Mechanisms for cell uptake of LDL/DNA complexes

细胞摄取 LDL/DNA 复合物的机制

• 批准号: 8586320
• 负责人: Natalia V. Guevara
• 金额: $ 0.36万
• 依托单位: UNIV/TEXAS BROWNSVILLE & SOUTHMOST COLL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-13 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8586320
• 关键词: Address; Affect; Apolipoprotein E; Apolipoproteins; Binding; Binding Sites; Capsid Proteins; Cell Line; Cell Nucleus; Cells; Chronic; Complex; Cytoplasm; DNA; DNA delivery; Diagnostic; E protein; Endocytosis; Event; Flaviviridae; Fluorescence; Fluorescence Microscopy; Genomic DNA; Goals; Hela Cells; Human; In Vitro; Infection; LDL-Receptor Related Protein 1; Label; Ligands; Lipoprotein Binding; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Malignant Neoplasms; Mediating; Molecular; Monitor; Monoclonal Antibodies; N-terminal; Neoplasm Metastasis; Nuclear Translocation; Nucleic Acid Binding; Nucleic Acids; Outcome; Participant; Pathway interactions; Peptides; Plasma; Plasmids; Play; Polysaccharides; Process; Protein Fragment; Proteins; Rattus; Reporting; Resolution; Role; Site; Testing; Therapeutic; Time; Transfection; Viral Cancer; Virus Diseases; analog; arginyllysine; base; env Gene Products; human disease; in vivo; insight; leucylarginine; particle; plasmid DNA; receptor; receptor binding; research study; residence; synthetic peptide; uptake; vector

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.

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以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（所有低密度脂蛋白颗粒的蛋白质成分）中包含的 LDL B/E 受体结合区域。 LDL 先前已被证明可以结合核酸，并已成功用于体外和体内的细胞转染实验。 LDL/核酸复合物 (LDL/NA) 进入细胞的机制尚不清楚。我们的具体目标 1 是确定 LDL/DNA 复合物细胞摄取的机制。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 LDL 过程中的参与情况。将确定 LDL/DNA 与内吞作用标记的共定位。 B/E 受体和糖胺聚糖 (GAG) 在 LDL/DNA 摄取中的贡献将通过各种单克隆抗体以及 LDL 受体缺陷和 GAG 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与载脂蛋白或其片段的复合物释放，并确定这些事件的时间。 我们的具体目标 3 是确定含有 apo E 受体配体类似物的富含赖氨酸/精氨酸的区域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 apo B100 上除了推定的 B/E 受体结合区之外的位点是否参与 LDL/DNA 的摄取。基于与 apo E 和黄病毒科蛋白相似的富含 R/K 的基序，apo B100 的三个区域（0014-0096、0583-0614 和 3182-3215）已被鉴定为潜在的 B/E 受体结合位点。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 apo B100 这些区域的单克隆抗体是否影响 LDL/DNA 的摄取。该项目将增进我们对 LDL 作为核酸递送载体的天然功能的理解。