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，在细胞中合成了小于50个氨基酸（称为µ蛋白）的蛋白质（称为µ蛋白），并在细菌和真核生物中履行重要的细胞功能。对于古细菌中的这些蛋白质知之甚少。因此，我们将在拟议的项目中实现两个主要目标：（1）鉴定卤素古老的haloferax火山的完整µ蛋白质组以及（2）（2）对已鉴定的µ蛋白的详细分析。在初步工作中，我们已经启动了µ蛋白的鉴定。通过质谱法分离并鉴定出细胞成长的细胞了解标准和两个应力条件的蛋白质。总共可以使用这种方法来鉴定34 µμ蛋白，并首先选择了四个µ蛋白进行深入研究。对于第四个基因，可以产生三个µ蛋白基因缺失菌株 - 我们无法删除 - 我们使用CRISPRI启动了基因表达。最初的投资显示了所有四种菌株有趣的表型，证实了他们在拟议项目中值得追求的。共同进行初步数据清楚地表明，Haloferax中存在µ蛋白质，而四种选定µ蛋白的投资的初始结果表明它们具有重要的细胞功能。在拟议的项目中，我们将通过使用针对Z1项目框架中的µ蛋白投资优化的方案来完成对完整haloferax µ蛋白质组的识别。作为识别µ蛋白的另一种方法，我们希望在Z2项目的框架中采用核糖体分析。为了进一步优化µ蛋白质识别，我们将使用生物信息学重新注释Haloferax基因组（在Z2 Project的框架中），产生完整的六框翻译。对我们开始对初步工作进行投资的四个蛋白基因的分析将继续揭示其功能。将对表型进行彻底检查的相对缺失和CRISPRI菌株。我们将监测四个蛋白在细胞上的过表达的影响。我们将通过使用Flag-Fusion蛋白来纯化并确定这些蛋白质的相互作用伙伴。我们将确定µ蛋白的细胞定位，并使用NMR阐明其结构。要研究的进一步有趣的候选者是由小调节性RNA编码的µ蛋白质。我们检测到两个可以用作µ蛋白的mRNA的SRNA。我们成功地为两个SRNA基因生成了缺失菌株，现在可以在拟议的项目中详细分析。