Mechanisms for cell uptake of LDL/DNA complexes

描述（由申请人提供）：我们的假设是低密度脂蛋白（LDL）是外源和内源核酸的天然载体。这些由LDL和核酸组成的复合物可能通过受体介导的途径进入细胞，该途径涉及载脂蛋白B100和载脂蛋白E中包含的LDL B/E受体结合区，载脂蛋白B100和载脂蛋白E是所有低密度脂蛋白颗粒的蛋白质成分。LDL先前已被证明与核酸结合，并已成功地用于体外和体内的细胞转染实验。低密度脂蛋白/核酸复合物（LDL/NA）进入细胞的机制尚不清楚。我们的特异性目标1是确定LDL/DNA复合物细胞摄取的机制。我们将评估以内吞作用为基础的低密度脂蛋白与质粒和人类基因组DNA复合物摄取机制的参与。LDL/DNA与内吞作用标记物的共定位将被确定。B/E受体和糖氨基聚糖（GAGs）在LDL/DNA摄取中的作用将通过各种单克隆抗体以及LDL受体缺陷和GAGs缺陷细胞系选择性阻断LDL-受体相互作用来表征。我们的具体目标2是研究LDL介导的细胞转染后LDL和DNA的细胞内分布，以解决内体逃逸的假设。我们将确定DNA是单独释放还是作为载脂蛋白或其片段的复合物释放，并确定这些事件的时间。我们的特异性目标3是确定含有载脂蛋白E受体配体类似物的赖氨酸/精氨酸丰富区域在核酸内吞作用和细胞内转运中的作用。我们将确定载脂蛋白B100上除假定的B/E受体结合区以外的位点是否参与LDL/DNA的摄取。载脂蛋白B100的三个区域（0014-0096,0583- 0614和3182-3215）已经被确定为潜在的B/E受体结合位点，基于存在与载脂蛋白E和黄病毒科蛋白相似的富含R/ k的基序。在我们的初步研究中，跨越Apo B100这些区域的合成肽被证明可以结合或进入HeLa细胞。我们将确定这些肽是否与LDL/DNA竞争进入细胞，以及载脂蛋白B100这些区域的单克隆抗体是否影响LDL/DNA的摄取。该项目将促进我们对低密度脂蛋白作为核酸传递载体的自然功能的理解。

DESCRIPTION (provided by applicant): Our hypothesis is that low-density lipoproteins (LDL) are natural vectors for foreign and endogenous nucleic acids. These complexes, comprised of LDL and nucleic acids, may gain cell entry through a receptor-mediated pathway that involves LDL B/E receptor binding regions contained in both apo B100 and apo E, protein constituents of all low-density lipoprotein particles. LDL were previously shown to bind nucleic acids and have been successfully used in cell transfection experiments both in vitro and in vivo. The mechanism for cell entry of LDL/Nucleic Acid complexes (LDL/NA) is not known. Our Specific Aim 1 is to determine the mechanism for LDL/DNA complex cell uptake. We will assess the involvement of endocytosis- based mechanisms in the uptake of LDL complexed to plasmid and human genomic DNA. Co- localization of LDL/DNA with the endocytosis markers will be determined. Contribution of the B/E receptor and glycosamino glycans (GAGs) in the LDL/DNA uptake will be characterized using selective blocking of LDL-receptor interaction by various monoclonal antibodies as well as LDL-receptor deficient and GAG-deficient cell lines. Our Specific Aim 2 is to study the intracellular distribution of both LDL and DNA after LDL-mediated cell transfection to address the hypothesis of endosomal escape. We will establish whether DNA is liberated separately or as a complex with the apolipoproteins or fragments thereof and determine the time of these events. Our Specific Aim 3 is to determine the role that Lysine/Arginine-rich regions which contain analogues of the apo E receptor ligand play in endocytosis and intracellular transit of nucleic acids. We will determine if sites on apo B100 other than putative B/E receptor-binding region are involved in the uptake of LDL/DNA. Three regions of apo B100 (0014-0096, 0583- 0614, and 3182-3215) have been identified as potential B/E receptor binding sites based on presence R/K-rich motifs similar to those in to apo E and in Flaviviridae proteins. Synthetic peptides spanning these regions of Apo B100 were shown to bind or enter HeLa cells in our preliminary studies. We will determine if these peptides compete with LDL/DNA for cell entry, and if monoclonal antibodies to these regions of apo B100 affect the uptake of LDL/DNA. This project will advance our understanding of the natural function of LDL as nucleic acid delivery vector. PUBLIC HEALTH RELEVANCE: The goal of this project is to determine the molecular basis and mechanism for human low-density lipoproteins' capacity in transport and delivery of DNA. Understanding the mechanism and role of low-density lipoproteins in the transfer of nucleic acids may provide new insights into important aspects of human disease, including viral infections and cancer.