Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702, USA.
Genome Modification Core, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.
Nat Commun. 2020 Mar 10;11(1):1291. doi: 10.1038/s41467-020-15073-7.
Regulated proteolysis by proteasomes involves ~800 enzymes for substrate modification with ubiquitin, including ~600 E3 ligases. We report here that E6AP/UBE3A is distinguished from other E3 ligases by having a 12 nM binding site at the proteasome contributed by substrate receptor hRpn10/PSMD4/S5a. Intrinsically disordered by itself, and previously uncharacterized, the E6AP-binding domain in hRpn10 locks into a well-defined helical structure to form an intermolecular 4-helix bundle with the E6AP AZUL, which is unique to this E3. We thus name the hRpn10 AZUL-binding domain RAZUL. We further find in human cells that loss of RAZUL by CRISPR-based gene editing leads to loss of E6AP at proteasomes. Moreover, proteasome-associated ubiquitin is reduced following E6AP knockdown or displacement from proteasomes, suggesting that E6AP ubiquitinates substrates at or for the proteasome. Altogether, our findings indicate E6AP to be a privileged E3 for the proteasome, with a dedicated, high affinity binding site contributed by hRpn10.
蛋白酶体的调控蛋白水解涉及约 800 种用于泛素化底物修饰的酶，包括约 600 种 E3 连接酶。我们在此报告，E6AP/UBE3A 通过其底物受体 hRpn10/PSMD4/S5a 在蛋白酶体上具有 12 nM 的结合位点，从而与其他 E3 连接酶区分开来。hRpn10 自身是无规则卷曲的，以前未被表征，其 E6AP 结合域锁定在一个明确定义的螺旋结构中，与 E6AP 的 AZUL 形成分子间的 4 螺旋束，这是该 E3 所特有的。因此，我们将 hRpn10 的 AZUL 结合域命名为 RAZUL。我们还在人细胞中发现，基于 CRISPR 的基因编辑缺失 RAZUL 会导致蛋白酶体中 E6AP 的缺失。此外，E6AP 敲低或从蛋白酶体中置换后，与蛋白酶体相关的泛素减少，这表明 E6AP 在蛋白酶体上或为蛋白酶体修饰底物。总之，我们的发现表明 E6AP 是蛋白酶体的一种特权 E3，其 hRpn10 贡献了一个专用的、高亲和力的结合位点。
