Towards a comprehensive protein interactome network in Schizosaccharomyces pombe

描述（由申请人提供）： 跨多个物种的基因组序列的急剧增加使得预测的具有未知功能的蛋白质的数量呈指数级增加。传统的“一次一个方法”表征因素始终是必不可少的，但它有助于能够对这些蛋白质之间的功能联系有一个全面的了解，以帮助生成特定的假设。研究蛋白质功能的一种重要方法是能够确定哪些其他蛋白质与其相关。在本提案中，我们的目标是在模型生物 S. pombe 中系统地生成蛋白质-蛋白质相互作用 (PPI) 数据。除了芽殖酵母之外，裂殖酵母具有所有真核模型生物中最容易操作的基因组，但它在进化上与酿酒酵母不同。我们的目标是使用双管齐下的平台生成裂殖酵母 PPI 图：酵母双杂交方法和共亲和纯化，然后采用质谱 (AP/MS) 方法。这项工作汇集了两个人（Yu 和 Krogan），他们共同使用 AP/MS 策略生成了迄今为止在任何生物体（酿酒酵母）中最全面的复合物 PPI 图谱（Krogan 等人，Nature，2006）。此外，最近，Yu 还生成了迄今为止最全面的双杂交数据集，也是在芽殖酵母中（Yu 等人，Science，2008）。因此，我们认为，在这两个小组之间，我们拥有实验和生物信息学专业知识来对裂殖酵母中的蛋白质相互作用组进行全面分析。从纯粹进化的角度来看，由于这两种酵母在大约 330-4.2 亿年前分化，通过比较这两种生物体的相互作用组网络可以获得有价值的信息。此外，对粟酒裂殖酵母相互作用网络的分析将提供无法从酿酒酵母研究中获得的新信息。例如，在其大型复杂的着丝粒结构、纺锤体结构对有丝分裂进入的限制、冗余和低效的复制起点、组蛋白甲基化和染色质异染色质蛋白的基因调节、RNAi途径的基因和转座子调节、端粒结合蛋白以及大多数基因中存在内含子方面，粟酒裂殖酵母比酿酒酵母更类似于后生动物。因此，该项目不仅将对裂殖酵母领域产生巨大影响，还将推动进化研究，并允许对裂殖酵母领域进行审问。 存在于高等生物中但不存在于芽殖酵母中的生物系统。 公共卫生相关性： 虽然我们对特定疾病致病途径的了解一直在迅速增加，但不同蛋白质和途径之间复杂的相互关联性质阻碍了我们利用这些知识制定治疗策略的能力。在这项研究中，我们的目标是在模型生物粟酒裂殖酵母中生成蛋白质-蛋白质相互作用数据集，该数据集将允许对不同物种之间的物理相互作用组进行进化比较，并将有助于对哺乳动物细胞中也存在的许多途径进行功能询问，其中许多途径在疾病状态下受到干扰。

DESCRIPTION (provided by applicant): The dramatic increase of genome sequences across multiple species is exponentially increasing the number of predicted proteins with unknown function. The traditional "one at a time approach" of characterizing factors will always be essential, but it helps to be able to have a global view of the functional connections between and among these proteins in order to aid in the generation of specific hypotheses. One important way to study protein function is to be able to ascertain which other proteins are associated with it. In this proposal, we aim to systematically generate a protein-protein interaction (PPI) data in the model organism, S. pombe. Next to budding yeast, the fission yeast S. pombe has the most easily manipulatable genome of any eukaryotic model organism, and yet it is evolutionarily distinct from S. cerevisiae. We aim to generate the fission yeast PPI map using a two-pronged platform: yeast two-hybrid methodology and co-affinity purification followed by mass spectrometry (AP/MS) approach. This work brings together two individuals (Yu and Krogan) who worked together to generate the most comprehensive co-complex PPI map to date in any organism (S. cerevisiae) using the AP/MS strategy (Krogan et al., Nature, 2006). Furthermore, more recently, Yu generated the most comprehensive two-hybrid dataset to date, also in budding yeast (Yu et al., Science, 2008). Therefore, we argue that, between the two groups, we have the experimental and bioinformatic expertise to carry out a comprehensive analysis of the protein interactome in fission yeast. From a purely evolutionary standpoint, valuable information would be obtained by comparing the interactome networks from both organisms since these two yeasts diverged around 330-420 million years ago. Additionally, analysis of interaction networks in S. pombe will provide novel information that cannot be obtained from studies performed with S. cerevisiae. For example, in its large complex centromere structure, the restriction of spindle construction to mitotic entry, redundant and inefficient origins of replication, gene regulation by histone methylation and chromodomain heterochromatin proteins, gene and transposon regulation by the RNAi pathway, telomere binding proteins, and the presence of introns in most genes, S. pombe is more similar to metazoans than is S. cerevisiae. Therefore, this project will not only have a huge impact on the fission yeast field but also will fuel evolutionary studies and will allow for interrogation of biological systems that are present in higher organisms but not in budding yeast. PUBLIC HEALTH RELEVANCE: While our knowledge of specific disease causing pathways has been increasing rapidly, our ability to harness this knowledge for therapeutic strategies has been hampered by the complex inter-connected nature of the different proteins and pathways. In this study, we aim to generate a protein-protein interaction dataset in the model organism S. pombe which will allow for evolutionary comparison of physical interactomes across different species and will facilitate the functional interrogation of many pathways that also exist in mammalian cells, many of which are perturbed in disease states.