SELF-SPLICING INTEINS: FUNCTION, EVOLUTION, APPLICATION

描述（申请人提供）：自剪接内含子和内含肽因其分子机制、遗传多样性、在基因组进化中的作用以及在研究、生物技术和医学中的应用而引起关注。对这些元件的兴趣源于它们在RNA水平（内含子）和蛋白质水平（内含肽）的自我剪接特性，以及它们在DNA水平作为移动的遗传元件的能力。在过去的资助期间，我们在这些自我剪接内含子和内含肽的结构和功能表征方面取得了相当大的进展。在下一个研究阶段，我们将集中在相对研究不足的内含肽。内含肽存在于 蛋白质化学、生物技术和分子进化等不同学科的交叉点。它们的自催化肽裂解和连接反应使它们成为现代化学生物学中的有用工具，而它们在重要细胞过程中关键蛋白质中的存在引发了关于它们在自然界中功能的挑衅性问题。 我们提出了以下三个具体的目标，在过去的资助期间所取得的发现的基础上：在第一个目标中，我们将分析侧翼宿主序列，外显蛋白，内含肽结构，剪接进化的作用。这项工作是通过我们与物理学家和结构生物学家的合作实现的。我们还将解决一个大胆的假设，内含肽坚持在特定的外显肽，因为他们赋予了选择性优势，他们的主机，通过适应性相互作用与侧翼外显肽残基。在第二个目标中，我们将研究内含肽抑制剂作为机制探针和抗菌剂。因此，我们将利用内含肽在微生物病原体的关键基因中的存在，探索内含肽作为细菌和真菌抗生素的新靶标。我们将进一步表征顺铂，化疗剂，我们确定为蛋白质剪接抑制剂。我们将研究顺铂对结核分枝杆菌感染的疗效 并测试其抑制隐球菌内含肽活性的能力。此外，我们的目标是分离小分子和肽抑制剂，以期比较它们的性质与对方和顺铂。在第三个目标中，我们将使用我们实验室以前开发的分子方法（氧化还原陷阱，荧光增益蛋白酶传感器和噬菌体展示选择），为生物技术和医学提供时尚工具。因此，我们将利用我们的能力，分离野生型内含肽前体的生物和化学应用，并构建传感器的蛋白酶在肉毒杆菌毒素诊断和检测结核病（TB）的生物标志物作为TB诊断。 我们再一次采取协作的跨学科方法，将遗传学、生物化学和微生物学与物理学和结构生物学联合收割机结合起来。通过这种方式，我们将提高我们对内含肽的结构，功能和进化的理解，作为一种手段，利用它们作为药物开发的潜在目标，并作为生物技术和医学诊断的新试剂。

DESCRIPTION (provided by applicant): Self-splicing introns and inteins attract attention for their molecular mechanisms, phylogentic diversity, role in genome evolution, and application in research, biotechnology and medicine. Interest in these elements stems from their self-splicing properties at the RNA level for introns and protein level for inteins, and from their ability to ac as mobile genetic elements at the DNA level. In the past funding period, we made considerable progress in structural and functional characterization of these self-splicing introns and inteins. For the next research phase, we will focus on the relatively understudied inteins. Inteins exist at the crossroads of the disparate disciplines of protein chemistry, biotechnology and molecular evolution. Their autocatalytic peptide cleavage and ligation reactions make them useful tools in modern chemical biology, whereas their existence within proteins critical to vital cellular processes raises provocative questions about their function in nature. We propose the following three specific aims, based on discoveries made in the past funding period: In the first aim, we will analyze the role of the flanking host sequences, the exteins, on intein structure, splicing an evolution. This work is enabled by our collaborations with physicists and structural biologists. We will also address a bold hypothesis, that inteins persist in specific exteins because they confer a selective advantage on their host, through adaptive interactions with flanking extein residues. In the second aim, we will study intein inhibitors as mechanistic probes and antimicrobials. Thus we will exploit the existence of inteins in critical genes of microbial pathogens, to probe inteins as novel targets for bacterial and fungal antibiotics. We will further characterize cisplatin, the chemotherapeutic agent, which we identified as a protein splicing inhibitor. We will investigate cisplatin's efficacy against infection by Mycobacterium tuberculosis and also test its ability to curtail activity of cryptococcal inteins. Additionally, we aim to isolte small-molecule and peptide inhibitors, with a view to comparing their properties with each other and with cisplatin. In the third aim, we will use molecular methodologies previously developed in our lab (redox traps, gain-of-fluorescence protease sensors, and phage display selections), to fashion tools for biotechnology and medicine. Thus, we will exploit our ability to isolate wild-typ intein precursors for biological and chemical applications, and construct sensors for proteases in a botulism toxin diagnostic and to detect tuberculosis (TB) biomarkers as a TB diagnostic. Once again we are taking collaborative, interdisciplinary approaches, which combine genetics, biochemistry and microbiology with physics and structural biology. In this way, we will enhance our understanding of the structure, function and evolution of inteins, as a means to exploit them as potential targets for drug development and as novel reagents in biotechnology and medical diagnostics.