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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Continuance Award (SC3)
Project #
1SC3GM099637-01
Application #
8214277
Study Section
Special Emphasis Panel (ZGM1-MBRS-9 (SC))
Program Officer
Krasnewich, Donna M
Project Start
2012-01-13
Project End
2014-11-30
Budget Start
2012-01-13
Budget End
2012-11-30
Support Year
1
Fiscal Year
2012
Total Cost
$87,384
Indirect Cost
$12,384
Name
University/Texas Brownsville & Southmost Coll
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
800187965
City
Brownsville
State
TX
Country
United States
Zip Code
78520