The long-term goal of this project is to characterize the structure and molecular mechanisms of lysophosphatidylcholine (LPC) transport catalyzed by the Na+-coupled LPC symporter 1 (NLS1). NLS1 is mainly expressed in the human blood-brain barrier (BBB) and the blood-retinal barrier (BRB), and can be targeted for the prevention and treatment of neurodegenerative and brain disorders. The current bottleneck for NLS1 studies is protein production. Here, we propose to work out the methods and develop protocols to obtain stable and functional NLS1 in large quantities, and to characterize NLS1 function. The fatty acid docosahexaenoic acid (DHA) is the major omega-3 fatty acid, enriched in brain and retina. This lipid plays important roles in the development and function of both neural tissues. NLS1 is responsible for DHA accumulation; however, NLS1 does not transport non-esterified DHA, but transports DHA-carrying LPC together with Na+ (cotransport). NLS1 has been also reported to suppress the transcytosis of BBB endothelial cells and function as a key regulator of BBB permeability, so a dual role in uptake of DHA and maintaining the BBB integrity has been proposed. These findings raise the possibility of using NLS1 for specific drug delivery into the brain or eyes, overcoming challenges in the treatment of brain tumors and other diseases, as well as for targeting LPC for the prevention and treatment of neurodegenerative diseases. To date, detailed biochemical/biophysical and structural biology studies that are essential to guide drug design and development are not available. In this R21 proposal, we aim to target the protein production barrier and establish the missing functional assays with purified NLS1 protein. We will: 1) develop and optimize the methodology to produce large amounts of purified recombinant human NLS1 that is stable and functional, and 2) establish assay methods to examine the function of purified NLS1. The production of NLS1 and functional assay methods will fill a critical gap for the characterization of NLS1 biophysically and structurally, and will impact future pharmacological developments.
The lysophosphatidylcholine transporter NLS1 is mainly expressed in brain and eyes, and can be targeted for the prevention and treatment of neurodegenerative and brain disorders. The current bottleneck for NLS1 studies is protein production, and this R21 exploratory/developmental proposal aims to target technical barriers for studies of omega-3 lipid transport by NLS1 through establishing methods to produce high-quality recombinant human NLS1 protein, and developing protocols to examine the function of purified NLS1. Success of the proposal will have significant impact by providing the material needed for detailed biophysical and structural studies that are essential to develop strategies for the prevention and treatment of a broad range of neurodegenerative diseases of the brain and eyes.
