PDF(Pubmed)
Abstract：
Kinetoplastid pathogens including Trypanosoma brucei, T. cruzi, and Leishmania species, are early diverged, eukaryotic, unicellular parasites. Functional understanding of many proteins from these pathogens has been hampered by limited sequence homology to proteins from other model organisms. Here we describe the development of a high-throughput deep mutational scanning approach in T. brucei that facilitates rapid and unbiased assessment of the impacts of many possible amino acid substitutions within a protein on cell fitness, as measured by relative cell growth. The approach leverages several molecular technologies: cells with conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants, degron-controlled stabilization of I-SceI meganuclease to mediate highly efficient transfection of a mutant allele library, and a high-throughput sequencing readout for cell growth upon conditional knockdown of wild-type gene expression and exclusive expression of mutant variants. Using this method, we queried the effects of amino acid substitutions in the apparently non-catalytic RNase III-like domain of KREPB4 (B4), which is an essential component of the RNA Editing Catalytic Complexes (RECCs) that carry out mitochondrial RNA editing in T. brucei. We measured the impacts of thousands of B4 variants on bloodstream form cell growth and validated the most deleterious variants containing single amino acid substitutions. Crucially, there was no correlation between phenotypes and amino acid conservation, demonstrating the greater power of this method over traditional sequence homology searching to identify functional residues. The bloodstream form cell growth phenotypes were combined with structural modeling, RECC protein proximity data, and analysis of selected substitutions in procyclic form T. brucei. These analyses revealed that the B4 RNaseIII-like domain is essential for maintenance of RECC integrity and RECC protein abundances and is also involved in changes in RECCs that occur between bloodstream and procyclic form life cycle stages.
摘要：
活动质体病原体，包括布鲁氏锥虫，T.Cruzi,和利什曼原虫物种，早期分歧，真核生物,单细胞寄生虫.对来自这些病原体的许多蛋白质的功能理解受到与来自其他模型生物的蛋白质的有限序列同源性的阻碍。在这里，我们描述了在T.brucei中高通量深度突变扫描方法的开发，该方法有助于快速和无偏见地评估蛋白质中许多可能的氨基酸取代对细胞适应性的影响。通过相对细胞生长来衡量。该方法利用了几种分子技术：具有感兴趣的野生型基因的条件表达和突变变体文库的组成型表达的细胞，degron控制的I-SceI大范围核酸酶的稳定，以介导突变等位基因文库的高效转染，和高通量测序读数，用于有条件敲除野生型基因表达和突变变体的排他性表达后的细胞生长。使用此方法，我们查询了KREPB4（B4）的明显非催化RNaseIII样结构域中氨基酸取代的影响，它是RNA编辑催化复合物（RECs）的重要组成部分，该复合物在布鲁氏菌中进行线粒体RNA编辑。我们测量了数千种B4变体对血流形式细胞生长的影响，并验证了含有单个氨基酸取代的最有害变体。至关重要的是，表型和氨基酸保守性之间没有相关性，证明了这种方法比传统的序列同源性搜索更强大，可以识别功能残基。血流形式细胞生长表型与结构模型相结合，RECC蛋白质接近度数据，并分析了顺环形式布氏T.brucei中的选定取代。这些分析表明，B4RNaseIII样结构域对于维持RECC完整性和RECC蛋白丰度至关重要，并且还参与在血流和前循环形式生命周期阶段之间发生的RECS变化。
