Project Details
Projekt
Molecular and functional characterization of two small proteins involved in nitrogen regulation in Methanosarcina mazei Gö1
Applicant
Professorin Dr. Ruth Anne Schmitz-Streit
Subject Area
Metabolism, Biochemistry and Genetics of Microorganisms
Term
since 2017
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 379644367
Small ORFs (sORF) encoded small proteins have been overlooked for a long time due to challenges in prediction, distinguishing between coding and non-coding sORFs and in their biochemical detection. Today new technologies are emerging, which enable their global profiling in genome-wide approaches. In the previous funding phase 40 small proteins were experimentally validated in Methanosarcina mazei with high confidence out of 1442 predicted sORFs in a comprehensive study in collaboration with the Z1 project (peptidomics platform). Two of them were selected and studied in detail since they are upregulated under nitrogen (N) starvation. Based on our findings we propose that both highly conserved small proteins, sP26 and sP36, play a crucial role in N regulation. We clearly showed that under N starvation sP26 interacts with the central enzyme of the N metabolism, glutamine synthetase (GlnA1), and induces its activity. We hypothesize that this induction is due to stabilizing the oligomeric state of GlnA1. In addition, sP26 might facilitate the interaction of GlnA1 with the glutamate synthase (GOGAT) which subsequently transfers the amino-group from glutamine to 2-OG to produce glutamate. sP36 was identified to be localized at the cytoplasmic membrane exclusively under N starvation. Combined with further results we hypothesize that sP36 has a regulatory function by affecting the ammonium transporter AmtB under N limitation. Consequently, we propose for the second phase to (i) analyze the GlnA1-sP26 interaction in detail with sophisticated biochemical tools (NMR, HDX-MS, crosslinking, size exclusions chromatography followed by LC-MS/MS) additionally including GlnK1 and glutamate synthase (GOGAT) as verified additional sP26 interacting partner; (ii) elucidate the physiological function of sP36 under N starvation by characterizing the interaction with AmtB by biochemical and genetic approaches complemented with structural analysis (NMR and crystal structure) and in vivo single molecular tracking, and (iii) discover genome-wide functional annotation of small proteins by advanced proteomic approaches in a functional and structural context within cells combined with machine learning in collaboration with Juri Rappsilber followed by experimental validation of the respective predictions.
DFG Programme
Priority Programmes
