Mechanism of Listeriolysin O in Cytosolic Delivery

描述(申请人提供)：细胞的胞液空间是药物输送系统和细胞内病原体的重要靶标。许多药物由于其大分子尺寸和亲水性而不需要膜，因此对其专门的、高效的胞液给药策略的需求比以往任何时候都更大。单核细胞增生性李斯特菌进入内细胞室，并利用致孔蛋白Listeriolysin O(LLO)的特殊机制，突破内体膜进入胞浆。LIO已被用于药物递送系统，并在产生模仿李斯特菌入侵的非病毒/非细菌递送系统以将外源大分子递送到细胞质中方面显示了其强大的效用。目前对LLO的分子机制还不是很清楚。具体地说，其pH依赖的活性及其分子调节，如唯一的半胱氨酸所需的还原，目前尚不清楚。目前赠款提案的目标是了解LLO活动的关键要素及其调控机制，可能取决于细胞类型和细胞分化和代谢状态，并利用这些知识更好地设计未来的胞浆递送和靶向策略。需要解决的关键问题是：(I)LLO与含胆固醇膜的相互作用、它的膜结合域、pH敏感元件、结合的开/关速率、寡聚率，以及LLO的哪些结构域以及如何调节这些过程；(Ii)LLO独特的半胱氨酸如何在内吞途径中被还原以激活LLO的活性及其调节方式；(Iii)LLO的递送策略如何受到LLO的各种调节因素的影响。由于内吞体内二硫键的减少在许多发病机制和药物传递系统中都是必不可少的，在调节LLO活性方面也是如此，该提案的重要部分致力于设计和表征探针，以监测内小体的减少，并研究调节减少过程的细胞因素。这项研究的结果和信息不仅对合理设计LLO介导的大分子递送和有效的胞液递送的长期策略，而且对于任何利用可逆二硫键的药物递送策略，以及对于阐明李斯特菌的入侵机制，都是极其重要和关键的。

DESCRIPTION (provided by applicant): The cytosolic space of cells is an important target for drug delivery systems and intracellular pathogens. Many drugs are membrane-impermeant because of their large molecular size and hydrophilic characteristics; therefore the need for their specialized, efficient cytosolic delivery strategy is greater than ever. Listeria monocytogenes enters endocytic compartments and utilizes the specialized mechanism of the pore-forming protein, Listeriolysin O (LLO), to breach the endosomal membrane to escape into the cytosol. LLO has been utilized in drug delivery systems and has demonstrated its powerful utility as an endosomolytic agent in generating non-viral/nonbacterial delivery systems that mimic the Listeria invasion to deliver exogenous macromolecules into the cytosol. The molecular mechanism of the necessary and sufficient agent, LLO, is still not clearly understood. Specifically, its pH-dependent activity and its molecular regulators, such as the required reduction of the unique single cysteine, are not clear at this point. The goal of the current grant proposal is to understand the key elements of LLO activity and its regulatory mechanisms, likely dependent on cell types and cellular differentiation and metabolic states, and to use this knowledge to better design cytosolic delivery and targeting strategies in the future. Key questions to be addressed are: (i) LLO interaction with cholesterol-containing membranes, its membrane-binding domain, pH sensitive elements, on/off rates of binding, oligomerization rate, and how and which domains of LLO regulate these processes, (ii) how the unique cysteine of LLO is reduced in the endocytic pathway to activate the LLO activity and how that is regulated, and (iii) how delivery strategies are affected by various regulatory factors of LLO. As the reduction of disulfide bonds in the endocytic compartment is essential in many cases of pathogenesis and drug delivery systems as much as in the regulation of LLO activity, a significant part of the proposal is dedicated to designing and characterizing probes to monitor the reduction in endosomes in general and to investigate cellular factors modulating the reduction processes. The results and information obtained from the proposed research will be extremely important and critical not only for the rational design of LLO-mediated macromolecule delivery and for the long-term strategy of efficient cytosolic delivery but also for any drug delivery strategy utilizing reversible disulfide bonds, as well as for the elucidation of the Listeria invasion mechanism.