טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHertz Rivka
SubjectProteins S-Nitrosylation and Dnmt2 Mediated Resistance
to Nitrosative Stress in the Parasite Entamoeba
histolytica
DepartmentDepartment of Medicine
Supervisor Professor Serge Ankri
Full Thesis textFull thesis text - English Version


Abstract

Nitric oxide (NO) works as an antimicrobial agent of many pathogens due to its reactivity as S-nitrosylating agent. In order to determine whether S-nitrosylation of proteins of the parasite, Entamoeba histolytica regulates their function, we used the resin-assisted capture method to identify S-nitrosylated proteins. We observed that S-nitrosylated proteins of E. histolytica are mainly involved in glycolysis/ gluconeogenesis, pyruvate metabolism, and ribosome synthesis. Indeed, NO inhibits many of the key enzymes involved in the metabolism and virulence of the parasite E. histolytica via their S-nitrosylation. Some of the S-nitrosylated proteins as peroxiredoxin and superoxide dismutase may be involved in the resistance to NO. However, very little information is available on the mechanism of resistance to NO in this parasite. We observed that exposure of the parasites to NO strongly reduces their viability and protein synthesis; however, the deleterious effects of NO were significantly reduced in trophozoites overexpressing the cytosine-5 methyltransferases of the Dnmt2 family (Ehmeth). In addition, these trophozoites exhibit high levels of tRNAAsp methylation suggesting that Ehmeth -mediated- tRNAAsp methylation is part of the resistance mechanism to NO.

We previously reported that enolase, another glycolytic enzyme, binds to Ehmeth and inhibits its activity. We observed that the amount of Ehmeth-enolase complex is significantly reduced in NO-treated E. histolytica which explains the aforementioned increase of tRNA methylation. Indeed, we demonstrated via site-directed mutagenesis that cysteine residues 228 and 229 in Ehmeth are susceptible to S-nitrosylation and are crucial for Ehmeth binding to enolase.

Our work provides the first global analysis of S-nitrosylated proteins in E.histolytica and indicates that Ehmeth plays a central role in the response of the parasite to NO and it contributes to the growing evidence towards NO as an emerging regulator of epigenetic mechanisms.