Molecular and functional characterization of two small proteins involved in nitrogen regulation in Methanosarcina mazei Gö1

长期以来，由于在预测、区分编码和非编码sORF及其生化检测方面的挑战，编码小ORF(SORF)的小蛋白一直被忽视。今天，新的技术正在涌现，这使得它们能够以全基因组的方法进行全球图谱分析。在之前的资助阶段，在与Z1项目(多肽平台)合作的综合研究中，对1442个预测的sORF中的40个小蛋白进行了实验验证，信任度很高。其中两个在氮(N)饥饿条件下表达上调，对它们进行了详细的研究。根据我们的发现，我们认为高度保守的小蛋白sP26和sP36在N调节中都发挥着关键作用。我们清楚地表明，在N饥饿下，sP26与N代谢的中心酶--谷氨酰胺合成酶(GlnA1)相互作用，并诱导其活性。我们假设这种诱导是由于稳定了GlnA1的寡聚态。此外，sP26可能促进GlnA1与谷氨酸合成酶(GOGAT)的相互作用，GOGAT随后将氨基从谷氨酰胺转移到2-OG以产生谷氨酸。在N饥饿条件下，sP36仅定位在细胞膜上。结合进一步的研究结果，我们推测在氮素限制下，sP36通过影响氨离子转运体AmtB而具有调节功能。因此，我们建议第二阶段(I)利用先进的生化工具(核磁共振、HDX-MS、交联、尺寸排除层析和LC-MS/MS)详细分析GlnA1-sP26的相互作用，另外还包括GlnK1和谷氨酸合成酶(GOGAT)作为额外的sP26相互作用伙伴；(Ii)通过生化和遗传学方法表征与AmtB的相互作用，辅之以结构分析(核磁共振和晶体结构)和活体单分子跟踪，阐明sP36在N饥饿下的生理功能；以及(Iii)通过先进的蛋白质组学方法在细胞内的功能和结构背景下结合机器学习发现小蛋白的全基因组功能注释，然后对各自的预测进行实验验证。

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.