Effect of Apolipoprotein Structural Adaptability

目前的MERIT奖延期将继续关注以下方面的卓越结构适应性： 载脂蛋白这类血浆蛋白执行基本功能，包括受体配体， 脂质代谢酶的调节剂和血浆脂蛋白的稳定。介绍新的方法 将通过改变rHDL脂质，引入 疏水性生物活性剂和束缚外来蛋白质。这些稳定的，自我的， 将研究组装的水溶性纳米尺寸颗粒。作为他们生理上的必然结果， 在脂质转运和胆固醇流出中的作用，使用“设计师”rHDL进行“替代疗法”应用 和生物活性剂输送是一个快速发展的研究前沿，我们的努力旨在验证 与蛋白质结合到rHDL及其递送到细胞相关的概念。在目标1中，假设 巨噬细胞模型中的戈谢氏病表型将通过与巨噬细胞一起孵育来校正。 富含磷脂酰丝氨酸的葡糖脑苷脂酶连接的rHDL。将通过流量评价酶摄取 流式细胞术/共聚焦荧光显微术。细胞裂解物中的葡糖脑苷脂酶活性，以及 将测量细胞葡糖脑苷脂含量的变化。在目标2中，我们假设细胞 通过apoE依赖性摄取的尼曼-匹克C2（NPC 2）蛋白质栓系的rHDL的递送 低密度脂蛋白受体家族将导致NPC 2递送至溶酶体，拯救尼曼-匹克 GM 18455成纤维细胞中的疾病表型。将NPC 2连接的含apoE的rHDL与 培养的GM 18455成纤维细胞和apoE促进GM 18455的结合、摄取和溶酶体递送的能力。 将确定NPC 2沿着对溶酶体胆固醇含量/细胞器形态的影响。在Aim中 3、研究激动剂诱导的巨噬细胞胆固醇流出的激活。我们假设 可以将一种或多种肝X受体/类维生素A X受体（LXR / RXR）激动剂递送至巨噬细胞， 与rHDL复合。进一步假设，核激素受体激活将诱导apoE /apoR。 ABCA 1表达，导致胆固醇流出能力增加。产生水溶性的能力， 能够将受体介导的蛋白质/生物活性剂递送至细胞的靶向rHDL代表了一种新的 疾病治疗策略。rHDL的脂质和蛋白质组分的固有多功能性允许 它们与附加组分的组装，产生可用作递送载体的稳定复合物

The present MERIT Award extension will maintain its focus on the remarkable structural adaptability of apolipoproteins. This class of plasma protein performs essential functions, including receptor ligands, modulators of lipid metabolic enzymes and stabilization of plasma lipoproteins. Methods to introduce novel functional properties into reconstituted HDL (rHDL) will be exploited by altering rHDL lipids, introducing hydrophobic bioactive agents and tethering extraneous proteins. The therapeutic utility of these stable, self- assembled, water-soluble nanoscale size particles will be investigated. As a corollary to their physiological role in lipid transport and cholesterol efflux, the use of "designer" rHDL for "replacement therapy" applications and bioactive agent delivery is a rapidly advancing research front Our efforts are designed to validate concepts related to protein binding to rHDL and their delivery to cells. In aim 1, it is hypothesized that the Gaucher's disease phenotype in a macrophage cell model will be corrected by incubation with a phosphatidylserine-enriched, glucocerebrosidase-tethered rHDL. Enzyme uptake will be evaluated by flow cytometry / confocal fluorescence microscopy. Glucocerebrosidase activity in cell lysates, as well as changes in the cellular glucocerebroside content, will be measured. In Aim 2, we hypothesize that cellular delivery of Niemann-Pick C2 (NPC2) protein-tethered rHDL via apoE dependent uptake by members of the low-density lipoprotein receptor family will result in NPC2 delivery to lysosomes, rescuing the Niemann-Pick disease phenotype in GM18455 fibroblasts. NPC2-tethered apoE-containing rHDL will be incubated with cultured GM18455 fibroblasts and the ability of apoE to promote binding, uptake and lysosomal delivery of NPC2 will be determined, along with effects on lysosomal cholesterol content / organelle morphology. In Aim 3, agonist-induced activation of macrophage cholesterol efflux will be investigated. We hypothesize that one or more liver X receptor / retinoid X receptor (LXR / RXR) agonist can be delivered to macrophages in complex with rHDL. It is further hypothesized that nuclear hormone receptor activation will induce apoE / ABCA1 expression, resulting in increased cholesterol efflux capacity. The ability to generate water soluble, targeted rHDL capable of receptor-mediated protein / bioactive agent delivery to cells represents a novel strategy for disease treatment. The intrinsic versatility of the lipid and protein components of rHDL allows for their assembly with additional components, yielding stable complexes that can be used as delivery vehicles