Identification of novel signal transducers in the mammalian unfolded protein response

在其他生物中产生的人类蛋白质，即细菌或哺乳动物细胞培养物，称为重组蛋白质或异源蛋白质。这些蛋白质在工业和医学上有许多应用。它们在临床上给药比从动物身上提纯的本地同类更安全。一种蛋白质的单点突变，即从猪身上分离出来的，如果偏离人类的序列，当给人类使用这种蛋白质时，可能会产生严重的副作用。此外，它们的翻译后修饰，即它们的糖基化模式，与在人类中发现的相似。同样，动物和人类蛋白质之间糖基化模式的微小偏差会严重影响蛋白质在人体内的表现。最后，与从动物中分离蛋白质相比，在重组蛋白质的生产过程中，蛋白质被感染剂污染的风险更容易控制。临床上使用的重组蛋白的生产过程依赖于哺乳动物细胞系。这些生产过程的生长条件和无菌要求使它们变得昂贵。此外，每年需要几千克规模的重组蛋白质的数量正在迅速增长，这就需要开发大规模(几立方米)的生产工艺。生产过程的成本效率通常决定了重组蛋白是否能够进入市场和进入临床。已经制定了几种策略来提高产品产量，进而提高这些生产过程的成本效益。到目前为止，重组蛋白生产的限速步骤是折叠成其天然的、活性的和非免疫原性构象。蛋白质折叠得到了辅助蛋白质的帮助，这些辅助蛋白质保护折叠蛋白质免受环境的影响，称为伴侣蛋白，并催化蛋白质折叠反应的子集，称为折叠酶。最近的基础研究发现，信号转导通路可以感知蛋白质的折叠状态，并激活这些辅助蛋白的表达，以提高蛋白质折叠的效率，以响应这种应激情况。这些信号通路的基因工程有望缓解重组蛋白生产中的蛋白质折叠瓶颈。然而，我们目前对这些信号转导途径的了解仍然有限。根据我们目前的知识，这些途径在重组蛋白生产上的工程结果是不可预测的。为了解决这个问题，我们建议在基因组水平上描述这些途径，通过使用最近发展的基因组技术来监测细胞的完整mRNA和蛋白质群体，以定义它们的完整底物谱。此外，我们将利用这些信号通路的独特生化特征，将研究重点放在有问题的通路上。通过这种方式，我们将针对三种独特的信号转导机制，即mRNAs的非剪接体剪接，当大多数mRNAs的翻译被抑制时优先翻译的mRNAs，以及内质网膜蛋白的蛋白水解性激活。我们期望识别参与这种对蛋白质展开反应的新蛋白质。我们将通过表征新基因在伴侣和折叠酶表达以及内质网应激生存中的作用来表征我们研究中确定的这些信号通路中涉及的新基因的作用。此外，通过定义这些通路的完整底物光谱，我们将能够对这些通路进行工程设计，并对细胞特异性蛋白质生产和细胞活力产生可预测的结果。

Human proteins produced in other organisms, i.e. bacteria or mammalian cell cultures are called recombinant or heterologous proteins. These proteins have many applications in industry and medicine. They are safer to administer in the clinic than their native counterparts purified from i.e. animals. Single point mutations in a protein, i.e. isolated from pigs, that deviate from the human sequence, can have serious side effects when the protein is administered to humans. Further, their posttranslational modifications, i.e. their glycosylation pattern, are similar to those found in humans. Again, minor deviations in the glycosylation pattern between an animal and a human protein can seriously affected the performance of the protein in the human body. Finally, the risk of contamination of the protein with infectious agents is much easier controlled in production processes for recombinant proteins compared to isolation of the protein from animals. Production processes for recombinant proteins for use in the clinic rely on mammalian cell lines. Growth conditions and sterility requirements for these production processes make them expensive. Further, the number of recombinant proteins for which there is a demand on a multikilogram per year scale is rapidly growing, requiring the development of large scale (several cubic metres) production processes. The cost efficiency of a production process often determines if a recombinant protein makes it to the market and to the clinic. Several strategies have been devised to improve product yield and in turn the cost effectiveness of these production processes. To date, the rate-limiting step for production of recombinant proteins is folding into their native, active, and non-immunogenic conformation. Protein folding is assisted by helper proteins that shield a folding protein from its environment, called chaperones, and that catalyse a subset of protein folding reactions, called foldases. Recent basic research has identified signal transduction pathways that sense the folding status of a protein, and that activate expression of these helper proteins to increase the efficiency of protein folding in response to this stress situation. Genetic engineering of these signalling pathways promises to alleviate the protein folding bottleneck in recombinant protein production. However, our current understanding of these signal transduction pathways is still limited. Based on our current knowledge the outcome of engineering of these pathways on recombinant protein production is unpredictable. To address this problem we propose to characterize these pathways on a genomic scale to define their complete substrate spectra by using recently developed genomic technologies to monitor the complete mRNA and protein population of a cell. In addition, we will exploit unique biochemical characteristics of these signalling pathways to focus our study on the pathways in question. In this way we will target three unique signal transduction mechanisms, non-spliceosomal splicing of mRNAs, preferentially translated mRNAs when translation of the majority of mRNAs is inhibited, and proteolytic activation of ER membrane proteins. We anticipate to identify new proteins involved in this response to protein unfolding. We will characterise the role of new genes involved in these signalling pathways identified in our study by characterising their role in chaperone and foldase expression, and survival of ER stress. Furthermore, through defining the complete substrate spectra of these pathways we will enable the engineering of these pathways with predictable outcomes on cell specific protein production and cell viability.