Specificity of cellular signaling scaffolds

细胞信号支架的特异性

• 批准号: RGPIN-2016-06318
• 负责人: Taipale, Mikko
• 金额: $ 4.3万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=616229
• 关键词: Specificity; cellular; signaling; scaffolds

14-3-3 proteins comprise a family of highly conserved and abundant regulatory molecules. They interact with a large number of target (client) proteins in a phosphorylation-dependent manner. By so doing, they are critical components of most eukaryotic signaling pathways. 14-3-3 clients are structurally and functionally highly diverse. They are involved in e.g. cell cycle, apoptosis, DNA damage, transcription, and cell motility. Intriguingly, the exact mode of action of 14-3-3 proteins varies depending on the client protein. They can regulate their clients’ enzymatic activity, conformation, assembly into complexes, subcellular localization, or mediate aggregation. Consequently, 14-3-3s represent an unusually versatile regulatory platform for signaling in eukaryotes. Proteomic studies have begun to unravel the client repertoire of 14-3-3 proteins. However, due to different experimental methods, cell lines, and assay sensitivity, the overlap between these studies is rather modest. Perhaps more importantly, however, standard non-quantitative approaches cannot address one of the major questions in the field: isoform specificity. Almost all organisms express multiple 14-3-3 isoforms, but their specificity is virtually identical in vitro. However, mouse studies have revealed striking isoform-specific phenotypes, suggesting that current proteomic methods are insufficiently sensitive and quantitative. I propose to combine quantitative state-of-the-art mass spectrometry with quantitative pairwise interaction assays to comprehensively characterize the client protein repertoire of all seven human 14-3-3 isoforms in isogenic cell lines. We will uncover novel client proteins, identify isoform-specific interactions, and dissect the basis of specificity at the molecular level. These experiments will be complemented with detailed functional follow-up studies. I will take advantage of my extensive experience with chaperones, which, like 14-3-3s, are abundant molecules with thousands of transiently associating client proteins. In particular, the high-throughput interaction assay LUMIER with BACON, which I developed as a postdoctoral fellow, provides a sensitive and quantitative readout for transient 14-3-3/client interactions. Taken together, our studies will lead to a much more comprehensive understanding of phosphorylation-dependent signaling pathways that are key mediators in development and misregulated in diverse pathologies.

14-3-3蛋白是一个高度保守和丰富的调控分子家族。它们以依赖于磷酸化的方式与大量的靶(客户)蛋白相互作用。通过这样做，它们是大多数真核信号通路的关键组成部分。14-3-3客户端在结构和功能上高度多样化。它们与细胞周期、细胞凋亡、DNA损伤、转录和细胞运动等有关。有趣的是，14-3-3蛋白的确切作用方式因客户蛋白而异。它们可以调节客户的酶活性、构象、组装成复合体、亚细胞定位或中介聚集。因此，14-3-3代表了真核生物信号转导的异常多功能的调控平台。 蛋白质组学研究已经开始揭开14-3-3蛋白质的客户库。然而，由于不同的实验方法、细胞系和检测敏感性，这些研究之间的重叠相当有限。然而，也许更重要的是，标准的非定量方法不能解决该领域的一个主要问题：异构体特异性。几乎所有的生物都表达多种14-3-3亚型，但它们的特异性在体外几乎是相同的。然而，小鼠的研究揭示了惊人的异构体特异性表型，这表明目前的蛋白质组学方法不够灵敏和定量。 我建议将最先进的定量质谱学与定量成对相互作用分析相结合，以全面表征同基因细胞系中所有七种人类14-3-3亚型的客户蛋白谱系。我们将发现新的客户蛋白，识别异构体特异性的相互作用，并在分子水平上剖析特异性的基础。这些实验将得到详细的功能后续研究的补充。我将利用我在伴侣方面的丰富经验，它像14-3-3S一样，是含有数千个瞬时结合的客户蛋白的丰富分子。特别是，我作为博士后开发的高通量相互作用分析LUMIER与培根，为14-3-3/客户的瞬时相互作用提供了灵敏和定量的读数。 综上所述，我们的研究将导致对磷酸化依赖的信号通路的更全面的理解，这些信号通路是发育中的关键中介，在不同的病理中受到错误调控。