Low‐Density Lipoprotein Receptor‐Related Protein 1 (LRP1) Is Degraded Primarily by The Lysosomal Degradation System in Hepatocellular Carcinoma (HepG2) Cells

LRP1 is a transmembrane protein responsible for binding and internalizing at least 50 different ligands, one of which is amyloid beta (Aβ). LRP1 has a role in clearance of brain and systemic Aβ. Understanding LRP1's role in Aβ degradation could have important implications for understanding Alzheimer's Disease (AD) pathogenesis. Since LRP1 in the liver is primarily responsible for systemic clearance of Aβ, its degradation could have important implications for AD pathogenesis. Normal degradation of proteins typically occurs through the ubiquitin proteasome system (UPS) and/or the lysosomal degradation system. To study LRP1 degradation through either of these two processes, one system was inhibited, then subsequent LRP1 output was analyzed. By inhibiting one degradation system at a time, we can analyze the protein output to quantify how much protein is degraded by each system. Bafilomycin A1 was used to inhibit the lysosomal degradation system, and MG132 was used to inhibit UPS. Finally, cycloheximide, a protein synthesis inhibitor, was used to determine LRP1 half‐life and to determine whether protein output is significantly affected by protein synthesis during inhibition of the protein degradation system (cycloheximide chase analysis). HepG2 cells were used to study LRP1 degradation in these experiments. The cells were treated with Bafilomycin A1, MG132, or dimethyl sulfoxide (DMSO; control group) and lysed at time points 0, 8, 16, and 24 hours to analyze LRP1 protein levels over time. Experiments with Bafilomycin A1 showed increasing levels of LRP1 over time, whereas experiments with MG132 showed unchanged, then decreasing levels of LRP1 over time. Cycloheximide was tested to determine the optimal concentration for HepG2 cells, which was found to be 30 μM. Then, experiments were performed with either cycloheximide and DMSO (as control), cycloheximide and Bafilomycin A1, or cycloheximide and MG132 at time points 0, 8, 16, and 24 hours to determine the half‐life of LRP1. Our results showed an extension of LRP1 half‐life by inhibition of lysosomal degradation system when using Bafilomycin A1, but half‐life was not extended when UPS was inhibited by MG132. Due to its significant increase in half‐life with Bafilomycin A1, but not with MG132, it's concluded that LRP1's degradation is primarily produced through the lysosomal degradation system. Support or Funding Information MCPHS University This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .