Mutagénèse site-dirigée dans la Laccase du Trametes versicolor et étude de la réactivité

Les Laccases sont des enzymes appartenant à la famille des oxydases multi-cuivre que l'on trouve dans de nombreuses plantes et champignons. Elles catalysent l'oxydation à un électron de substrats aromatiques riches en électrons (phénols ou amines), couplée à la réduction à quatre électrons du dioxygène en eau. Des mutations ponctuelles de la laccase du champignon Trametes versicolor nous ont permis, d'une part, d'acquérir de nouvelles informations sur les interactions entre le substrat et le site actif de l enzyme et, d'autre part, d oxyder un plus large spectre de composés. Notre étude montre que l'efficacité d'oxydation de la laccase ne dépend pas uniquement du potentiel d'oxydo-réduction du substrat. Elle dépend aussi de la facilité d'approche du substrat de l'His458 qui transfère ensuite les électrons au site Cu(T1). L'utilisation de la mutagenèse dirigée pour remplacer l'unique résidu polaire du site actif de la laccase, un acide aspartique en position 206, ainsi que la détermination de la réactivité d'une série homologue de phénols para susbtitués à deux valeurs de pH ont apporté des éléments expérimentaux sans ambiguïté quant à la stabilisation électrostatique impliquant ce résidu anionique (Asp206). Un autre type de mutation a concerné une série de phénylalanines qui limite l accessibilité de la poche catalytique. Plusieurs résidus phénylalanines ont été mutés en résidus alanine plus petits, soit successivement, soit par couple. Nous nous attendions à une augmentation de l'efficacité de l'oxydation des substrats encombrants. Malheureusement les mutants n'ont pas tous montré une réactivité supérieure à celle de l'enzyme non mutée envers la série choisie de phénolsLaccases are a family of multi-copper oxidases, found in many plants and fungi. They catalyse single electron oxidation of electron-rich aromatic substrates, usually phenols or amines, coupled to the four-electron reduction of dioxygen to water. Single-point mutations in Trametes versicolor Laccase have enabled us to acquire novel information about the profitable interactions of the substrate in the enzymatic pocket and to design and express mutated enzymes hopefully apt to oxidize a larger spectrum of substrates. Our study shows that the oxidation efficiency of laccase does not uniquely respond to the redox potential of a putative substrate. It also reflects the ease of approach of the substrate to T1 Cu through its His458 electron relay. The use of site-directed mutagenesis to replace the unique polar residue in laccase active site (Asp206) with Glu, Ala and Asn residues, along with the determination of the enzymatic reactivity at two different pH values, has given unambiguous experimental support to the electrostatic stabilization brought about by this anionic 206-residue. The use of an homologous series of X-substituted phenols has enabled us to shed light on this facet of the oxidation reactivity. The other mutation concerned a series of phenylalanines which confers size-discriminating ability to the enzyme. Upon mutating these phenylalanine residues in single and double mutations with smaller alanine residues we expected an increase in the oxidation efficiency of the mutants towards bulky substrates. Unfortunately not all the Phe mutants turned out to be more reactive than the non-mutated TvL towards a series of model phenols endowed with increasing size.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceItalyFRI

Influence of 9p21.3 Genetic Variants on Clinical and Angiographic Outcomes in Early-Onset Myocardial Infarction

