Abstract：
A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it\'s membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies.
摘要：
增加治疗性肽跨细胞膜转运的广泛策略是将脂质部分连接到肽主链(脂化)以增强它们的内在膜相互作用。体外和体内研究脂化特征与肽膜易位效率之间的相关性的努力传统上依赖于终点读出测定和基于试错的优化策略。因此,治疗性肽脂化如何影响其膜渗透和易位机制的分子细节仍未解决。在这里，我们采用鲑鱼降钙素作为模型治疗肽，并合成了9种具有不同脂质链长度的双脂化类似物。我们使用单个巨大的单层囊泡（GUV）钙黄绿素流入延时荧光显微镜来确定调整脂化长度如何导致全或无GUV填充机制，指示肽介导的孔形成。最后,我们使用含有GUV的内部GUV测定法来证明,只有能够诱导孔形成的肽类似物显示出有效的膜易位.我们的数据提供了有关治疗性肽脂化如何影响其膜扰动机制的第一个机理细节，并证明了微调脂化参数可以诱导内在的孔形成能力。为研究结构-功能关系而引入的这些见解和基于显微镜的工作流程对于优化未来的肽设计策略至关重要。
