Characterisation of the secretion mechanisms of microbial cell factories using organelle proteomics strategies.

我们建议采用蛋白质组学方法来研究两种酵母的蛋白质分泌和周转：微生物细胞工厂毕氏酵母和模式真核生物酿酒酵母。我们将使用基于蛋白质组学的方法来观察，在这两个物种中，异源蛋白从其合成位点到细胞外部的运输动力学。在这两个宿主中，我们将使用重组人溶菌酶（hly）作为测试对象。先前的工作已经证明，使用一系列溶菌酶突变体，HuLY的展开程度是决定其分泌量的主要因素(1)。高度未折叠的变体显示出较差的分泌量并触发未折叠蛋白反应（UPR）。我们将使用全局蛋白质组学方法来确定从内质网到细胞外部溶菌酶及其变体的分泌途径中的何处积累。高度未折叠的蛋白可以诱导内质网应激和未折叠蛋白反应（UPR）。我们采用全局方法来解剖酵母中异源蛋白的分泌途径的基本原理是，不仅需要确定这些蛋白质积累的亚细胞位置，而且需要确定每个特定位置内它们的结合伙伴。使用诱饵免疫沉淀捕获蛋白质复合物的标准方法不能获得关于中间产物的区隔化和区隔内结合伙伴的动态性质的信息。因此，我们将利用Lilley实验室开发的最先进的技术，通过定量蛋白质组学方法结合复杂的统计工具确定亚细胞区室在密度梯度上的分布模式，可以准确地将蛋白质分配到亚细胞位置(2)。在这个项目中，我们将与Waters蛋白质组学主任Jim Langridge合作，进一步发展这种方法，采用最新的无标签蛋白质组学方法，同时确定数千种蛋白质的分布。这种无标签的方法是由沃特斯首创的，比利利实验室的方法有很多优点。迄今为止，常用的是体外等压稳定同位素标签。Lilley实验室最近的研究。已经表明，这些标签，如iTRAQ，在其精度和准确性方面都存在重大问题(3)。稳健的无标记方法已被证明不会遭受iTRAQ标签相同的缺点（4,5），并且它们在确定密度梯度内细胞器蛋白质分布模式时更有可能导致这种模式的精确测量，从而更好地解决与不同亚细胞结构相关的模式。此外，所采用的无标记方法MSE还可以估计不同组分中蛋白质的绝对数量，从而可以测量复合物中蛋白质的化学计量，作为蛋白质种类在每个隔室中蛋白质分子的绝对分布。通过进一步开发无标签定量蛋白质组学方法来确定蛋白质及其结合伙伴的精确亚细胞位置，我们将重点研究重组蛋白的区隔化。我们将对其与其他蛋白质的关联进行全局分析，包括unfold -protein chaperone， Kar2p（一个BiP同源物）和蛋白酶体。我们将对淀粉样蛋白版本的HuLy （I156T）特别感兴趣，并将通过表达阿尔茨海默病蛋白Abeta来验证我们的结果，包括其天然形式和Abeta42 -GFP融合体。1. 熊田，JR .等（2006）地球物理学报，第2卷第4期：711- 720。Dunkley， T等人，（2006）Proc。国家的。科学通报，30(5):357 - 357。Karp， NA等（2010）Mol. Cell Prot。请按4。Silva， JC等人（2005），Anal Chem. 1；77(7): 2187 - 200 5。Stapel, M et al. (2010) Sci Signal.2；3 (111)

We propose to take a proteomics approach to the study of protein secretion and turnover in two yeasts: the microbial cell factory, Pichia pastoris, and the model eukaryote, Saccharomyces cerevisiae. We shall use a proteomics based approach to look, in both species, at the dynamics of transport of a heterologous protein from the site of its synthesis to the cell exterior. In both hosts we shall use recombinant human lysozyme (HuLy) as the test object. Previous work has demonstrated, using a series of lysozyme mutants, that the degree of unfolding of HuLY is a major factor in determining its secreted yield (1). Highly unfolded variants show poor secretion yield and trigger the unfolded protein response (UPR). We shall use a global proteomics approach to determine where in the secretion pathway from the ER to the cell exterior lysozyme and its variants accumulate. Highly unfolded proteins are known to induce both ER stress and the unfolded protein response (UPR). Our rationale for taking a global approach to dissect the secretion pathways of heterologous proteins in yeasts, is based on the need to determine not only the subcellular locations at which such proteins accumulate, but also their binding partners within each specific location. Standard methodologies to capture protein complexes using immunopreciptation of a bait do not yield information about compartmentalisation of intermediates and the dynamic nature of binding partners within compartments. We will thus make use of state-of-the-art technologies developed in the Lilley laboratory which allow accurate assignment of proteins to subcellular locations using distribution patterns of subcellular compartments on density gradients as determined by quantitation proteomics methods coupled with sophisticated statistical tools (2). In this project we will work with Jim Langridge who is Director of Proteomics at Waters to further develop this method employing up-to-date label-free proteomics methodologies to determine the distribution of thousands of proteins simultaneously. This label-free approach has been pioneered by Waters and has many advantages over the methods that the Lilley lab. has used to date, namely isobaric stable isotope in vitro labels. Recent work by the Lilley lab. has shown that these labels, such as iTRAQ, have significant problems regarding both their precision and accuracy (3). Robust label-free approaches have been shown not to suffer from the same shortcomings as the iTRAQ tags (4,5) and their use in determining the distribution patterns of organelle proteins within density gradients are more likely to lead to accurate measurement of such patterns and thus better resolution of the patterns associated with different sub cellular structures. Moreover, the label-free method to be employed, MSE, also estimates the absolute amount of proteins within different fractions, enabling measurement of stoichiometeries of proteins in complexes, as absolute distributions of protein species in terms of molecules of protein per compartment. Having further developed label free quantitative proteomics approaches to determine methods accurate subcellular locations of proteins and their binding partners, we will focus on examining the compartmentalisation of the recombinant protein. We will carry out global analysis of its association with other proteins including the unfolded-protein chaperone, Kar2p (a BiP ortholog) and the proteasome. We shall be particularly interested in the amyloidogenic version of HuLy (I156T) and will validate our results using this variant by expressing the Alzheimer's protein Abeta, both in its native form and as Abeta42 -GFP fusions. 1. Kumita, JR et al (2006) FEBS J273(4):711-20 2. Dunkley, T et al, (2006)Proc. Natl. Acad. Sci 103(17):6518-23 3. Karp, NA et al (2010) Mol. Cell Prot. in press 4. Silva, JC et al (2005), Anal Chem. 1;77(7):2187-200 5. Stapel, M et al (2010) Sci Signal.2;3(111)