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

细胞已经进化出特定的压力传感器来维持细胞平衡。一种这样的传感器是一种称为IRE 1的酶，它可以监测细胞中的蛋白质产生，并在蛋白质没有正常产生时发出信号。IRE 1传感器的调节缺陷与多种人类疾病有关，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。IRE 1使用两种不同的功能来维持蛋白质质量控制，这两种功能要么破坏要么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历史中的不同点进化。虽然来自哺乳动物的IRE 1执行两种功能，但来自微生物如酵母的IRE 1仅执行一种功能。这就引出了一个问题：IRE 1的原始功能是什么--破坏还是编辑？为了回答这个问题，该提案试图使用遗传和生物信息学方法深入挖掘IRE 1传感器的进化历史，以表征IRE 1在变形虫中的功能，变形虫与哺乳动物和酵母有着共同的祖先。此外，该项目还研究了IRE 1是否也作为一种古老的细胞防御有害细菌的功能。这项研究旨在整合到一个基于课程的本科生研究经验（CURE）和程序，促进在生物学中的代表性不足的群体的参与，培养和多样化的未来STEM劳动力。肌醇需要酶1（IRE 1）是一种高度保守的压力传感器在真核细胞中，可以检测错误折叠的蛋白质在内质网（ER）的积累。IRE 1使用两种基本上不同的核糖核酸酶（RNase）活性：mRNA降解和mRNA剪接来维持ER中的蛋白质质量控制。虽然哺乳动物IRE 1执行两种RNA酶活性，但来自某些酵母物种的IRE 1仅执行一种。本研究旨在揭示IRE 1独特功能的进化历史和机制，通过表征来自与酵母和哺乳动物共享遥远共同祖先的模式变形虫Dictyosteelium discoideum的IRE 1。这将通过遗传方法来实现，该方法突变阿米巴IRE 1的氨基酸残基，这些氨基酸残基在阿米巴，酵母和哺乳动物之间是保守的，然后测试这些突变体是否可以在破坏ER中蛋白质生产的生长条件下存活。此外，将来自不同种属酵母的IRE 1替换为来自变形虫的IRE 1，以检测微生物之间的功能冗余。同时，将使用生物信息学管道鉴定阿米巴IRE 1的候选mRNA靶标，然后使用体内和体外RNA酶测定法进行验证。最后，该提案旨在通过利用细菌和阿米巴之间的天然宿主-病原体相互作用来揭示IRE 1作为古老宿主防御细胞内病原体的作用。由此产生的数据将阐明一个重要的细胞传感器的进化历史和功能，在所有真核细胞中维持细胞内稳态，并确定它如何可以被差异调节，以控制细胞fate.This奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.