Ubiquitin Proteasome System Inhibition Reduces LRP1 Protein Levels in HepG2 Cells

Abstract

Ubiquitin proteasome system (UPS) is a conserved proteolytic process involved in the degradation of damaged and misfolded proteins. Proteins undergoing this event must be conjugated to one or more monomers of ubiquitin, a process known as ubiquitination. UPS inhibition produces protein aggregation which causes cytotoxicity. Low‐density lipoprotein receptor‐related protein‐1 (LRP1) is a transmembrane receptor composed of two subunits, α (515 kDa) and β (85 kDa) located at extracellular and intracellular levels, respectively. This receptor is involved in the uptake of molecules via clathrin for either transcellular transport or lysosomal degradation. UPS inhibition by MG132 increases LRP1 half‐life in HepG2 cells as reported in the literature. Conversely, protein aggregation might have a role in LRP1 reduction.The purpose of this research was to demonstrate that MG132, a UPS inhibitor, induces cytotoxicity, protein aggregation and reduces LRP1 protein levels in HepG2 cells. These cells were treated for 24 hours with different concentrations of MG132 (0.03 – 3.0 μM) reducing cell viability in a concentration‐dependent manner determined by 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS assay). Whole cell lysates were extracted and separated on SDS‐PAGE. Immunoblotting was used to determine K48‐linked ubiquitinated proteins and LRP1. MG132 at 0.3 and 1 μM increased K48‐linked ubiquitinated proteins and reduced LRP1 protein levels in a concentration‐dependent manner.These results suggest that UPS inhibition promotes protein aggregation, which can be the cause of cytotoxicity. In addition, MG132 reduced LRP1 protein levels which might be a consequence of protein aggregate accumulation. Further experiments are required to elucidate the LRP1 reduction mechanism when UPS is inhibited.