Objectives: The purpose of this study was to test whether the 9p21.3 variant rs1333040 influences the occurrence of new cardiovascular events and coronary atherosclerosis progression after early-onset myocardial infarction. Background: 9p21.3 genetic variants are associated with ischemic heart disease, but it is not known whether they influence prognosis after an acute coronary event. Methods: Within the Italian Genetic Study of Early-onset Myocardial Infarction, we genotyped rs1333040 in 1,508 patients hospitalized for a first myocardial infarction before the age of 45 years who underwent coronary angiography without index event coronary revascularization. They were followed up for major cardiovascular events and angiographic coronary atherosclerosis progression. Results: Over 16,599 person-years, there were 683 cardiovascular events and 492 primary endpoints: 77 cardiovascular deaths, 223 reoccurrences of myocardial infarction, and 383 coronary artery revascularizations. The rs1333040 genotype had a significant influence (p = 0.01) on the primary endpoint, with an adjusted hazard ratio of 1.19 (95% confidence interval [CI]: 1.08 to 1.37) for heterozygous carriers and 1.41 (95% CI: 1.06 to 1.87) for homozygous carriers. Analysis of the individual components of the primary endpoints provided no significant evidence that the rs1333040 genotype influenced the hazard of cardiovascular death (p = 0.24) or the reoccurrence of myocardial infarction (p = 0.57), but did provide significant evidence that it influenced on the hazard of coronary revascularization, with adjusted heterozygous and homozygous ratios of 1.38 (95% CI: 1.17 to 1.63) and 1.90 (95% CI: 1.36 to 2.65) (p = 0.00015), respectively. It also significantly influenced the angiographic endpoint of coronary atherosclerosis progression (p = 0.002). Conclusions: In early-onset myocardial infarction, the 9p21.3 variant rs1333040 affects the progression of coronary atherosclerosis and the probability of coronary artery revascularization during long-term follow-up

Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants.

We conducted a genome-wide association study testing single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) for association with early-onset myocardial infarction in 2,967 cases and 3,075 controls. We carried out replication in an independent sample with an effective sample size of up to 19,492. SNPs at nine loci reached genome-wide significance: three are newly identified (21q22 near MRPS6-SLC5A3-KCNE2, 6p24 in PHACTR1 and 2q33 in WDR12) and six replicated prior observations (9p21, 1p13 near CELSR2-PSRC1-SORT1, 10q11 near CXCL12, 1q41 in MIA3, 19p13 near LDLR and 1p32 near PCSK9). We tested 554 common copy number polymorphisms (>1% allele frequency) and none met the pre-specified threshold for replication (P < 10(-3)). We identified 8,065 rare CNVs but did not detect a greater CNV burden in cases compared to controls, in genes compared to the genome as a whole, or at any individual locus. SNPs at nine loci were reproducibly associated with myocardial infarction, but tests of common and rare CNVs failed to identify additional associations with myocardial infarction risk

Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants

We conducted a genome-wide association study testing single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) for association with early-onset myocardial infarction in 2,967 cases ad 3,075 controls. We carried out replication in an independent sample with an effective sample size of up to 19,492. SNPs at nine loci reached genome-wide significance: three are newly identified (21q22 near MRPS6-SLC5A3-KCNE2, 6p24 in PHACTR1 and 2q33 in WDR12) and six replicated prior observations(1-4) (9p21, 1p13 near CERSL2-PSRC1-SORT1, 10q11 near CXCL12, 1q41 in MIA3, 19p13 near LDLR and 1p32 near PCSK9). We tested 554 common copy number polymorphisms (> 1% allele frequency) and none met the pre-specified threshold for replication (P < 10(-3)). We identified 8,065 rare CNVs but did not detect a greater CNV burden in cases compared to controls, in genes compared to the genome as a whole, or at any individual locus. SNPs at nine loci were reproducibly associated with myocardial infarction, but tests of common and rare CNVs failed to identify additional associations with myocardial infarction risk

Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants

We conducted a genome-wide association study testing single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) for association with early-onset myocardial infarction in 2,967 cases and 3,075 controls. We carried out replication in an independent sample with an effective sample size of up to 19,492. SNPs at nine loci reached genome-wide significance: three are newly identified (21q22 near MRPS6-SLC5A3-KCNE2, 6p24 in PHACTR1 and 2q33 in WDR12) and six replicated prior observations (9p21, 1p13 near CELSR2-PSRC1-SORT1, 10q11 near CXCL12, 1q41 in MIA3, 19p13 near LDLR and 1p32 near PCSK9). We tested 554 common copy number polymorphisms (>1% allele frequency) and none met the pre-specified threshold for replication (P < 10(-3)). We identified 8,065 rare CNVs but did not detect a greater CNV burden in cases compared to controls, in genes compared to the genome as a whole, or at any individual locus. SNPs at nine loci were reproducibly associated with myocardial infarction, but tests of common and rare CNVs failed to identify additional associations with myocardial infarction risk