Mechanism of Listeriolysin O in Cytosolic Delivery

描述（由申请人提供）：细胞的胞质空间是药物递送系统和细胞内病原体的重要靶点。许多药物是膜不渗透的，因为它们的大分子尺寸和亲水特性;因此，需要其专门的，有效的胞质递送策略比以往任何时候都更大。单核细胞增生李斯特菌进入内吞区室，并利用孔形成蛋白李斯特菌溶血素O（LLO）的专门机制，突破内体膜逃逸到胞质溶胶中。LLO已用于药物递送系统中，并且已证明其作为内体溶解剂在产生模拟李斯特菌入侵以将外源性大分子递送到胞质溶胶中的非病毒/非细菌递送系统中的强大效用。必要和充分剂LLO的分子机制仍然不清楚。具体而言，其pH依赖性活性及其分子调节剂，例如所需的独特单个半胱氨酸的还原，目前尚不清楚。目前拨款提案的目标是了解LLO活性及其调控机制的关键要素，可能取决于细胞类型和细胞分化和代谢状态，并利用这些知识在未来更好地设计胞质递送和靶向策略。需要解决的关键问题是：（i）LLO与含胆固醇的膜的相互作用、其膜结合结构域、pH敏感元件、结合的开/关速率、寡聚化速率以及LLO的哪些结构域如何调节这些过程，以及（iii）交付策略如何受LLO的各种监管因素的影响。由于内吞隔室中二硫键的还原在发病机制和药物递送系统的许多情况下以及在LLO活性的调节中是必不可少的，因此该提案的重要部分致力于设计和表征探针以监测内体中的还原，并研究调节还原过程的细胞因子。从拟议的研究中获得的结果和信息将是非常重要和关键的，不仅合理设计的LLO介导的大分子传递和有效的胞质传递的长期战略，而且任何药物传递策略利用可逆二硫键，以及阐明李斯特菌的入侵机制。

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.