BRC-BIO: The evolution of cellular stress responses and host defenses to bacterial pathogens

细胞进化出了特定的压力传感器来维持细胞平衡。其中一种传感器是一种被称为IRE1的酶，它可以监测细胞中的蛋白质生产，并在蛋白质生产不正常时发出信号。IRE1传感器的调节缺陷与几种人类疾病有关，包括癌症、糖尿病和神经系统疾病；因此，确定调节这种重要传感器的新方法可能会导致新疗法的创新设计。IRE1通过两种不同的功能来维持蛋白质质量控制，这两种功能可以破坏或编辑构建蛋白质所需的分子指令。这两种功能在这种酶的进化史上的不同阶段进化而来。来自哺乳动物的IRE1具有这两种功能，而来自微生物（如酵母）的IRE1仅具有一种功能。这就引出了一个问题：IRE1最初的功能是什么——破坏还是编辑？为了回答这个问题，本提案试图深入挖掘IRE1传感器的进化史，使用遗传和生物信息学方法来表征IRE1在变形虫中的功能，变形虫与哺乳动物和酵母有共同的祖先。此外，本项目还研究了IRE1是否也作为一种古老的细胞防御有害细菌的功能。这项研究旨在整合基于课程的本科研究经验（CURE）和促进代表性不足群体参与生物学的项目，以培养和多样化未来的STEM劳动力。肌醇要求酶1 （IRE1）是真核细胞中高度保守的应激传感器，可以检测内质网（ER）中错误折叠蛋白的积累。IRE1通过两种完全不同的核糖核酸酶（RNase）活性：mRNA降解和mRNA剪接来维持内质网中的蛋白质质量控制。哺乳动物的IRE1具有两种RNase活性，而某些酵母的IRE1只具有一种RNase活性。本研究旨在通过表征与酵母和哺乳动物有着遥远共同祖先的模型阿米巴Dictyostelium disideum的IRE1，揭示IRE1独特功能的进化历史和机制。这将通过突变阿米巴原虫IRE1氨基酸残基的遗传方法来实现，这些氨基酸残基在阿米巴原虫、酵母和哺乳动物之间是保守的，然后测试这些突变体是否能在破坏内质网蛋白质产生的生长条件下存活。此外，来自不同种类酵母的IRE1将被来自变形虫的IRE1所取代，以测试生物体之间的功能冗余。与此同时，阿米巴IRE1的候选mRNA靶点将通过生物信息学管道进行鉴定，然后通过体内和体外RNase检测进行验证。最后，本研究试图通过利用细菌和变形虫之间的自然宿主-病原体相互作用来揭示IRE1作为古代宿主防御细胞内病原体的作用。由此产生的数据将阐明在所有真核细胞中维持细胞稳态的重要细胞传感器的进化史和功能，并确定如何通过差异调节来控制细胞命运。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The cell has evolved specific stress sensors that maintain cellular balance. One such sensor is an enzyme known as IRE1, which can monitor protein production in the cell, and signal when proteins are not being made properly. Defects in the regulation of the IRE1 sensor have been implicated in several human diseases including cancer, diabetes, and neurological disorders; therefore, identifying new ways to regulate this vital sensor could lead to innovative designs of new therapeutics. IRE1 maintains protein quality control using two distinct functions that either destroys or edits the molecular instructions needed to build proteins. These two functions have evolved at different points in the evolutionary history of this enzyme. While IRE1 from mammals performs both functions, the IRE1 from microorganisms, such as yeast, exclusively performs only one function. This begs the question: what was the original function of IRE1 – destruction or editing? To answer this question, this proposal seeks to dig deeper into the evolutionary history of the IRE1 sensor using genetic and bioinformatic approaches to characterize the function of the IRE1 in amoeba, which share a common ancestor with mammals and yeast. Furthermore, this project investigates whether IRE1 also functions as an ancient cellular defense against harmful bacteria. This research is designed to integrate into a course-based undergraduate research experience (CURE) and programs that promote the participation of underrepresented groups in biology to train and diversify the future STEM workforce.Inositol-requiring enzyme 1 (IRE1) is a highly conserved stress sensor in eukaryotic cells that can detect the accumulation of misfolded proteins in the endoplasmic reticulum (ER). IRE1 maintains protein quality control in the ER using two fundamentally distinct ribonuclease (RNase) activities: mRNA degradation and mRNA splicing. While mammalian IRE1 performs both RNase activities, IRE1 from certain species of yeast exclusively perform only one. This research aims to reveal the evolutionary history and mechanisms that underlie the distinct functions of IRE1 by characterizing IRE1 from the model amoeba, Dictyostelium discoideum, which shares a distant common ancestor with both yeast and mammals. This will be accomplished through genetic approaches that mutate amino acid residues of amoeba IRE1 that are conserved between amoeba, yeast, and mammals, then testing whether these mutants can survive growth conditions that disrupt protein production in the ER. In addition, IRE1 from different species of yeast will be replaced with IRE1 from amoeba to test for functional redundancy between organisms. In parallel, candidate mRNA targets of amoeba IRE1 will be identified using a bioinformatic pipeline, then validated using both in vivo and in vitro RNase assays. Lastly, this proposal seeks to uncover the role of IRE1 as an ancient host defense against intracellular pathogens by exploiting a natural host-pathogen interaction between bacteria and amoeba. The resulting data will elucidate the evolutionary history and function of a vital cellular sensor that maintains cellular homeostasis in all eukaryotic cells and determine how it can be differentially regulated to control cell fate.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.