Mechanisms of amphipathic and cargo delivery peptides

描述(申请人提供)：这项研究的长期目标是开发合理的方法来设计和改进穿膜两亲性多肽，这些多肽是抗生素或细胞溶解的，或者可以携带其他有毒分子作为货物进入细胞。实现这一目标的关键知识是确定这些多肽的膜渗透机制(S)。获得这一信息的一个重要步骤是建议在这一应用中进行实验动力学研究，并结合我们开发的全球理论分析，研究一组已知的细胞毒性或抗菌两亲性肽的膜渗透。已知这类多肽具有相当大的靶向性，这似乎源于多肽与靶细胞膜脂双层的相互作用，而不需要蛋白质受体的干预。这种广泛存在的生物防御系统的机制目前还不清楚。选择了长度(14到37个氨基酸)、电荷(0到+6)和特异性(抗生素、抗真菌或溶血)不同的四个肽来测试一组当前的肽机制模型。此外，还将对嵌合构建的转运蛋白TP10进行检测，以确定其作为药物输送载体的机制和用途。研究表明，转运蛋白甚至能够将绿色荧光蛋白等大型蛋白质运送到细胞内。将确定货物的附着是否以及如何改变多肽的作用机制。为了用作运输货物进入细胞的载体，多肽必须在不永久插入双层的情况下转移到囊泡内部。这项研究将指出所研究的哪种类型的多肽是构建这种多肽载体的最佳模板。载物肽可以用来将药物输送到真核细胞中，或者将抗生素输送到细菌细胞中。克服细胞屏障，包括细胞内的隔间，是使用抗生素的主要困难。

DESCRIPTION (provided by applicant): The long-term objective of this research is the development of rational methods for the design and improvement of membrane-penetrating, amphipathic peptides that are antibiotic or cytolytic, or can carry other toxic molecules as cargo into cells. Critical knowledge in reaching this objective is the determination of the mechanism(s) of membrane penetration by these peptides. An important step in obtaining this information is the proposal in this application of an experimental kinetic study, combined with a global theoretical analysis that we have developed, of membrane penetration by a set of known cytotoxic or antibacterial amphipathic peptides. Peptides in this class are known to exhibit considerable target specificity which appears to derive from the interaction of the peptides with the lipid bilayer of the target cell membrane without the intervention of protein receptors. The mechanism of this widespread biological defense system is presently not understood. Four peptides that differ in length (from 14 to 37 amino acids), charge (0 to +6), and specificity (antibiotic, antifungal, or hemolytic), were selected to test a set of current models of peptide mechanisms. In addition, the chimeric construct transportan TP10 will also be examined in order to determine its mechanism and usefulness as a drug delivery vehicle. Transportans have been shown to be able to transport even large proteins, such as green fluorescent protein, into cells. It will be determined whether and how attachment of cargo modifies the mechanism of the peptide action. In order to be useful as vehicles to transport cargo into cells, peptides must translocate into the interior of vesicles without getting permanently inserted into the bilayer. This study will indicate which of the types of peptides examined is the best template to build such a peptide vehicle. Cargo-carrying peptides can be used to transport drugs into eukaryotic cells or antibiotics into bacterial cells. Surmounting cellular barriers, including intracellular compartments, is a major difficulty in the use of antibiotics.