residues presumably participating directly in pY binding (R307G); 2) structural mutations that, via conformational change, not only impair pY binding, but severely derange the structure of the SH2 domain and possibly interfere with the overall conformation of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural-functional mutations, which contain features from both categories above (Y361C).
Assuntos
Agamaglobulinemia/enzimologia , Agamaglobulinemia/genética , Mutação de Sentido Incorreto , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/genética , Domínios de Homologia de src/genética , Tirosina Quinase da Agamaglobulinemia , Substituição de Aminoácidos/genética , Arginina/genética , Dicroísmo Circular , Ligação Genética , Glicina/genética , Histidina/genética , Humanos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Conformação Proteica , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Solubilidade , Relação Estrutura-Atividade , Cromossomo X/genéticaRESUMO
X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled and the recent update lists 368 entries from 318 unrelated families showing 228 unique molecular events. In addition to mutations the database lists also some polymorphisms and site-directed mutations. Each patient is given a unique patient identity number (PIN). Information is provided regarding the phenotype including symptoms. Mutations in all the five domains of BTK have been noticed to cause the disease, the most common event being missense mutations. The mutations appear almost uniformly throughout the molecule and frequently affect CpG sites forming arginine residues. These hot spots have generally pyrimidines 5'and purines 3'to the mutated cytosine. A decreased frequency of missense mutations was found in the TH, SH3 and the upper lobe of the kinase domain. The putative structural implications of all the missense mutations are given in the database showing 228 unique molecular events, including a novel missense mutation causing an R28C substitution as previously seen in the Xid mouse.
Assuntos
Agamaglobulinemia/genética , Bases de Dados Factuais , Mutação , Proteínas Tirosina Quinases/genética , Cromossomo X/genética , Tirosina Quinase da Agamaglobulinemia , Fosfatos de Dinucleosídeos/genética , Éxons/genética , Ligação Genética , Humanos , Estrutura Secundária de Proteína , Proteínas Tirosina Quinases/químicaRESUMO
X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled and the recent update lists 463 mutation entries from 406 unrelated families showing 303 unique molecular events. In addition to mutations, the database also lists variants or polymorphisms. Each patient is given a unique patient identity number (PIN). Information is included regarding the phenotype including symptoms. Mutations in all the five domains of BTK have been noticed to cause the disease, the most common event being missense mutations. The mutations appear almost uniformly throughout the molecule and frequently affect CpG sites that code for arginine residues. The putative structural implications of all the missense mutations are given in the database. The improved version of the registry having a number of new features is available at http://www. helsinki.fi/science/signal/btkbase.html
Assuntos
Agamaglobulinemia/genética , Bases de Dados Factuais , Mutação , Proteínas Tirosina Quinases/genética , Cromossomo X , Tirosina Quinase da Agamaglobulinemia , Sequência de Aminoácidos , Redes de Comunicação de Computadores , Humanos , Armazenamento e Recuperação da Informação , Dados de Sequência MolecularRESUMO
Primary immunodeficiencies are intrinsic defects of immune systems. Mutations in a large number of cellular functions can lead to impaired immune responses. More than 80 primary immunodeficiencies are known to date. During the last years genes for several of these disorders have been identified. Here, mutation information for 23 genes affected in 14 immunodefects is presented. The proteins produced are employed in widely diverse functions, such as signal transduction, cell surface receptors, nucleotide metabolism, gene diversification, transcription factors, and phagocytosis. Altogether, the genetic defect of 2,140 families has been determined. Diseases with X-chromosomal origin constitute about 70% of all the cases, presumably due to full penetrance and because the single affected allele causes the phenotype. All types of mutations have been identified; missense mutations are the most common mutation type, and truncation is the most common effect on the protein level. Mutational hotspots in many disorders appear in CPG dinucleotides. The mutation data for the majority of diseases are distributed on the Internet with a special database management system, MUTbase. Despite large numbers of mutations, it has not been possible to make genotype-phenotype correlations for many of the diseases.