Identification of novel signal transducers in the mammalian unfolded protein response

在其他生物体（如细菌或哺乳动物细胞培养物）中产生的人类蛋白质称为重组蛋白或异源蛋白。这些蛋白质在工业和医学上有许多应用。在诊所使用它们比从动物中纯化的天然对应物更安全。一种蛋白质的单点突变，即从猪身上分离出来的，偏离人类序列的，当这种蛋白质用于人类时，可能会产生严重的副作用。此外，它们的翻译后修饰，即糖基化模式，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差异会严重影响蛋白质在人体内的表现。最后，在重组蛋白的生产过程中，与从动物中分离蛋白质相比，感染因子污染蛋白质的风险更容易控制。用于临床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昂贵。此外，每年需要数公斤的重组蛋白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能否进入市场和临床。已经制定了若干战略，以提高产品产量，进而提高这些生产过程的成本效益。迄今为止，重组蛋白生产的限速步骤是折叠成它们的天然、活性和非免疫原性构象。蛋白质折叠是由辅助蛋白协助的，这些辅助蛋白保护折叠蛋白不受其环境的影响，称为伴侣蛋白，并催化蛋白质折叠反应的一个子集，称为折叠酶。最近的基础研究已经确定了信号转导途径，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，以应对这种应激情况。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理解仍然有限。根据我们目前的知识，这些重组蛋白生产途径的工程结果是不可预测的。为了解决这个问题，我们建议在基因组尺度上表征这些途径，通过使用最近开发的基因组技术来监测细胞的完整mRNA和蛋白质群，以定义其完整的底物光谱。此外，我们将利用这些信号通路的独特生化特征，将我们的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，mrna的非剪接剪接，当大多数mrna的翻译被抑制时优先翻译mrna，以及ER膜蛋白的蛋白水解激活。我们期望发现新的蛋白质参与这种蛋白质展开的反应。我们将通过描述新基因在伴侣蛋白和折叠酶表达以及内质网应激存活中的作用来描述参与我们研究中发现的这些信号通路的新基因的作用。此外，通过定义这些途径的完整底物光谱，我们将使这些途径的工程具有可预测的细胞特异性蛋白质生产和细胞活力的结果。

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.