Investigate The Mechanism Of PPARγ Proteasomal Degradation Through CRL4B^AhR-mediated Ubiquitination

BackgroundIn the process of protein ubiquitination,ubiquitin activating enzyme E1,ubiquitin conjugating enzyme E2 and the ubiquitin ligase E3 work together to transfer ubiquitin molecules to a specific substrate.The specificity of this process is mainly determined by the ubiquitin ligase E3.Cullin-RING E3 ligases(CRLs)represent the biggest E3 ligase family.CUL4B,a type of cullin protein,acts as a scaffold to assemble Cullin-RING E3 ligase.CUL4B associates with DNA damage binding protein 1(DDB1)on its N-terminus and RING finger proteins ROC1 on its C-terminus to form the CUL4B-RING E3 ligase complex(CRL4B).CRL4B mainly mediates ubiquitination of substrate proteins and plays an essential role in epigenetic process.In the process of substrate ubiquitination,CRL4B relies on the CUL4-DDB1 associated factors(DCAFs)for specific substrate recognition.DCAF is a type of protein containing WD40 repeats.Recent studies showed that there are several DCAFs interacting with DDB1,such as WDTC1,COP1,CDT2,AhR,and so on.These DCAFs have been found to target specific substrate proteins for ubiquitination.Peroxisome proliferator-activated receptor γ(PPARγ)is a ligand-dependent nuclear receptor and is widely studied.There are two isoforms expressed differently across tissue.PPARyl is expressed in all tissues except muscle,whereas PPARγ2 is highly expressed in adipose tissue.In this thesis,the isoform of PPARγ that we study is PPARγ2.PPARγ has a variety of biological functions.It has been demonstrated to regulate essential genes in cell differentiation and various molecular process,especially lipid and glucose homeostasis.PPARγ has been shown to be associated with type 2 diabetes and hypertension.The activation of PPARγ can increase the sensitivity to insulin.In addition,PPARγ can also mediate NFκB/p65 degradation as an E3 ubiquitin ligase.Meanwhile,the physiological function of PPARγ is affected by post-translational modifications.In 2014,it was found that PPARy is regulated by MKRN1 E3 ubiquitin ligase,and K184 and K185 are two lysine sites where PPARy is ubiquitinated by MKRN1.In 2016,it was found that FBX09 is an E3 ubiquitin ligase of PPARγ,which regulates PPARγ activity and protein stability.In 2017,Assistant Professor Peishan Li found that PPARy can be ubiquitinated and degraded by CRL4B E3 ubiquitin ligase,but the detailed mechanism of ubiquitination remains unclear.Aryl hydrocarbon receptor(AhR),a member of the family of basic helix-loop-helix transcription factors,is a ligand activated transcription factor.AhR is commonly reported as a receptor for many polycyclic aromatic hydrocarbons such as TCDD in environmental toxins.AhR and ARNT form heterodimers,then heterodimers enter the nucleus and bind to exogenous chemical regulatory elements and initiate downstream cytochrome P450 family gene expression.AhR also plays a role in the development of tumor.In 2017,AhR was found as a potential tumor suppressor in pituitary adenomas.In addition,the ligand activated AhR can also be used as a DCAF element in the CRL4BAhR complex to specifically recognize estrogen receptor a and mediating its degradation process.ObjectiveThis study investigates the molecular mechanism of PPARγ ubiquitination mediated by the CRL4B,clarifies the corresponding DCAF that recognizes the substrate PPARγ,investigates the interaction between DCAF and PPARy,and investigates the lysine site where the substrate PPARγ is ubiquitinated by the CRL4B.Methods and results1 We examined the expression level of COP1,CDT2,WDTC1,and AhR across mouse tissues,we also used co-immunoprecipitation experiments to screen DCAFs that bind PPARγ in 3T3-L1 cells.Half-life experiments showed that AhR affects the stability of PPARγ protein.The addition of AhR can shorten the half-life of PPARγ.2 We found that AhR participates in the ubiquitination modification of PPARγ by CRL4B E3 ubiquitin ligase.Co-immunoprecipitation experiments further demonstrated that AhR is an integral part of the CRL4B E3 ligase complex and interacts with PPARγ in 3T3-L1 cells.Intracellular and extracellular ubiquitination experiments show that AhR can promote PPARγ ubiquitination level.Ubiquitination assays in HEK293T cells demonstrated that PPARγ is mainly degraded by the ubiquitin-proteasome pathway.3 The co-immunoprecipitation in HEK293T cells revealed that the PPARγ and AhR interaction region was located within 1-232 amino acids region of PPARy.4 The ubiquitination site of PPARγ was located in the region of 233-505 amino acids and was located near the K347 site of PPARγ by ubiquitination assay in HEK293T cells.OutlookThis study further explored the molecular mechanism of PPARγ ubiquitination by the CRL4B,which may provide new ideas for inhibiting the ubiquitination and degradation of PPARγ,increasing or stabilizing PPARγ protein levels,and enhancing PPARγ function.In this study,we also clarified the DCAF for PPARγ,which may serve as a new potential drug target for the specific treatment of type 2 diabetes and hypertension.