毕赤酵母核糖体蛋白功能及其对外源蛋白合成的调控

Work published in the past year has shown that changes to the ribosome core can affect the mechanism of translation, which potentially leads to specific changes in the relative efficiencies with which different proteins are made. However, few of the translational regulation of heterologous protein synthesis were reported because of the diversity of ribosomal proteins (RPs) and complexity of ribosome structure. The methylotrophic yeast Pichia pastoris is a powerful eukaryotic platform for the production of heterologous protein. Recent publication of the Pichia pastoris genome has facilitated strain development toward biopharmaceutical and environmental science applications, developed the study on transcriptomics and proteomics. On quantitative iTRAQ LC-MS/MS proteomics of the cellular response to heterologous protein overexpression and the regulation of HAC1 in Pichia pastoris, we observed that over 30 of RPs exited the different expression and accompanied with the slower of cells growth. This result is hypothesized that the different expression of RPs would impact the ribosome structure and translational efficiency, especial translation of heterologous protein. To investigate functional characterization of RPs from Pichia pastoris and regulation on heterologous protein synthesis, the ribosome structure with polyribosome analysis and translational efficiency using ribosome-sequence will be carried on. The further research will be developed to RPs gene deletion strains to validate its ribosome translational function and non-ribosome function,containing the protection of ER stress,enhancing scretion of heterologous protein, and so on. It will expand the database of RPs genes and the investigation of ribosome engineering.

由于细胞体内核糖体蛋白的多样性及核糖体结构的复杂性，人们较少在核糖体蛋白及核糖体翻译水平上研究细胞外源蛋白合成调控机制。毕赤酵母为重要外源蛋白表达工业菌株，我们关于毕赤酵母外源蛋白表达及蛋白质组分析中发现：重组毕赤酵母核糖体蛋白下调与细胞生长及外源蛋白过量表达密切相关。本课题拟采用iTRAQ LC-MS/MS分析重组毕赤酵母核糖体蛋白及能量代谢途径的差异表达，结合基于Ribo-seq定量分析核糖体蛋白差异表达对核糖体翻译效率的影响，尤其是对外源蛋白mRNA翻译的影响，分析多核糖体构成与分布变化，研究核糖体蛋白对毕赤酵母细胞生长及外源蛋白合成的调控规律。并通过对表达下调的核糖体蛋白基因缺失构建毕赤酵母突变菌株，分析核糖体蛋白对细胞核糖体翻译、内质网胁迫耐受及蛋白合成分泌等功能的影响，深入探讨核糖体蛋白对毕赤酵母细胞生长及外源蛋白合成的分子调控机制，为毕赤酵母核糖体优化与定制提供基础。

研究选择毕赤酵母非必需核糖体蛋白基因进行敲除和筛选，构建核糖体蛋白缺陷库。通过构建毕赤酵母核糖体蛋白突变株，并研究其生长变化等表型，分析其对蛋白质合成的影响。完成了27株核糖体蛋白缺陷菌株的构建，并初步测定其蛋白质表达量。发现缺失核糖体蛋白可提高蛋白质过量表达，这一现象在缺失60S核糖体蛋白菌株中表现更为明显。研究表明40S控制翻译起始效率，而60S则更多影响翻译的延伸速率，并且缺失核糖体蛋白可使40S、60S组装受到影响，从而使其含量发生变化。本研究表明，rpl38∆、rpl9a∆、rps25∆可增加Phy折叠稳定性，提高Phy的合成质量，减少其在胞内积累的聚集效应，使其蛋白质产量更高。研究核糖体蛋白对毕赤酵母细胞生长和外源蛋白合成的影响，在翻译水平研究外源蛋白合成的分子调控机制，丰富蛋白质合成“中心法则”中外源蛋白翻译调控的内容。 通过毕赤酵母核糖体蛋白突变菌株库的构建、核糖体结构与外源蛋白表达的分析发现，非必需大亚基核糖体蛋白缺少并不影响外源蛋白基因的转录与翻译，但减慢了60s核糖体大亚基的组装，有利于外源蛋白的折叠与活性表达，表明毕赤酵母外源蛋白表达同样存在“翻译暂停”机制。与暨南大学张弓教授合作在国际上首次建立了毕赤酵母翻译组学研究平台。. 此外，研究发现外源蛋白过量表达将引起内质网的扩张，同时引起核糖体蛋白表达的抑制，敲除WSC1（细胞壁完整性胁迫响应因子）核糖体蛋白表达回复，外源蛋白表达进一步提升，使内质网扩增达到一个新的稳态平衡。此外，发现在毕赤酵母中，p180一种犬类来源的核糖体结合蛋白具有与核糖体结合的功能，过表达p180，在内质网表面起到招募核糖体作用，促进外源蛋白的表达。基于外源蛋白分泌表达与内质网扩张线性关系构建了外源蛋白高效分泌表达的筛选技术平台，申请专利2件。.