CRL4B^AhR-mediated Ubiquitination Of PPARγ And Identification Of Modification Sites

BackgroundIn China,obesity and type 2 diabetes are becoming the most common diseases that seriously affect human,health and quality of life.High blood glucose with insulin resistance is the main cause of blood pressure and hyperlipidemia.Adipocyte differentiation is the physiological basis of normal fat storage.Abnormal proliferation and differentiation of adipocyte can cause excessive accumulation in adipose tissue,imbalance in energy metabolism,and a series of diseases related to obesity.Peroxisome proliferator-activated receptors(PPARs)belong to nuclear receptor superfamily,which includes PPARα,PPARβ,and PPARy.They are highly expressed in adipocytes and play important roles in adipose differentiation,energy balance,and lipid biosynthesis.Among them,PPARγ is a key regulator of adipocyte differentiation.There are two isoforms of PPARy:PPARy 1 and PPARγ2.Compared to PPARγ1,PPARγ2 has additional 28 amino acids in mice and additional 30 amino acids in human.The expression and activity of PPARγ are regulated by post-translational modifications.These post-translational modifications include phosphorylation,ubiquitination,acetylation,and glycosylation.CUL4B plays an important role in tumorigenesis and development.CUL4B has many cellular roles,such as:participating in cell cycle regulation,maintaining genome stability,participating in immune regulation,and regulating epigenetic modification.In addition,CUL4B functions as a scaffold protein in ubiquitin ligase E3.Its N-terminus binds DDB1,and C-terminus binds ROC1 to form the core structure of CUL4B-RING ubiquitin ligase(CRL4B).DDB1 specifically recruits substrates receptors,named DCAFs(DDB1 and CUL4-associated factors)which directly interact with substrates.In recent years,we found that PPARy is ubiquitinated by CRL4B,which affects adipocyte differentiation and body sensitivity to insulin.However,the detailed ubiquitination mechanism of PPARy is unclear,such as the ubiquitinated lysine site of PPARy and the relationship between PPARγ ubiquitination and acetylation.ObjectiveUbiquitination sites will help us to understand the ubiquitination degradation mechanism of PPARγ and provide a theoretical basis for the treatment of obesity-related diseases.We investigate the lysine sites of PPARγ modified by CRL4B ubiquitination,clarify the relationship between PPARy ubiquitination and acetylation,and illustrate the effect of AhR localization on the ubiquitination level of PPARy.Research methodsUsing the proximity ligation assay to detect the interactions between PPARγ and AhR in mouse adipose tissue;using site-directed mutagenesis and ubiquitination assays to illustrate the ubiquitination modification mechanism of PPARγ facilitated by AhR;using protein mass spectrometry analysis and protein stability assay to identify the ubiquitination modification sites on PPARγ.Results1.AhR negatively regulates fat differentiation.In order to study the effect of AhR on fat differentiation,it was found that the level of PPARγ protein in mouse adipose tissue increased when cu14b was deleted;the interaction between PPARy and AhR was also significantly enhanced when cul4b was deleted using the ortho-linking technique;During the process of fat differentiation,the level of AhR protein generally showed a downward trend.2.AhR is involved in the ubiquitination modification of PPARγ.To study the effect of AhR on PPARy ubiquitination modification,it was found that CRL4BAhR-mediated PPARγ ubiquitination modification occurred in the nucleus,and AhR agonist 3MC significantly enhanced PPARγ ubiquitination level.Discovery of point mutation(AhR F287A)and nuclear inhibitor(MDL-28170)reduces PPARy ubiquitination3.The two ubiquitination modification sites of PPARγ are K268 and K293,and there is site competition between the acetylation and ubiquitination of these two sites.In order to study the two ubiquitination modification sites of PPARγ,protein mass spectrometry was used to screen potential ubiquitination sites.According to the results of mass spectrometry,different types of mutant plasmids were constructed,and it was found that the two lysines on PPARγ were modified by ubiquitination,namely K268 and K293.These two sites are also PPARγ acetylation sites.By adding the deacetylase inhibitor NAM or knocking down AhR,it is found that there is site competition between acetylation and ubiquitination.ConclusionIn the CRL4B ligase complex,AhR as a substrate receptor protein recruits PPARγand participates in mediating PPARγ ubiquitination.Both ubiquitination modification and protein degradation occur in the nucleus.Besides,the ubiquitination of PPARγ is affected by its acetylation.