Background: 3OxoC12 acylhomoserine lactone (3OxoC12) is the master regulator of Quorum Sensing (QS) regulating the expression of several bacterial virulence. Paraoxonase2 (PON2) has lactonase and anti-oxidant activities that are inhibited by 3OxoC12. PON2, expressed in all tissues, appears involved in many areas of defence of the organisms, including bacterial infections, inflammation, oxidative stress, and innate immune systems. PON2 polymorphic sites (148A/G and 311 S/C) have been previously described to be related to cardiac problems and diabetes type 2. The hydrolysis of 3OxoC12 can be a rational approach to the cure of CF. We focused on the comprehension of the transcriptional and the posttranslational regulation of PON2 expression. Materials and methods: We produced in E.coli an engineered version of PON2, with activities matching those described for the native protein, used to generate mutants to understand its structural and biochemical details. We set up qRT-PCRs to highlight the presence of the most abundant mRNA isoforms and to genotype for the two most common PON2 SNP coding variants p.Ala148Gly and p.Ser311Cys. We investigated the regulation of expression of PON2 mRNA, by checking an hypothesis of control via an "mRNA operon", through the silencing of target genes. Results: We focused to identify the post translational modification and we showed a 3OxoC12-dependent ubiquitination (UBQ) (LYS 168) of PON2 nearby a polymorphic site, able to influence the lactonase activity. Recently a second UBQ site, nearby a second polymorphic in position 311, has been identified by us; then we have produced mutants of the two PON2 polymorphic sites (148A/G and 311 S/C) to study the activities and relationships with PTMs. Studying the mechanism of control of PON2 regulation, we identified a RNA binding protein and a E3 ubiquitin ligase that, when silenced, is able to increase the expression of the PON2 gene. Moreover we genotyped for the SNP 311 several cell lines identifying normal, heterozygotes and also mutant homozygotes. Conclusions: Considering the impact of this gene on the defence against gram-bacterial, its involvement in the inflammation, the indication that the polymorphisms could dictate the severity of disease -as already demonstrated for other pathology- it could be useful to analyze CF patients to understand if the gene can have a role of modifier in relation to the presence of SNPs.
Understanding the network of the regulation of PON2 expression
Giovanna Cardiero;Giuseppina Lacerra;Giuseppe Manco
2018
Abstract
Background: 3OxoC12 acylhomoserine lactone (3OxoC12) is the master regulator of Quorum Sensing (QS) regulating the expression of several bacterial virulence. Paraoxonase2 (PON2) has lactonase and anti-oxidant activities that are inhibited by 3OxoC12. PON2, expressed in all tissues, appears involved in many areas of defence of the organisms, including bacterial infections, inflammation, oxidative stress, and innate immune systems. PON2 polymorphic sites (148A/G and 311 S/C) have been previously described to be related to cardiac problems and diabetes type 2. The hydrolysis of 3OxoC12 can be a rational approach to the cure of CF. We focused on the comprehension of the transcriptional and the posttranslational regulation of PON2 expression. Materials and methods: We produced in E.coli an engineered version of PON2, with activities matching those described for the native protein, used to generate mutants to understand its structural and biochemical details. We set up qRT-PCRs to highlight the presence of the most abundant mRNA isoforms and to genotype for the two most common PON2 SNP coding variants p.Ala148Gly and p.Ser311Cys. We investigated the regulation of expression of PON2 mRNA, by checking an hypothesis of control via an "mRNA operon", through the silencing of target genes. Results: We focused to identify the post translational modification and we showed a 3OxoC12-dependent ubiquitination (UBQ) (LYS 168) of PON2 nearby a polymorphic site, able to influence the lactonase activity. Recently a second UBQ site, nearby a second polymorphic in position 311, has been identified by us; then we have produced mutants of the two PON2 polymorphic sites (148A/G and 311 S/C) to study the activities and relationships with PTMs. Studying the mechanism of control of PON2 regulation, we identified a RNA binding protein and a E3 ubiquitin ligase that, when silenced, is able to increase the expression of the PON2 gene. Moreover we genotyped for the SNP 311 several cell lines identifying normal, heterozygotes and also mutant homozygotes. Conclusions: Considering the impact of this gene on the defence against gram-bacterial, its involvement in the inflammation, the indication that the polymorphisms could dictate the severity of disease -as already demonstrated for other pathology- it could be useful to analyze CF patients to understand if the gene can have a role of modifier in relation to the presence of SNPs.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.