RESUMO
Terminal deoxynucleotidyltransferase (TdT), which template-independently synthesizes DNA during V(D)J recombination in lymphoid cells, is ubiquitylated by a BPOZ-2/Cul3 complex, as the ubiquitin ligase, and then degraded by the 26 S proteasome. We show here that TdT is ubiquitylated by the Cul3-based ubiquitylation system in vitro. Because TdT could also be ubiquitylated in the absence of Cul/BPOZ-2, we determined that it could also be directly ubiquitylated by the E2 proteins UbcH5a/b/c and UbcH6, E3-independently. Furthermore, the ubiquitylated TdT inhibited its nucleotidyltransferase activity.
Assuntos
DNA Nucleotidilexotransferase/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Ubiquitinação/genética , Animais , Bovinos , Proteínas Culina/genética , Proteínas Culina/metabolismo , DNA Nucleotidilexotransferase/genética , Biblioteca Gênica , Células HeLa , Humanos , Fígado/citologia , Fígado/metabolismo , Plasmídeos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Timo/citologia , Timo/metabolismo , Transfecção , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
AIM: Bilirubin, a final degradation product of heme produced mainly in the spleen, is carried to the liver through its binding to albumin in the blood circulation. After its transport to hepatocytes, ligandin (glutathione S-transferase; GST) carries bilirubin to the endoplasmic reticulum (ER). uridine 5'-diphosphate-glucuronosyltransferase 1A1 (UGT1A1) glucuronidates bilirubin for solubilization in the ER. METHODS: By GST pull-down and co-immunoprecipitation assays, GSTA2, a member of the alpha-class of GST, was observed to directly bind to UGT1A1 through the region present inside the ER. RESULTS: GSTA2 was detected in the microsomal fraction together with the cytosolic fraction after hepatocyte fractionation. CONCLUSION: These results strongly suggest that bilirubin is directly delivered to UGT1A1 from ligandin for glucuronidation.
