Polyclonal Antibody Preparation Of Adp And Substrate Screening Of CRL4^Adp E3 Ligase

Adp is an evolutionarily conserved gene,which appears to inhibit fat formation in a dosage-sensitive manner.The anti-obesity function of Adp is conserved from flies to mammals.Adp heterozygous flies and Adp heterozygous mutant mice are obese and insulin resistant,and the fat biology of Adp heterozygous mutant mice intermediate between wild-type and Adp nulls.Adp is expressed in many tissues,and a transient transgenic increase in Adp activity in fat tissues reduces fat accumulation,but it is not clear how this gene acts in other tissues.Distupting the interaction between Adp and DDB1 leads to a loss of adipogenic suppression by Adp,increased triglyceride accumulation and adipogenic gene expression.The nuclear fraction of Adp blocks adipogenesis while the cytoplasm fraction not and in some conditions even stimulated adipogenesis,like a dominant negative Adp.The differentiation of preadipocytes into adipocyte is regulated by an elaborate network of transcription factors that coordinate expression of hundreds of proteins responsible for establishing the mature fat-cell phenotype.At the center of this network are the two principal adipogenic factors,C/EBPa and PPAR?,which oversee the entire terminal differentiation process.The first wave is initiated by adipogenic stimuli that activate several early adipogenic factors including C/EBP?,C/EBP?,KLFs,CREB,Krox20,SREBP-1c.These transcription factors in turn induce expression of the second wave of transcription factors,of which C/EBPa and PPARy are the most important,and these key adipogensis factors then induce expression of the gene program that leads to the mature adipocyte phenotype.First of all,we expressed recombinant Adp using prokaryotic expression system and prepared rabbit polyclonal antibodies of Adp.After that,the molecular mechanism of Adp against obesity was revealed by 3T3-L1 cell culture model of adipogenesis.Adp may function as a substrate receptor to recruit transcription factor or coactivitor for ubiquitination by the CRL4 ligases.Screening the transcription factors associated with adipocyte differentiation,we did not find the ubiquitinated substrate for CRL4Adp E3 ligase.On the one hand,RT-qPCR experiments showed that Adp exerts its anti-obesity function by influencing the late stages of adipocyte differentiation.On the other hand,the expression of late transcription factors was regulated by early transcription factors and its coactivitors.Since C/EBP? is the most important transcription factor in adipocyte differentiation,it is likely that its coactivator is regulated by ubiquitination and thus affects the expression of late transcription factors.Thus we chose C/EBP? pull down assays to search the substrate of CRL4Adp E3 ligase.Thro?gh the Candidate screening strategy and protein degradation experiments,it was finally found that Protein C was degraded by the CRLs ligase ubiquitination system.In a word,those results provide a potential of Adp in therapy for metabolic disease.