Accumulation of unfolded antibody chains in the ER triggers ER stress that may lead to reduced productivity in therapeutic antibody manufacturing processes. We identified UBR4 and UBR5 as ubiquitin E3 ligases involved in HC ER-associated degradation. Knockdown of UBR4 and UBR5 resulted in intracellular accumulation, enhanced secretion, and reduced ubiquitination of HC. In concert with these E3 ligases, PDIA3 was shown to cleave ubiquitinated HC molecules to accelerate HC dislocation. Interestingly, UBR5, and to a lesser degree UBR4, were down-regulated as cellular demand for antibody expression increased in CHO cells during the production phase, or in plasma B cells. Reducing UBR4/UBR5 expression before the production phase increased antibody productivity in CHO cells, possibly by redirecting antibody molecules from degradation to secretion. Altogether we have characterized a novel proteolysis/proteasome-dependent pathway involved in degradation of unfolded antibody HC. Proteins characterized in this pathway may be novel targets for CHO cell engineering.

中文翻译：

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UBR E3 连接酶和 PDIA3 蛋白酶通过 ERAD 控制未折叠抗体重链的降解

内质网中未折叠抗体链的积累会引发内质网应激，从而可能导致治疗性抗体制造过程的生产率降低。我们确定 UBR4 和 UBR5 是参与 HC ER 相关降解的泛素 E3 连接酶。UBR4 和 UBR5 的敲低导致 HC 细胞内积累、分泌增强和泛素化减少。与这些 E3 连接酶配合，PDIA3 可以裂解泛素化的 HC 分子，从而加速 HC 错位。有趣的是，随着生产阶段 CHO 细胞或血浆 B 细胞中抗体表达的细胞需求增加，UBR5 和较小程度的 UBR4 被下调。在生产阶段之前减少 UBR4/UBR5 表达可增加 CHO 细胞中的抗体生产率，这可能是通过将抗体分子从降解重定向到分泌来实现的。总之，我们描述了一种参与未折叠抗体 HC 降解的新型蛋白水解/蛋白酶体依赖性途径。该途径中表征的蛋白质可能是 CHO 细胞工程的新靶标。