July 30, 2019 -- A new system using reporter proteins has been developed by researchers at Carnegie, Johns Hopkins University, and the Mayo Clinic that uses high-throughput genetic and small-molecule screening, as well as whole-organism imaging to find new drugs for the treatment of cardiovascular disease. These results which provide insights into heart disease were published in Nature Communications on July 31, 2019.
Cardiovascular disease is a leading cause of death in the world and is caused from plaques, or a build up of cholesterol, within blood vessels. Cholesterol and other lipids are shuttled around the body primarily by apolipoprotein B (ApoB). Understanding more about how ApoB functions will help scientists determine how to reduce plaque formation and lower the risk of cardiovascular disease.
Researchers developed the LipoGlo system which uses a protein tag (NanoLuc) to track atherogenic lipoproteins attached to ApoB (ApoB-LP). In this system, NanoLuc, a genetically engineered luciferase reporter, is fused with the endogenous ApoB gene such that each atherogenic lipoprotein (LP) is tagged with a light-emitting molecule (ApoB-LP). The luciferase reporter generates a quantitative chemiluminescent signal through processing of its substrate molecule, furimazine. LipoGlo allows scientists to monitor the movement of ApoB complexes in larval zebrafish. With this system, they were able to directly observe the concentration, size, and distribution of lipoproteins in vanishingly small samples of material so that they can eventually elucidate ways to fight the risks of heart disease.
Traditional methods of isolating LPs from plasma using assays for triglyceride and cholesterol content include: unable to detect lipoproteins outside of the bloodstream, limited information on the size or abundance of LPs, not conducive to high-throughput screening. The researchers state that it has been difficult to identify drugs that modulate ApoB-LP size and abundance because the current models used for high-throughput drug screening do not replicate the complex multi-organ physiology responsible for ApoB-LP homeostasis.
High-throughput screening: an automated testing of large numbers of chemical and/or biological compounds for a specific biological target. It is used to rapidly identify active compounds, antibodies, or genes that modulate a particular biomolecular pathway of interest that can be used as a starting point for drug design.
Using the LipoGlo system, researchers were able to confirm localization patterns corresponding primarily to lipoprotein-producing tissues (such as the liver, intestine, and yolk-syncytial layer) and the circulatory system in zebrafish. Moreover, larval zebrafish lipoprotein profiles were similarly affected as humans by changes in: genetic mutations (apoc2−/− and mtp−/−), pharmaceuticals (Lomitapide), and dietary manipulations (fasting and high-fat feeding). This suggests that they highly conserved lipoprotein processing pathways between these two species.
Additionally, researchers discovered a mysterious gene called pla2g12b, which has a huge impact on both the size and number of ApoB-containing lipoproteins. Therefore, they determined that pla2g12b is a potent regulator of lipoprotein size. This information can also be leveraged in the drug discovery process.
These proteins are “essential to fighting the global epidemic of cardiovascular disease,” said lead author James Thierer, a graduate student at Johns Hopkins University.
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