Ubiquitin And SUMO Post-Translational Modifications In Development And Disease

为了确定泛素介导的蛋白质降解在正常发育中起关键作用的靶标，以及当失调时可能导致肿瘤发生的靶标，我们对E3泛素连接酶Nedd4的相互作用伙伴进行了酵母双杂交筛选。该筛选鉴定出一种新的发育表达蛋白N4BP1。先前的研究表明，N4BP1也与相关的E3连接酶ITCH相互作用，但不是ITCH介导的泛素化的底物。相反，N4BP1与ITCH结合，负向调节ITCH E3活性，直接针对其底物，包括p53相关的肿瘤抑制蛋白p73和p63，以及c-Jun。这些结果表明，N4BP1可能在调节肿瘤进展和癌细胞对化疗的反应中发挥作用。许多蛋白质可以与泛素和小泛素相关修饰物（SUMO）结合，这两种翻译后修饰物之间的串扰有助于调节蛋白质的功能和稳定性。我们之前确定N4BP1是E3泛素连接酶Nedd4单泛素化的底物。我们现在发现N4BP1经历了nedd4介导的多泛素化和蛋白酶体降解。此外，我们发现N4BP1可以与SUMO1偶联，这可以消除N4BP1的泛素化。与此一致的是，内源性N4BP1在来自脱氧酶SENP1突变体的原代胚胎成纤维细胞中是稳定的，显示出稳态sumo化水平的增加。我们将内源性N4BP1主要定位在原代细胞的核仁上。然而，在早幼粒细胞白血病（PML）核体（NBs）中发现了一小部分。在SENP1缺失的细胞或蛋白酶体抑制剂MG132处理的野生型细胞中，我们发现PML NBs中有相当多的N4BP1积累。这些发现表明亚核室之间的动态相互作用，以及泛素和SUMO在核仁蛋白周转调节中的翻译后修饰作用。

To identify targets of ubiquitin mediated protein degradation playing key roles in normal development, and that might potentially contribute to neoplasia when dysregulated, we carried out a yeast two hybrid screen for interaction partners of the E3 ubiquitin ligase Nedd4. This screen identified N4BP1, a novel developmentally expressed protein. Previous work has shown that N4BP1 also interacts with the related E3 ligase, ITCH, but is not a substrate for ITCH mediated ubiquitination. Rather, N4BP1 binding to ITCH, negatively regulates ITCH E3 activity directed toward its substrates, including the p53 related tumor suppressor proteins p73 and p63, as well as c-Jun. These results suggest that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy. A number of proteins can be conjugated with both ubiquitin and the small ubiquitin-related modifier (SUMO), with crosstalk between these two post-translational modifications serving to regulate protein function and stability. We previously identified N4BP1 as a substrate for monoubiquitylation by the E3 ubiquitin ligase Nedd4. We have now found that N4BP1 undergoes Nedd4-mediated polyubiquitylation and proteasomal degradation. In addition, we found that N4BP1 can be conjugated with SUMO1 and that this abrogates N4BP1 ubiquitylation. Consistent with this, endogenous N4BP1 is stabilized in primary embryonic fibroblasts from mutants of the desumoylating enzyme SENP1, which show increased steady-state sumoylation levels. We localized endogenous N4BP1 predominantly to the nucleolus in primary cells. However, a small fraction was found at promyelocytic leukemia (PML) nuclear bodies (NBs). In cells deficient for SENP1 or in wild-type cells treated with the proteasome inhibitor MG132, we found considerable accumulation of N4BP1 at PML NBs. These findings suggest a dynamic interaction between subnuclear compartments, and a role for post-translational modification by ubiquitin and SUMO in the regulation of nucleolar protein turnover.