Evolution of new protein function in the multi-protein, multi-functional Toll-like receptor 4 complex

摘要 了解蛋白质功能的进化是进化生物化学的中心目标。最机械化的 关于这个问题的工作主要集中在具有单一功能的单一蛋白质上；然而，蛋白质通常作为 多功能、多蛋白质复合体的成员。进化如何优化一种蛋白质的功能 而不打碎另一个吗？新的蛋白质是如何整合成多蛋白质复合体的？回答这些问题并 其他问题需要对多功能、多蛋白质的自然进化进行机械研究。 复合体。 为了回答这些问题，我们建议研究Toll样受体4(TLR4)的进化-一种多蛋白质， 脊椎动物体内发现的多功能蛋白质复合体。这个复合体是动物反应的中心 对感染和组织损伤都有影响。这是一个引起人们极大兴趣的主题，从基础生物学的角度来看 并作为治疗炎症性疾病的药物靶点。该建筑群在数亿年的时间里连续进化。 在这项工作中，我们将提出-并回答-三个问题：1)复合体如何识别亲- 炎症信号在过去的3亿年中发生了变化？2)进化的力量和过程是什么 这导致了这些功能的变化？3)陆地动物的祖先是如何进化出一种全新的蛋白质的？ 并融入到这个建筑群中？为了回答这些问题，我们将结合使用 系统发育分析，祖先序列重建，高通量蛋白质表征， 生物化学/生物物理研究，仔细的功能表征，以及斑马鱼的活体实验。 这项工作将提供对生化和进化机制的前所未有的理解。 这为多功能蛋白质复合体带来了新的功能。通过专门研究进化 导致人类TLR4复合体的过程，这项工作也将提供一个镜头，通过它我们可以解释 先天免疫的动物模型研究，并将其结果应用于人类生物学。最后，一个 进化方法是剖析蛋白质如何工作的有力手段。我们的结果将为我们提供深刻的见解 研究这种重要的蛋白质复合体如何发挥作用，以及如何被操纵以实现更好的人类健康 结果。

SUMMARY Understanding the evolution of protein function is a central goal in evolutionary biochemistry. Most mechanistic work on this problem has focused on single proteins with single functions; however, proteins often work as members of multi-functional, multi-protein complexes. How can evolution optimize one protein function without breaking another? How are new proteins integrated into multi-protein complexes? Answering these and other questions requires mechanistic studies of the natural evolution of multi-functional, multi-protein complexes. To answer these questions, we propose studying the evolution of Toll-like receptor 4 (TLR4)—a multi-protein, multi-functional protein complex found in vertebrate animals. This complex is central to how animals respond to both infection and tissue damage. It is the subject of intense interest, both from the perspective a basic biology and as a drug target for inflammatory disorders. The complex evolved in serial over hundreds of millions of years. In this work, we will pose—and answer—three questions: 1) How did the ability of the complex to recognize pro- inflammatory signals change over the last 300 million years? 2) What were the evolutionary forces and processes that led to these functional changes? 3) How did a completely new protein evolve in the ancestor of land animals and become integrated into the complex? To answer these questions, we will employ a combination of phylogenetic analysis, ancestral sequence reconstruction, high-throughput protein characterization, biochemical/biophysical studies, careful functional characterization, and in vivo experiments in zebrafish. This work will provide unprecedented understanding of the mechanisms—both biochemical and evolutionary— that lead to new function in multi-functional protein complexes. By specifically studying the evolutionary process that led to the human TLR4 complex, this work will also provide a lens through which we can interpret animal model studies of innate immunity and apply their findings to human biology. And, finally, an evolutionary approach is a powerful means to dissect how proteins work. Our results will provide deep insight into how this important protein complex functions and can be manipulated to achieve better human health outcomes.