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（此处称为 µ 蛋白质）在细胞中合成，并在细菌和真核生物中发挥重要的细胞功能，因此我们对古细菌中的这些蛋白质知之甚少。拟议项目：(1) 鉴定嗜盐古菌 Haloferax volcanii 的完整 µ 蛋白质组，以及 (2) 对初步选定的已鉴定 µ 蛋白质进行详细分析。我们已经开始鉴定 µ 蛋白质组。使用质谱法分离并鉴定了在标准和两种应激条件下生长的细胞中的蛋白质，并且首先我们选择了四种 µ 蛋白质。对于三个 µ 蛋白基因删除菌株，我们无法删除第四个基因，我们使用 CRISPRi 启动基因抑制，初步研究显示所有四个菌株都具有有趣的表型，证实它们是值得的。初步数据清楚地表明，Haloferax 中存在 µ 蛋白，并且对四种选定 µ 蛋白的初步研究结果表明，它们具有重要的细胞功能。在拟议项目中，我们将完成完整 Haloferax 的鉴定。 µ 蛋白质组，通过使用针对 Z1 项目框架中 µ 蛋白质研究而优化的方案作为鉴定 µ 蛋白质的另一种方法，我们希望在 Z2 项目框架中采用核糖体分析。进一步优化 µ 蛋白鉴定，我们将使用生物信息学重新注释 Haloferax 基因组（在 Z2 项目的框架中），生成我们在我们将继续开展初步工作，以阐明各自的缺失和 CRISPRi 菌株的表型，我们将监测四种 µ 蛋白的过度表达对细胞的影响。我们将通过 FLAG 融合蛋白确定 µ 蛋白的细胞定位，并使用 NMR 阐明其结构。我们检测到了两种可以由小调节 RNA 编码的 µ 蛋白。作为 µ 蛋白的 mRNA，我们成功地生成了两个 sRNA 基因的缺失菌株，现在可以在拟议的项目中对其进行详细分析。