细胞穿膜肽跨膜转导过程的动力学机制研究

Cell-penetrating peptides (CPPs) have the ability to directly penetrate through membranes into the interior of cells via the mechanism of transduction. Such a mechanism of CPPs is regarded as one of the key factors of virus infection, and on the other hand, promises them as perfect carriers for the transmembrane delivery of gene and drugs into targeted cells. Unfortunately, due to the limit in techniques, previous experiments mainly focus on the “initial” and “final” states of the CPP-membrane interactions, via analyzing the mean status of a large amount of molecules in system. However, direct and/or single-molecular proofs on the dynamic interaction process are still lacking. In this thesis, the applicant plans to work from a new perspective of the changes in “motion modes” of the peptide/lipid molecules due to their changing conformations/states during the CPP-membrane interactions. A new method of single-molecular 3D tracking and (sub-) diffusion analysis will be developed and used, to realize the real-time tracking and physical analysis of the movement behaviors of peptides and lipids during the CPP-membrane interactions. In combination with small-angle X-ray scattering (SAXS) and molecular dynamic (MD) simulations, the temporal and spatial relationship among many complex motion modes, as well as conformational changes, of peptides and lipids will be specifically investigated. Consequently, the dynamic mechanism of transduction process of CPPs will be clarified. The relationship between key residues and transduction efficiency of peptides will be acquired. Based on these, new types of drug carriers will be developed via residue regulation. This work will provide effective method for the researches on peptide (or protein)-cell membrane interfacial dynamics, as well as useful suggestions on designing new types of peptide-based drug-carriers with high efficiency.

细胞穿膜肽（CPP）跨生物膜转导过程是病毒感染细胞的关键步骤，也为靶向于细胞的基因及药物输运提供了理想的候选途径；但由于技术限制，传统实验大多局限于对CPP-膜相互作用中大量分子平均效果的分析或对作用“首”“末”状态的表征，而对其单分子行为及动力学过程缺乏直接的实验观测和物理认识。本项目拟从分子“运动模式”直接反映其构象及状态变化这一新角度出发，开发一种对单个多肽/脂分子进行三维动态追踪和扩散行为分析的方法；结合小角X射线散射实时表征、分子动力学模拟，揭示CPP及脂膜不同运动模式及构象变化之间的时间空间关联性，从而阐明CPP跨膜转导的动力学机制，解决CPP关键氨基酸残基如何影响其跨膜转导效率这一科学问题，进而从氨基酸序列设计的角度调控CPP转导过程和设计高效药物载体。本项目将发展一类针对大分子-细胞膜界面相互作用机制研究的普适手段，为多肽基-高效率药物载体设计提供切实指导。

细胞穿膜肽（以及蛋白质）和细胞膜之间的相互作用对于实现许多基本细胞功能以及药物研发至关重要，然而，对其相互作用机制的理解一直是一个巨大的挑战。究其原因，主要在于相互作用的复杂性，其中许多影响因素在不同的时间和空间尺度上发挥作用，特别是以动态的方式发挥作用。本项目聚焦细胞穿膜肽跨生物膜转导的动力学过程这一核心问题，从三个层次开展研究并取得了创新性研究成果：发展了单脂分子追踪的技术手段、揭示了多种多肽及多肽类纳米药物与细胞膜的界面作用分子机制、进而指导细胞膜靶向的纳米药物设计。具体研究成果包括：1. 通过追踪和分析不同浓度膜活性多肽（蜂毒肽、As-CATH4，TAT）与细胞膜相互作用的过程中，单个脂质分子在细胞膜中的扩散行为，揭示了脂质运动的非高斯特性，并将其定量分解为“normal-diffusion”和“slow-diffusion”两种扩散运动模式；基于此，获得了多肽和膜之间变化的亚稳态相互作用状态的定量信息。这项工作建立了单脂质扩散行为与膜活性肽功能实现之间的关联，从“单脂质分子运动”的角度为研究细胞膜界面复杂动力学过程提供了一种方法。2.从单分子运动的角度，揭示了蜂毒肽通过构象变化实现高效跨膜成孔的动力学过程，揭示了PGLa和MAG2两种多肽在跨膜成孔过程中复杂的协作机制；继而，揭示了脂质纳米粒与细胞膜之间发生识别和结合过程中的两步机制和熵驱动机制。3.基于对多肽膜作用分子机制的深刻理解，设计了一系列以天然多肽为基础的物理攻击型抗耐药菌药物及策略，实现了抗菌效率20倍以上的提高。总之，本项目的实施，发展了一类研究多肽与细胞膜界面作用的生物物理方法，揭示了多种典型多肽的膜作用分子机制，进而为靶向细胞膜的抗菌药物及药物载体的设计提供指导和帮助。项目发表第一/共通讯论文25篇，IF之和>140，授权专利6项，培养学生4名。完成了预期目标。