Regulation der Funktion/Aktivität von Proteinen in Pflanzen durch S-Nitrosylierung
Final Report Abstract
In animals, protein S-nitrosylation, the covalent attachment of nitric oxide (NO) to the thiol group of cysteine residues, is an intensively investigated posttranslational modification, which regulates many different processes. Growing body of evidence suggests that this type of redoxbased regulation mechanism plays a pivotal role in plants, too. Recently, three enzymes of the methylation cycle were identified as candidates for protein S-nitrosylation, including cobalaminindependent methionine synthase, S-adenosylhomocysteinase and S-adenosylmethionine synthetase, assuming that this pathway is regulated by NO. The methylation cycle provides S-adenosylmethionine (SAM) - the most important methyl donor in transmethylation reactions, including the methylation of DNA, RNA, proteins, and secondary metabolites. Additionally, SAM is substrate for the biosynthesis of polyamines and the plant hormone ethylene. The coding sequences of these three enzymes, including all their isoforms, were isolated and recombinant proteins were produced in E. coli. The differential inhibition of Arabidopsis SAMS by protein S-nitrosylation is already described. In detail, the activity of SAMSl is inhibited by GSNO, while the activities of the other two isoforms are not affected by this treatment. To reveal the physiological function of NO-dependent inhibition of SAMSl I analysed an Arabidopsis SAMSl knock-out mutant for its ethylene production, polyamine production, and DNA-methylation status in comparison to wild type plants. While ethylene emission and DNA-methylation status is not changed in the SAMSl knock-out mutant, the synthesis of the polyamines spermidin and spermin is reduced in the mutant. Interesting, polyamines - especially spermidin and spermin - induce rapid biosynthesis of NO in Arabidopsis. Probably the physiological function of S-nitrosylation of SAMSl is a mechanism to control NO production. Under high NO concentrations SAMSl is S-nitrosylated and SAM production will be reduced. A reduced SAM content will result in reduction of spermidin and spermin production and as a consequence of this NO biosynthesis will be diminished. Further experiments are already planned to verify this hypothesis. Additionally, it could be demonstrated that both isoforms of SAHC, but non of the MS enzymes are inhibited by GSNO. To further characterize the GSNO-dependent inhibition of SAHC mutants were generated, where cysteine residues are replaced by serine residues to identify GSNO-sensitive cysteine residue(s). Unfortunately, using this approach no GSNO-sensitive cysteine residue could be identified. Probably, more than one cysteine residue have to be modified (S-nitrosylated) at the same time to get an inhibition of the enzyme activity. Until now it just can be speculated about the physiological function of the NO-dependent inhibition of SAHC. It is already known that the regulation of SAH level is very important for DNA methylation status, since SAH inhibits methyltransferases. A reduced SAH activity will result in an increased SAH content and as consequence in inhibition of methylation reactions and demethylation of genomic DNA. Taken together, investigation of the NO-dependent regulation of the methylation cycle will contribute very important information for a better understanding of protein S-nitrosylation in plants.
