µ proteins in Haloferax volcanii: an important part of the proteome

Haloferax volcanii 中的 µ 蛋白质：蛋白质组的重要组成部分

• 批准号: 379070156
• 负责人: Professorin Dr. Anita Marchfelder
• 金额: --
• 依托单位: Institut für Molekularbiologie und Biotechnologie der Prokaryoten
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/379070156?language=en
• 关键词: proteins; Haloferax; volcanii; important; part

Recent data clearly show that proteins smaller than 50 amino acids (termed µ proteins here) are synthesised in the cell and fulfil important cellular functions in bacteria and eukaryotes. Very little is known about these proteins in archaea. Therefore we will pursue two major goals in the proposed project: (1) the identification of the complete µ proteome of the halophilic archaeon Haloferax volcanii as well as (2) the detailed analysis of selected already identified µ proteins. In the preliminary work we already initiated the identification of the µ proteome. Proteins from cells grown under standard and two stress conditions were isolated and identified using mass spectrometry. Altogether 34 µ proteins could be identified with this approach and to begin with we selected four µ proteins for in depth investigations. For three of the µ protein genes deletion strains could be generated, for the fourth gene -which we could not delete- we initiated gene repression using CRISPRi. Initial investigation showed for all four strains interesting phenotypes, confirming that they are worth pursuing in the proposed project. Taken together preliminary data clearly show that there are µ proteins present in Haloferax and initial results of the investigation of four selected µ proteins suggest that they have important cellular functions. In the proposed project we will complete the identification of the complete Haloferax µ proteome by using a protocol optimised for the investigation of µ proteins in the frame of the Z1 project. As an additional approach to identify µ proteins we want to employ ribosome profiling in the frame of the Z2 project. To further optimise the µ protein identification we will use bioinformatics to re-annotate the Haloferax genome (in the frame of the Z2 project), generating a complete six-frame translation. Analysis of the four µ protein genes for which we started the investigation in the preliminary work will be continued to unravel their functions. The respective deletion and CRISPRi strains will be examined thoroughly for phenotypes. We will monitor the effect of overexpression of the four µ proteins on the cell. We will co-purify and identify interaction partners of these proteins by using FLAG-fusion proteins. We will determine the cellular localisation of the µ proteins and elucidate their structure using NMR. Further interesting candidates to be studied are µ proteins encoded by small regulatory RNAs. We detected two sRNAs that could act as mRNAs for µ proteins. We successfully generated deletion strains for both sRNA genes that can now be analysed in detail in the proposed project.

最近的数据清楚地表明，小于50个氨基酸的蛋白质(这里称为µ蛋白质)是在细胞中合成的，并在细菌和真核生物中履行重要的细胞功能。人们对古生菌中的这些蛋白质知之甚少。因此，我们将在拟议的项目中追求两个主要目标：(1)鉴定嗜盐考古菌Haloferax Volcanii的完整µ蛋白质组，以及(2)对选定的已鉴定的µ蛋白质进行详细分析。在前期工作中，我们已经开始了对µ蛋白质组的鉴定。分离和鉴定在标准和两种胁迫条件下生长的细胞中的蛋白质。用这种方法总共可以鉴定出34个微蛋白，首先我们选择了四个微蛋白进行深入的研究。对于其中三个µ蛋白基因，我们可以产生缺失菌株，对于第四个基因--我们无法删除--我们使用CRISPRi启动了基因抑制。初步调查显示，所有四个菌株都有有趣的表型，证实它们值得在拟议的项目中继续研究。综上所述，初步数据清楚地表明，在Haloferax中存在µ蛋白，对选定的四种µ蛋白的初步研究结果表明，它们具有重要的细胞功能。在拟议的项目中，我们将使用一种为研究Z1项目框架中的µ蛋白质而优化的方案，完成对完整的Haloferaxµ蛋白质组的鉴定。作为识别µ蛋白的另一种方法，我们希望在Z2项目的框架内使用核糖体图谱。为了进一步优化µ蛋白质的鉴定，我们将使用生物信息学来重新注释Haloferax基因组(在Z2项目的框架内)，生成一个完整的六帧翻译。我们将继续对我们在前期工作中开始调查的四个微蛋白基因进行分析，以揭示它们的功能。将对各自的缺失和CRISPRi菌株进行彻底的表型检查。我们将监测四种微蛋白的过表达对细胞的影响。我们将使用FLAG融合蛋白来共同纯化和鉴定这些蛋白的相互作用伙伴。我们将确定µ蛋白的细胞定位，并使用核磁共振阐明它们的结构。有待研究的另一个有趣的候选是由小调节RNA编码的微蛋白。我们检测到两个sRNA，它们可以作为µ蛋白的mRNAs。我们成功地产生了两个sRNA基因的缺失菌株，现在可以在拟议的项目中进行详细分析。