Mechanisms for cell uptake of LDL/DNA complexes

细胞摄取 LDL/DNA 复合物的机制

基本信息

批准号：
8214277
负责人：
Natalia V. Guevara
金额：
$ 8.74万
依托单位：
UNIV/TEXAS BROWNSVILLE & SOUTHMOST COLL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-01-13 至 2014-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8214277
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 Genomics 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

项目摘要

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.

描述（由申请人提供）：我们的假设是低密度脂蛋白（LDL）是外源和内源性核酸的天然向量。这些由LDL和核酸组成的复合物可以通过受体介导的途径获得细胞进入，该途径涉及APO B100和APO E中包含的LDL B/E受体结合区，这是所有低密度脂蛋白颗粒的蛋白质成分。先前显示LDL结合核酸，并已在体外和体内成功地用于细胞转染实验。 LDL/核酸复合物（LDL/NA）的细胞进入机制尚不清楚。我们的特定目的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的参与，使其摄取摄入质粒和人类基因组DNA的LDL摄取。将确定LDL/DNA与内吞作用标记的共同定位。 LDL/DNA摄取中B/E受体和糖胺聚糖（GAGS）的贡献将使用各种单克隆抗体以及LDL受体不足和缺乏GAG缺乏的细胞系对LDL受体相互作用的选择性阻断进行表征。我们的具体目的2是研究LDL介导的细胞转染后LDL和DNA的细胞内分布，以解决内体逃生的假设。我们将确定DNA是单独解放还是与载脂蛋白或其碎片的复合物，并确定这些事件的时间。我们的具体目的3是确定赖氨酸/精氨酸富含apo e受体配体在内吞作用和核酸内细胞内转透中包含apo e受体配体类似物的作用。我们将确定除推定的B/E受体结合区域以外的APO B100的位点是否参与LDL/DNA的摄取。 APO B100的三个区域（0014-0096、0583-0614和3182-3215）已被鉴定为基于存在与APO E和FLAVIVIRIDAE蛋白质相似的r/K-富基序，为潜在的B/E受体结合位点。在我们的初步研究中，跨越这些Apo B100区域的合成肽显示与HELA细胞结合或进入HELA细胞。我们将确定这些肽是否与LDL/DNA竞争细胞进入，以及对这些APO B100这些区域的单克隆抗体是否影响LDL/DNA的摄取。该项目将促进我们对LDL作为核酸递送载体的自然功能的理解。公共卫生相关性：该项目的目的是确定人类低密度脂蛋白在DNA运输和传递能力的分子基础和机制。了解低密度脂蛋白在核酸转移中的机制和作用可能会为人类疾病的重要方面（包括病毒感染和癌症）提供新的见解。