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传递系统和细胞内病原体的重要靶点。许多药物由于其大分子大小和亲水特性而不含膜；因此，需要他们的专业化，高效的细胞质输送策略比以往任何时候都大。单核增生李斯特菌进入内吞室，利用成孔蛋白李斯特菌溶素O （LLO）的特殊机制，突破内体膜逃逸到细胞质中。LLO已被用于药物递送系统，并已证明其作为一种内溶剂的强大功能，可以产生模拟李斯特菌入侵的非病毒/非细菌递送系统，将外源性大分子递送到细胞质中。必要和充分剂LLO的分子机制尚不清楚。具体而言，其ph依赖性活性及其分子调节因子，如所需的独特单半胱氨酸的减少，目前尚不清楚。目前的资助提案的目标是了解LLO活性的关键要素及其调控机制，可能依赖于细胞类型、细胞分化和代谢状态，并利用这些知识在未来更好地设计细胞质递送和靶向策略。需要解决的关键问题是：(i) LLO与含胆固醇膜的相互作用，其膜结合结构域，pH敏感元件，结合的开/关率，寡聚化率，以及LLO的哪些结构域如何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减少以激活LLO活性以及如何调节，以及（iii） LLO的各种调节因子如何影响递送策略。由于内吞室中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，就像在LLO活性的调节中一样，该提案的一个重要部分是致力于设计和表征探针，以监测内体的减少，并研究调节还原过程的细胞因子。该研究的结果和信息不仅对合理设计低分子量纤维素介导的大分子传递和有效的胞质传递的长期策略，而且对利用可逆二硫键的任何药物传递策略以及阐明李斯特菌的侵袭机制都是非常重要和关键的。

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.