Mechanisms for cell uptake of LDL/DNA complexes

描述(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。这些由低密度脂蛋白和核酸组成的复合体可能通过受体介导的途径进入细胞，该途径涉及所有低密度脂蛋白颗粒的蛋白质成分载脂蛋白B100和载脂蛋白E中包含的低密度脂蛋白B/E受体结合区。低密度脂蛋白以前被证明可以与核酸结合，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/核酸复合体(LDL/NA)进入细胞的机制尚不清楚。我们的具体目标1是确定低密度脂蛋白/DNA复合体细胞摄取的机制。我们将评估基于内吞作用的机制在摄取与质粒和人类基因组DNA复合的低密度脂蛋白中的作用。将确定低密度脂蛋白/DNA与内吞作用标志物的共同定位。B/E受体和糖胺聚糖(GAG)在低密度脂蛋白/DNA摄取中的作用将通过各种单抗以及低密度脂蛋白受体缺陷和低聚糖低聚糖细胞系选择性地阻断低密度脂蛋白受体相互作用来表征。我们的具体目标2是研究低密度脂蛋白介导的细胞转染后低密度脂蛋白和DNA在细胞内的分布，以解决内体逃逸的假说。我们将确定DNA是单独释放的，还是与载脂蛋白或其片段形成复合体的，并确定这些事件的时间。我们的具体目标3是确定含有载脂蛋白E受体配体类似物的赖氨酸/精氨酸富集区在核酸的内吞作用和细胞内转运中所起的作用。我们将确定载脂蛋白B100上的B/E受体结合区以外的其他位点是否参与了低密度脂蛋白/DNA的摄取。ApoB100的三个区域(0014-0096、0583-0614和3182-3215)已被确定为潜在的B/E受体结合位点，其存在的富含R/K的基序与ApoE和黄病毒科的蛋白相似。在我们的初步研究中，跨越载脂蛋白B100这些区域的合成肽被证明结合或进入HeLa细胞。我们将确定这些多肽是否与低密度脂蛋白/DNA竞争进入细胞，以及针对载脂蛋白B100这些区域的单抗是否影响低密度脂蛋白/DNA的摄取。这个项目将促进我们对低密度脂蛋白作为核酸递送载体的天然功能的理解。

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.