In a study published in Science Advances, the research group led by Dr. OUYANG Bo from Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science of the Chinese Academy of Sciences and Dr. WANG Shuqing from Tianjin Medical University revealed a unique role of cholesterol in regulating PD-L1 surface level, which provides insights into the development of new drugs for anti-cancer treatment.
Cholesterol is one of the major components of cell membranes that regulates the structural integrity, thickness, fluidity and permeability of membranes. Moreover, recent studies have demonstrated that cholesterol participates in modulating the cell function and signaling through direct binding to many transmembrane proteins, and several cholesterol binding motifs have been identified.
Cholesterol levels are tightly regulated to maintain the cell functions in normal cells, while a high level of cholesterol is required in cancer cells to meet the needs of rapid proliferation and invasion. Programmed death ligand-1 (PD-L1) is a highly expressed protein on cancer cell membranes that binds to programmed cell death 1 (PD-1) receptor on the surface of immune T cells and keeps T cells from killing cancer cells. Protein sequence analysis showed that the transmembrane domain of PD-L1 contains two canonical CRAC cholesterol-binding motifs. Dr. OUYANG's group proposed that a direct link may exist between the high levels of cholesterol and high expression of PD-L1 in cancer cells.
To examine the possibility, researchers first added simvastatin (a cholesterol-lowering drug) and methyl-β-cyclodextrin (a cholesterol removal reagent) to reduce the cholesterol level in cancer cells and found that reduced cholesterol level lowered PD-L1 level on the cell membranes and enhanced PD-L1 degradation.
They then solved the structure of PD-L1 transmembrane and cytoplasmic domain (PD-L1-TC) using nuclear magnetic resonance (NMR) technology. NMR titrations and paramagnetic relaxation enhancement (PRE) experiments further identified that the two CRAC motifs existing in PD-L1-TC can directly bind to cholesterol and form a sandwich-like architecture to stabilize PD-L1 from the downstream degradation. Molecular dynamic simulations confirmed that the binding of two molecules of cholesterol to PD-L1-TC is thermodynamically stable. Mutations of the CRAC motifs disrupted the PD-L1–cholesterol interactions and accelerated the degradation of PD-L1.
The cholesterol dual binding mode observed in this study proposes a special molecular mechanism for regulating the stability of PD-L1 and leads to a potential therapeutic strategy for cancer treatment.