Characterization of the structural determinants of the ubiquitin-dependent proteasomal degradation of human hepatic tryptophan 2,3-dioxygenase.
Characterization of the structural determinants of the ubiquitin-dependent proteasomal degradation of human hepatic tryptophan 2,3-dioxygenase.
复制标题
DOI：
10.1042/bcj20210213
复制
发表时间：
2021-05-28
期刊：
影响因子：
--
通讯作者：
Correia MA
中科院分区：
文献类型：
--
作者：
Liu Y;Kim SM;Wang Y;Karkashon S;Lewis-Ballester A;Yeh SR;Correia MA
人肝色氨酸2，3-双加氧酶（hTDO）是一种同源四聚体血红素蛋白。它是降解最快的肝蛋白之一，半衰期（t1/2）约为2.3 h，而总肝蛋白的平均t1/2约为2-3天。hTDO寿命不长的分子机制仍然难以捉摸。以前，我们发现hTDO可以识别和泛素化的两个E3泛素（Ub）连接酶，gp 78/AMFR和CHIP，随后通过Ub依赖的蛋白酶体降解途径降解。此外，我们确定了15个泛素化K位点，并证明色氨酸结合到一个外位点阻碍其蛋白水解降解。在这里，我们进一步建立了自噬-溶酶体降解作为细胞hTDO降解的替代备用途径。此外，与蛋白激酶A和C，我们确定了13个磷酸化的丝氨酸/苏氨酸（pS/pT）的网站。在hTDO表面上定位这些pS/pT位点揭示了它们与酸性Asp/Glu（D/E）残基的接近性，从而在整个蛋白质表面上的泛素化K位点附近产生带负电荷的DEpSpT簇。通过gp 78的带正电荷的补丁的定点诱变，先前记录与其他靶蛋白中的DEpSpT簇相互作用，我们揭示了DEpSpT簇在gp 78和可能的其他E3 Ub连接酶对hTDO的分子识别中的可能作用。此外，环己酰亚胺追逐分析揭示了关键的结构相关性的无序的N-和C-末端不仅在Ub-连接酶的识别，而且在蛋白酶体的参与。一起，表面DEpSpT簇和N-和C-末端构成hTDO生理周转的内在二分降解决定子。
Human hepatic tryptophan 2,3-dioxygenase (hTDO) is a homotetrameric hemoprotein. It is one of the most rapidly degraded liver proteins with a half-life (t1/2) of ~2.3 h, relative to an average t1/2 of ~2–3 days for total liver protein. The molecular mechanism underlying the poor longevity of hTDO remains elusive. Previously, we showed that hTDO could be recognized and ubiquitinated by two E3 ubiquitin (Ub) ligases, gp78/AMFR and CHIP, and subsequently degraded via Ub-dependent proteasomal degradation pathway. Additionally, we identified 15 ubiquitination K-sites and demonstrated that Trp-binding to an exosite impeded its proteolytic degradation. Here, we further established autophagic-lysosomal degradation as an alternative back-up pathway for cellular hTDO degradation. In addition, with protein kinases A and C, we identified 13 phosphorylated Ser/Thr (pS/pT) sites. Mapping these pS/pT sites on the hTDO surface revealed their propinquity to acidic Asp/Glu (D/E) residues engendering negatively charged DEpSpT clusters vicinal to the ubiquitination K-sites over the entire protein surface. Through site-directed mutagenesis of positively charged patches of gp78, previously documented to interact with the DEpSpT clusters in other target proteins, we uncovered the likely role of the DEpSpT clusters in the molecular recognition of hTDO by gp78 and plausibly other E3 Ub-ligases. Furthermore, cycloheximide-chase analyses revealed the critical structural relevance of the disordered N- and C-termini not only in the Ub-ligase recognition, but also in the proteasome engagement. Together, the surface DEpSpT clusters and the N- and C-termini constitute an intrinsic bipartite degron for hTDO physiological turnover.