title: Proteomic analysis of 4-hydroxynonenal and nitrotyrosine modified proteins in RTT fibroblasts.
abstract: Rett syndrome (RTT) is a pervasive developmental disorder, primarily affecting girls with a prevalence of 1 in every 10,000 births. A clear etiological factor present in more than 90% of classical RTT cases is the mutation of the gene encoding methyl-CpG-binding protein 2 (MECP2). Recent work from our group was able to shown a systemic oxidative stress (OxS) in these patients that correlates with the gravity of the clinical features. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have performed a two-dimensional gel electrophoresis in order to evidence the oxidative modifications of proteins with special focus on the formation of protein adducts with 4-hydroxynonenal (4-HNE PAs)-a major secondary product of lipid peroxidation- and Nitrotyrosine, a marker derived from the biochemical interaction of nitric oxide (NO) or nitric oxide-derived secondary products with reactive oxygen species (ROS). Then, oxidatively modified spots were identified by mass spectrometry, LC-ESI-CID-MS/MS. Our results showed that 15 protein spots presented 4-HNE PAs and/or nitrotyrosine adducts in fibroblasts proteome from RTT patients compared to healthy control cells. Post-translationally modified proteins were related to several functional categories, in particular to cytoskeleton structure and protein folding. In addition, clear upregulated expression of the inducible NO synthase (iNOS) with high nitrite levels were observed in RTT fibroblasts, justifying the increased nitrotyrosine protein modifications. The present work describes not only the proteomic profile in RTT fibroblasts, but also identifies the modified proteins by 4-HNE and nitrotyrosine. Of note, for the first time, it appears that a dysregulation of NO pathway can be associated to RTT pathophysiology. In conclusion, the evidence of a wide range of proteins able to forms adducts with 4-HNE, Nitrotyrosine or with both confirms the possible alteration of several aspects of cellular functions that well correlates to the complex clinical features of RTT patients.
authors: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy; Child Neuropsychiatry Unit, University Hospital, Azienda Ospedaliera Universitaria Senese (AOUS), Siena, Italy.
authors: Alessandra
authors: A
authors: Pecorelli
authors: Department of Biomedical and Specialist Surgical Sciences, University of Ferrara, Ferrara, Italy.
authors: Carlo
authors: C
authors: Cervellati
authors: Department of Medical Biotechnologies, University of Siena, Siena, Italy.
authors: Alessio
authors: A
authors: Cortelazzo
authors: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.
authors: Franco
authors: F
authors: Cervellati
authors: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy.
authors: Claudia
authors: C
authors: Sticozzi
authors: Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy.
authors: Cristiana
authors: C
authors: Mirasole
authors: Department of Medical Biotechnologies, University of Siena, Siena, Italy.
authors: Roberto
authors: R
authors: Guerranti
authors: Department of Biomedical and Specialist Surgical Sciences, University of Ferrara, Ferrara, Italy.
authors: Alessandro
authors: A
authors: Trentini
authors: Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy.
authors: Lello
authors: L
authors: Zolla
authors: Department of Medical, Surgery and Neurosciences, University of Siena, Siena, Italy.
authors: Vinno
authors: V
authors: Savelli
authors: Child Neuropsychiatry Unit, University Hospital, Azienda Ospedaliera Universitaria Senese (AOUS), Siena, Italy.
authors: Joussef
authors: J
authors: Hayek
authors: Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy. Electronic address: giuseppe.valacchi@unife.it.
authors: Giuseppe
authors: G
authors: Valacchi
mesh_terms: D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D016022:Case-Control Studies; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D020543:Proteome; D040901:Proteomics; D060888:Real-Time Polymerase Chain Reaction; D015518:Rett Syndrome / Q000235:genetics / Q000503:physiopathology*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D000447:Aldehydes; D011506:Proteins; D020543:Proteome; C002744:3-nitrotyrosine; D014443:Tyrosine; C027576:4-hydroxy-2-nonenal
keywords: 4-hydroxynonenal; Inducible nitric oxide synthase; Nitric monoxide; Nitrotyrosine; Oxidative stress; Post-translational modification