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

甲烷八叠球菌 Gö1 中参与氮调节的两种小蛋白的分子和功能表征

• 批准号: 379644367
• 负责人: Professorin Dr. Ruth Anne Schmitz-Streit
• 金额: --
• 依托单位: Institut für Allgemeine Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/379644367?language=en
• 关键词: Molecular; functional; characterization; two; small

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.

由于预测、区分编码和非编码sORF及其生化检测方面的挑战，编码小蛋白的小ORF（sORF）长期以来一直被忽视。今天，新技术正在出现，使其能够在全基因组方法中进行全球分析。在之前的资助阶段，在与Z1项目（肽组学平台）合作的一项综合研究中，在1442个预测的sORF中，40个小蛋白质在Mazei甲烷八叠球菌中得到了实验验证。选择其中两种并详细研究，因为它们在氮（N）饥饿下上调。基于我们的研究结果，我们提出，这两个高度保守的小蛋白，sP26和sP36，在N调节中发挥着至关重要的作用。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（GlnA 1）相互作用，并诱导其活性。我们推测，这种诱导是由于稳定的GlnA 1的寡聚状态。此外，sP26可能促进GlnA 1与谷氨酸合酶（GOGAT）的相互作用，该谷氨酸合酶随后将氨基从谷氨酰胺转移到2-OG以产生谷氨酸。在氮饥饿条件下，sP36只定位于细胞质膜。结合进一步的结果，我们假设sP36在氮限制下通过影响铵转运蛋白AmtB而具有调节功能。因此，我们建议第二阶段（i）用复杂的生化工具详细分析GlnA 1-sP26相互作用（NMR，HDX-MS，交联，尺寸排阻色谱法，然后是LC-MS/MS），另外包括GlnK 1和谷氨酸合酶（GOGAT）作为验证的另外的sP26相互作用配偶体;（ii）通过生物化学和遗传学方法结合结构分析来表征sP36与AmtB的相互作用，从而阐明sP36在N饥饿下的生理功能（NMR和晶体结构）和体内单分子跟踪，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与Juri Rappsilber合作结合机器学习，发现小蛋白质的全基因组功能注释，然后对各自的预测进行实验验证。