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

最近的数据清楚地表明，小于50个氨基酸的蛋白质（这里称为微蛋白）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。我们对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追求两个主要目标：(1)鉴定嗜盐古菌Haloferax volcanii的完整微蛋白质组，以及(2)对已鉴定的选定微蛋白质进行详细分析。在前期工作中，我们已经开始了微蛋白质组的鉴定。从标准和两种应激条件下生长的细胞中分离出蛋白质，并用质谱法进行鉴定。用这种方法总共可以鉴定34个µ蛋白，首先我们选择了4个µ蛋白进行深入研究。对于其中3个µ蛋白基因，我们可以产生缺失菌株，对于第4个基因（我们不能删除），我们使用CRISPRi启动基因抑制。初步调查显示，所有四种菌株都具有有趣的表型，证实了它们值得在拟议的项目中进行研究。综上所述，初步数据清楚地表明，在Haloferax中存在微蛋白，对四种选定的微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，我们将通过使用Z1项目框架中优化的微蛋白研究方案来完成完整Haloferax微蛋白组的鉴定。作为鉴定µ蛋白的另一种方法，我们希望在Z2项目的框架内采用核糖体分析。为了进一步优化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），生成一个完整的六框架翻译。我们将继续对我们在前期工作中开始调查的4个µprotein基因进行分析，以揭示其功能。将对各自的缺失和CRISPRi菌株进行彻底的表型检查。我们将监测这四种µ蛋白的过表达对细胞的影响。我们将利用flag融合蛋白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NMR阐明其结构。进一步值得研究的候选蛋白是由小调控rna编码的µ蛋白。我们检测到两个sRNAs可以作为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详细分析。

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.