UNS: Engineering Cell-penetrating Peptides to Transport Molecular Cargo into Fungal Cells

PI：Karlsson，Amy J.Proposal number：1511718治疗系统性真菌疾病是一项艰巨的任务，因为可以有效消除真菌感染而不伤害患者的抗真菌药物数量有限。该项目旨在使抗真菌药物的开发具有更好的能力，在不伤害人类细胞的情况下选择性杀死真菌细胞。为了完成这项任务，将研究多肽作为针对真菌细胞的工具，并将分子携带穿过细胞屏障进入真菌细胞。该项目将揭示多肽的性质，这些多肽对于特定靶向真菌细胞而不是人类细胞很重要，并将提供有关可携带到细胞内的分子大小限制的信息。这个项目将使用合理的设计策略和分子生物学工具，通过(1)鉴定能够穿过白念珠菌细胞壁和细胞膜的穿透肽，(2)确定穿透肽介导的进入真菌细胞的货物系链和大小限制，以及(3)改造穿透肽以改善转位和选择性，从而了解细胞穿透肽(CPPs)与真菌细胞，特别是真菌病原体白色念珠菌的相互作用。之前被证明进入哺乳动物细胞的CPPs将被筛选其转运到真菌细胞的能力，并将利用CPPs与蛋白质货物的基因融合来研究货物大小的限制以及货物如何与CPP捆绑在一起。为了提高CPPs的转位和选择性，将设计具有不同疏水性和净电荷的多肽，并对其在白念珠菌和哺乳动物细胞中的转位进行分析，以确定一套设计CPPs的规则，以实现高水平的转位到真菌细胞和低水平的转位到哺乳动物细胞。本项目将阐明多肽的结构与其在真菌细胞中作为CPP的功能之间的关系，并为设计靶向抗真菌药物和为真菌细胞生物学研究提供生物活性分子提供新的平台。该项目将提供设计具有治疗系统性真菌病所需的特异性和有效性的改进的抗真菌药物所需的知识，该项目中开发的方法将可用于开发治疗其他疾病的输送系统。此外，该项目将通过(1)让高中生、本科生和研究生参与拟议的研究项目，(2)为马里兰日创造活动，向孩子和他们的父母介绍生物分子工程，以及(3)将研究技术和成果纳入本科课程，为学生提供未来生物分子工程职位的培训。该提案由化学、生物工程、环境和运输系统部门的生物医学工程计划和颗粒和多阶段过程计划共同资助。

PI: Karlsson, Amy J.Proposal Number: 1511718Treating systemic fungal disease is a difficult task, due to the limited number of antifungal agents that can effectively eliminate fungal infections without harming the patient. This project seeks to enable the development of antifungal agents with an improved ability to selectively kill fungal cells without harming human cells. To accomplish this task, peptides will be studied as tools to target fungal cells and to carry molecules across cellular barriers and into fungal cells. This project will reveal properties of the peptides that are important for specifically targeting fungal cells over human cells and will provide information on limitations on the size of molecules that can be carried into the cells. This information could then be used to design improved antifungal agents and will also contribute to knowledge of fungal cell biology.This project will use rational design strategies and molecular biology tools to build an understanding of the interaction of cell-penetrating peptides (CPPs) with fungal cells, specifically the fungal pathogen Candida albicans, by (1) identifying CPPs able to cross the cell wall and cell membrane of C. albicans, (2) determining cargo tethering and size limitations for CPP-mediated transport into fungal cells, and (3) engineering CPPs for improved translocation and selectivity. CPPs previously shown to enter mammalian cells will be screened for their ability to translocate into fungal cells, and limitations on the size of cargo and how the cargo is tethered to CPPs will be studied using genetic fusions of CPPs to protein cargo. To improve the translocation and selectivity of CPPs, peptides will be designed with varied hydrophobicity and net charge, and the translocation into both C. albicans and mammalian cells will be assayed to determine a set of rules for designing CPPs for high levels of translocation into fungal cells and low levels of translocation into mammalian cells. This project will elucidate the relationship between a peptide's structure and its function as a CPP in fungal cells and provide a new platform for designing targeted antifungal agents and delivering bioactive molecules for fungal cell biology studies. This project will provide knowledge needed to design improved antifungal agents with the specificity and efficacy needed to treat systemic fungal disease, and the approaches developed in this project will be transferable to developing delivery systems for treating other diseases. In addition, this project will provide students with training for future positions in biomolecular engineering by (1) involving high school, undergraduate, and graduate students in the proposed research project, (2) creating activities for Maryland Day that introduce children and their parents to biomolecular engineering, and (3) incorporating research techniques and results into undergraduate courses. This proposal is co-funded by the Biomedical Engineering Program and by the Particulate and Multiphase Processes Program, both in the Chemical, Bioengineering, Environmental and Transport Systems Division.