The N-recognin UBR4 of the N-end rule pathway is required for neurogenesis and homeostasis of cell surface proteins

The N-end rule pathway is a proteolytic system in which single N-terminal amino acids of proteins act as a class of degrons (N-degrons) that determine the half-lives of proteins. We have previously identified a family of mammals N-recognins (termed UBR1, UBR2, UBR4/p600, and UBR5/EDD) whose conserved UBR boxes bind N-degrons to facilitate substrate ubiquitination and proteasomal degradation via the ubiquitin-proteasome system (UPS). Amongst these N-recognins, UBR1 and UBR2 mediate ubiquitination and proteolysis of short-lived regulators and misfolded proteins. Here, we characterized the null phenotypes of UBR4-deficient mice in which the UBR box of UBR4 was deleted. We show that the mutant mice die around embryonic days 9.5–10.5 (E9.5–E10.5) associated with abnormalities in various developmental processes such as neurogenesis and cardiovascular development. These developmental defects are significantly attributed to the inability to maintain cell integrity and adhesion, which significantly correlates to the severity of null phenotypes. UBR4-loss induces the depletion of many, but not all, proteins from the plasma membrane, suggesting that UBR4 is involved in proteome-wide turnover of cell surface proteins. Indeed, UBR4 is associated with and required to generate the multivesicular body (MVB) which transiently store endocytosed cell surface proteins before their targeting to autophagosomes and subsequently lysosomes. Our results suggest that the N-recognin UBR4 plays a role in the homeostasis of cell surface proteins and, thus, cell adhesion and integrity.

N端规则通路（N-end rule pathway）是一类蛋白水解系统，蛋白质的单个N端氨基酸可作为一类降解基序（degrons，又称N-降解基序（N-degrons）），决定蛋白质的半衰期。我们此前已鉴定出哺乳动物的一类N识别蛋白（N-recognins），分别为UBR1、UBR2、UBR4/p600及UBR5/EDD，其保守的UBR盒（UBR box）可结合N-降解基序，进而通过泛素-蛋白酶体系统（ubiquitin-proteasome system, UPS）介导底物的泛素化与蛋白酶体降解。在这类N识别蛋白中，UBR1与UBR2可介导短命调控蛋白及错误折叠蛋白的泛素化与蛋白水解过程。本研究针对UBR4缺陷小鼠（其UBR盒被敲除）的纯合缺失表型进行了系统表征。结果显示，该突变小鼠会在胚胎发育第9.5至10.5天左右（E9.5–E10.5）死亡，并伴随神经发生、心血管发育等多种发育进程的异常。上述发育缺陷在很大程度上可归因于细胞完整性与细胞黏附维持能力的丧失，该表型与纯合缺失小鼠的症状严重程度显著相关。UBR4的缺失会导致多数（而非全部）质膜蛋白的丰度降低，提示UBR4参与了细胞表面蛋白的全蛋白质组周转过程。事实上，UBR4可与多泡体（multivesicular body, MVB）结合，且对其生成不可或缺；多泡体可短暂储存经内吞作用获取的细胞表面蛋白，随后将这些蛋白靶向至自噬体，最终转运至溶酶体。我们的研究结果表明，作为N识别蛋白的UBR4可参与细胞表面蛋白的稳态维持，进而调控细胞黏附与细胞完整性。