Regulation Of Cholesterol Metabolism By The Ubiquitin Ligases RNF145 And AL1

Cholesterol plays an important role in mammalian cells.Cholesterol is the component of membranes,and is required for regulating the fluidity of membranes and maintaining the function of lipid rafts.Cholesterol is also the precursor of steroid hormones and bile acids.Cholesterol also functions in signal transduction in cells.Cholesterol is synthesized in cells by a multi-enzymatic cascade.The synthesis pathway is tightly regulated in cells.HMGCR(3-Hydroxy-3-Methylglutaryl-Co A Reductase)is the rate-limiting enzyme of cholesterol synthesis pathway which catalyzes the conversion of HMG-Co A to mevalonic acid.The stability of HMGCR is tightly regulated by cellular cholesterol levels.Elevated cholesterol level leads to rapid ubiquitination and proteasomal degradation of HMGCR,which is mediated by the ubiquitin ligases-gp78 and TRC8.However,experiments conducted by another group showed that knockdown of gp78 and TRC8 together in some cell lines did not affect the sterol-induced degradation of HMGCR,indicating the existence of other ubiquitin ligases in mediating HMGCR degradation.In this thesis,we identified a novel ubiquitin ligase called RNF145,mediated the sterol-induced degradation of HMGCR.Knockout of RNF145 and gp78 together in CHO cells largely blunted the sterol-induced degradation of HMGCR.RNF145 interacted with Insig proteins through its transmembrane domain.Point-mutations of RNF145 which lost the ubiquitin ligase activity or the binding capacity with Insig proteins failed to induce HMGCR degradation.LDLR is the receptor of low-density lipoprotein(LDL)and is important for plasma LDL level.Loss of function of LDLR leads to familial hypercholesterolemia.LDLR binds to LDL at the cell surface,after which the LDL/LDLR complex undergoes endocytosis pathway to enter the cells.LDL dissociates with LDLR in the acidic condition of endosomes,after which LDLR is transported back to the plasma membrane and LDL goes to lysosome for degradation.The mechanism of LDLR cycling in cells is not clear.In this thesis,a novel ubiquitin ligase called AL1 was identified.We observed that knockdown of AL1 in cells induced the expression of many SREBP-2 target genes including LDLR.Knockdown of AL1 also caused the accumulation of LDLR proteins in endosome.Moreover,knockdown of AL1 impaired the suppression of LDL on SREBP-2 pathway.Also we found that AL1 bound to LDLR and co-localized with LDLR in cells.Further experiments showed that AL1 was an ER-localized transmembrane protein with a C-terminal cytosolic HECT ubiquitin ligase domain.At last,we found that AL1 mediated the membrane contact between ER and some acid organelles,indicating a new function of ER.