Regulation Of Lipid Biosynthesis Through Proteasome Degradation:Mechanism And Application

3-Hydroxy-3-methylglutaryl coenzyme reductase(HMG-Co A Reductase or HMGCR)is an eight-transmembrane protein in the endoplasmic reticulum(ER)and is the rate-limiting enzyme that catalyzes the conversion of cholesterol into methylhydroxyglutarate in the biosynthesis pathway.Statins are classical drug to treat dyslipidemia.It includes the most famous atorvastatin(Liptor?;Pfizer)have been used as HMGCR inhibitors for decades in clinic.Although statins inhibit cholesterol biosynthesis by inhibiting HMGCR,compensatory expression of HMGCR is often encountered in cultured cells,as well as in the liver of some animal species,including humans.Although it is not surprising,because lower cholesterol and other product armour hydroxyl pentanoic acid can reduce the enzyme in the form of multivalent normal feedback inhibition,it did limit the maximum effectiveness of the drug,therefore,It’s urgent to develop new inhibition and degradation of HMGCR.PROTAC are currently receiving a lot of attention.Of all the four E3 ubiquitin ligases that PROTAC can recruit to target proteins,cereblon(CRBN)is of particular interest because it binds directly to pomalidomide、thalidomide and lenalidomide.And it mediates the antiproliferative activity of these immunomodulatory drugs(IMi Ds).PROTAC compounds based on thalidomide and its analogues have achieved remarkable success in the field of cancer research,inducing rapid degradation of multiple oncogenic target proteins.These encouraging results have stimulated our interest in investigating whether crbn-based PROTAC can be effectively used to degrade HMGCR proteins and thus replace statins in inhibiting cholesterol synthesis.In this study,we designed and synthesized a series of PROTAC molecules,where atorvastatin and pomalidomide were linked by chemical reaction,and polyethylene glycol(PEG)with different lengths between them was used to connect.We found that compound P22 A were most effective at degrading endogenous HMGCR proteins in hamster ovary(CHO)cells lacking Insig1 and Insig2(called SRD15 cells).P22 A induces HMGCR ubiquitination in a CRBN-dependent manner.Degradation of HMGCR mediated by P22 A requires the formation of HMGCR/PROTAC/CRBN complex,which reduces the accumulation of cholesterol caused by atorvastatin in huh7 cells and reduces the synthesis of cholesterol.Insig-2 is an ER(endoplasmic reticulum)membrane protein that negatively controls lipid biosynthesis through blocking SREBP processing and promoting HMGCR degradation.It is long-lived in cultured fibroblasts but undergoes rapid degradation in mouse liver in an E3 gp78-dependent manner.Little is known about the mechanisms regulating Insig-2 stability.Here we found that the protein level of Insig-2 was dramatically increased in the tissues including liver,but unaltered in the muscle of whole-body gp78-deficient mice.In cultured hepatocytes and undifferentiated C2C12 myoblasts,Insig-2 was ubiquitylated on Cys215 by gp78 and degraded rapidly.However,the C215 residue became oxidized by elevated reactive oxygen species(ROS)level during C2C12 myoblasts differentiating into myotubes,preventing Insig-2 from ubiquitylation and degradation.The stabilized Insig-2 downregulated lipogenesis through inhibiting the SREBP pathway,helping to shift the carbon flux to ATP generation and protecting myotubes from lipid over-accumulation.Evolutionary analysis showed that the YECK(in which C represents Cys215 in human Insig-2)tetrapeptide sequence in Insig-2 is highly conserved in terrestrial vertebrates but not in aquatic amphibians and fishes,suggesting it may have been shaped by divergent selection.Further,Insig-2 proteins containing the YECK sequence from numerous species across the vertebrate phylogeny can all be stabilized by ROS.Together,this study suggests that competitive oxidation-ubiquitylation on Cys215 of Insig-2 senses ROS and ameliorates lipotoxicity from excess cholesterol in muscle cells.This mechanism might play an important role during vertebrate transition from water to land in response to high oxygen in the air.