Inteins: Expanding Biological Roles and Biotechnological Applications

内含子：扩大生物学作用和生物技术应用

基本信息

批准号：
10286202
负责人：
Christopher William Lennon
金额：
$ 39.92万
依托单位：
MURRAY STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10286202
关键词：
Active Sites Archaea Area Attenuated Bacteria Behavior Biochemical Biological Biomedical Research Biomedical Technology Biotechnology Chemistry Communities Cryptococcus neoformans DNA biosynthesis Development Drug Targeting Elements Engineering Environment Environmental Risk Factor Essential Genes Exteins Genes Genetic Goals Human In Vitro Investigation Measures Mediating Methods Mobile Genetic Elements Molecular Monitor Mycobacterium leprae Mycobacterium tuberculosis Nature Organism Oxidative Stress Parasites Peptides Play Post-Translational Regulation Process Protein Engineering Protein Splicing Proteins RNA Splicing Reaction Recombinant Proteins Regulation Regulatory Element Ribosomes Role Signal Transduction Stress System Techniques Technology Time Translating Translations Work Zinc antimicrobial applied biomedical research attenuation base biological adaptation to stress experimental study high risk human pathogen improved in vivo inhibitor/antagonist intein microbial mycobacterial novel pathogen pathogenic bacteria pathogenic fungus pathogenic microbe polypeptide protein activation protein expression protein folding protein purification recombinational repair response sensor tool

项目摘要

PROJECT SUMMARY/ABSTACT The long-term goals of this application are to better understand the biological importance of inteins as post- translational regulatory elements, their emerging role in pathogen stress response, and to harness the power of their unique chemistry to invent useful technologies for the biomedical research community. Inteins, or intervening proteins, are self-catalytic, mobile genetic elements removed from host genes through protein splicing. From the applied perspective, the ability of inteins to shuffle peptide bonds in highly specific ways has proven exceptionally useful in protein engineering, leading to the development of numerous technologies. While intein applications have dominated their investigation, and new intein-based technologies are developed frequently, the biological importance of inteins in nature is poorly understood. Inteins are abundant mobile genetic elements in the microbial world, found in approximately one-half of archaea and one-quarter of bacteria. Contrary to long-standing assumption that inteins are molecular parasites, mounting recent evidence suggests that some intein-containing proteins have evolved to couple splicing, and thus host protein activation, to environmental signals. This represents a novel and potentially widespread form of post-translational regulation. Further, given inteins are absent in humans and located within essential genes of several pathogens, understanding the environmental factors that regulate protein splicing inform possible antimicrobial development. We propose the following two aims, building upon recent discoveries, as well as expanding into new arenas. Aim 1 will for the first time investigate the role inteins play as nascent chains, broadly determining whether splicing is possible prior to release from the ribosome, as well as examining possible stress response strategies of bacterial and fungal pathogens. Aim 2 seeks to develop two novel intein-based technologies. First, a general strategy to improve expression of misfolding-prone proteins in bacteria and second, a zinc- controlled self-removing protein purification tag for use in both bacterial and mammalian systems. Through these Aims, this work will enhance our understanding of the roles these exciting and understudied elements play in nature, their role in pathogen stress response, and will lead to new intein-based technologies for protein engineering.

项目概要/摘要该应用的长期目标是更好地了解内含肽作为后蛋白质的生物学重要性。翻译调控元件，它们在病原体应激反应中的新兴作用，并利用其力量利用其独特的化学特性为生物医学研究界发明有用的技术。内含子，或干预蛋白是自催化的、可移动的遗传元件，通过蛋白质从宿主基因中去除拼接。从应用的角度来看，内含肽以高度特异性的方式重组肽键的能力已经事实证明，它在蛋白质工程中非常有用，导致了许多技术的发展。虽然内含肽应用主导了他们的研究，并且开发了新的基于内含肽的技术通常，人们对内含肽在自然界中的生物学重要性知之甚少。内含子具有丰富的可移动性微生物世界中的遗传元素，存在于大约一半的古细菌和四分之一的古细菌中细菌。与内含肽是分子寄生虫的长期假设相反，最近的证据越来越多表明一些含内含肽的蛋白质已经进化为耦合剪接，从而激活宿主蛋白质，环境信号。这代表了一种新颖且可能广泛传播的翻译后形式规定。此外，给定的内含肽在人类中不存在，并且位于多种必需基因内病原体，了解调节蛋白质剪接的环境因素为可能的抗菌药物提供信息发展。基于最近的发现并扩展到新的领域，我们提出以下两个目标。目标 1 将首次研究内含肽作为新生链的作用，广泛确定是否在从核糖体释放之前可以进行剪接，并检查可能的应激反应细菌和真菌病原体的策略。目标 2 寻求开发两种新型的基于内含肽的技术。首先，改善细菌中容易错误折叠的蛋白质表达的一般策略，其次是锌- 受控自移除蛋白质纯化标签，适用于细菌和哺乳动物系统。通过这些目标，这项工作将增强我们对这些令人兴奋和未充分研究的元素的作用的理解在自然界中发挥作用，它们在病原体应激反应中发挥作用，并将带来新的基于内含肽的蛋白质技术工程。