Tracing the Origins of the Chaperonin (CCT) Complex in Eukaryotes

通过最近的元基因组分析，人们已经清楚地看到，真核生物的根存在于古生物界的微生物中。这个项目将探索一个古老的蛋白质家族的起源，它有助于蛋白质的折叠和稳定，即伴侣蛋白(CPN)，以追踪它们向真核生物的进化。最近对所谓的阿斯加德古生菌基因组中的CPN编码基因的鉴定将被用来获得对这些蛋白质的生化功能的结构洞察，这些蛋白质以前由于生物的不可培养而无法获得。该项目将利用基因合成和表达来自元基因组组装基因组(MAG)的蛋白质。主要的科学利益将是通过应用合成生物学来洞察它们祖先的蛋白质折叠功能，以及从它们的原核生物祖先发展成真核生物。正确的蛋白质折叠对所有生命过程都是必不可少的，对健康和疾病都有影响。更广泛的影响将通过培训一名工作人员科学家和大学本科生以及通过外联活动来实现。该项目的主要目标是发现对新发现的CPN60系统发育树最深分支中蛋白质功能的定量生化洞察。来自古菌最深处分支的快速积累的基因组序列和单扩增基因组(SAG)揭示了随着基因组复杂性和基因复制的增加而产生的谱系。研究人员已经在阿斯加德古生菌中发现了不同的CPN60基因，这些基因似乎是现存真核生物中发现的蛋白质的CCT亚单位的祖先。古生物和真核生物的CPN60都表现出协同变构效应，以响应专有的折叠底物，如微管蛋白。为了探索真核细胞CCT复合体的功能进展，本工作将从ATP结合、ATPase活性和蛋白质折叠能力方面评估这些古老的CPN基因和复杂伴侣的功能。由于在Asgard Archaea单扩增基因组中也存在微管蛋白同源物，该项目将解决令人兴奋的假设，即古生物蛋白平衡是真核生物中发现的详细伴侣/客户蛋白质相互作用网络的祖先试验场，并将追踪伴侣复合体的功能进展，从具有基本生化特征的假定祖先到现代真核生物共享的高度专业化和差异化的真核CCT复合体。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Through recent metagenomic analysis, it has become clear that the root of eukaryotes lies among microbes in the kingdom archaea. This project will explore the origins of an ancient protein family that assists with protein folding and stability, the chaperones (CPNs), to track their evolution into the eukaryotes. Recent identification of CPN encoding genes within the genomes of the so-called Asgard archaea will be utilized in order to gain structural insights into the biochemical function of these proteins, which have previously not been available due to the unculturability of the organisms. The project will utilize gene synthesis and expression of protein from metagenomic assembled genomes (MAGs). The main scientific benefit will be through the application of synthetic biology to gain insights into their ancestral protein folding functions and the development of eukaryotes from their prokaryotic ancestors. Proper protein folding is essential to all life processes and has implications in health and disease. Broader impacts will be through the training of a staff scientist and and undergraduates, as well as through outreach activities. The main goal of the project is the discovery of quantitative biochemical insights into the functions of proteins in the newly discovered deepest branch of the CPN60 phylogenetic tree. Rapidly accumulating genome sequences and Single Amplified Genomes (SAGS) from the deepest branches of the archaea reveal lineages with increases in genome complexity and gene duplication. The investigators have identified divergent CPN60 genes in the ASGARD archaea which appear to be ancestral to the CCT subunits of the proteins found in extant eukarya. CPN60 from both the archaea and eukaryotes show cooperative allosteric effects in response to an obligate folding substrate such as tubulin. To explore the functional progression towards the eukaryotic CCT complex, this work will assess the function of these ancient CPN genes and the complex chaperones in terms of their ATP binding and ATPase activity and protein folding ability. Because there are also tubulin homologs in the Asgard Archaea Single Amplified Genomes, the project will address the exciting hypothesis that archaeal proteostasis was the ancestral proving ground for the detailed chaperone/client protein interaction networks found in eukaryotes, and will trace the functional advances of the chaperone complex from a putative ancestor with rudimentary biochemical features to the highly specialized and differentiated eukaryotic CCT complex shared by modern eukaryotes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.