Functional Analysis And Mechanistic Study On ZRANB1 And SENP1 Promoting The Stability Of LDLR Protein

Cholesterol is an essential component of mammalian cell membrane contributing to the fluidity and phase transition of membrane bilayer.It is also the precursor of several biological molecules including steroid hormones and bile acids,and plays an important role in the metabolic regulation.Cholesterol is mainly transported as lipoprotein particles in the blood.Low-density lipoprotein(LDL)binds to LDL receptor(LDLR)on the cell surface and enters the cell through receptor-mediated endocytosis.Loss-of-function mutations in LDLR elevate circulating LDL levels,leading to cardiovascular diseases such as atherosclerosis.Therefore,elucidating the mechanism promoting LDLR stability lays an important foundation in the development of lipid?lowering drugs.LDLR is subjected to sterol-regulatory element binding protein(SREBP)-mediated regulation at the transcriptional level and pro-protein convertase subtilize kexin 9(PCSK9)-and inducible degrader of the LDL receptor(IDOL)-mediated regulation at the post-transcriptional level.As an E3 ligase,IDOL ubiquitinates K832 and C841 of the LDLR cytoplasmic tail and mediates LDLR degradation in lysosomes.However,whether deubiquitinating enzymes(DUBs)can antagonize IDOL-mediated LDLR ubiquitination is poorly understood.Previous work of our lab identified two LDLR-stablizing protein from 95 DUB families,namely ZRANB1(Zinc Finger RANBP2-Type Containing 1)and SENP1(Sentrin-specific protease 1).Overexpression of ZRANB1 or SENP1 increased LDLR level,whereas knockdown of ZRANB1 or SENP1 decreased LDLR level.Deletion of SENP1 using CRISPR-Cas9 technique lowered LDLR level and LDL uptake.Further,overexpression of SENP1 in mouse liver using adeno-associated virus resulted in elevated hepatic LDLR level accompanied by reduced plasma total cholesterol and increased hepatic total cholesterol.Notably,ZRANB1 and SENP1 failed to block IDOL-or PCSK9-induced LDLR degradation.Together,these results suggest that ZRANB1 and SENP1 regulate cholesterol metabolism by promoting LDLR stability,but the detailed molecular mechanisms remain to be characterized.