The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here, we show that its lysosomal degradation is dependent on ubiquitylation at lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal deubiquitylase, USP8, interacts with CTLA4, and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells, and cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype but shows distinct properties vis-à-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive nor a localization-compromised ΔMIT domain mutant can rescue delayed degradation of CTLA4 or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.

中文翻译：

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CTLA4 的快速周转与可逆泛素化的复杂结构有关。


免疫检查点调节因子 CTLA4 是一种异常短寿命的膜蛋白。在这里，我们表明其溶酶体降解依赖于赖氨酸残基 203 和 213 的泛素化。放线菌酮处理后，抑制 v-ATP 酶可部分恢复 CTLA4 水平，但也揭示了外泌体中分泌的部分。内体去泛素化酶 USP8 与 CTLA4 相互作用，其缺失增强了癌细胞、小鼠 CD4+ T 细胞和癌细胞衍生外泌体中的 CTLA4 泛素化。USP8 接头蛋白 HD-PTP 的耗竭（而不是 ESCRT-0）的耗竭概括了这种细胞表型，但显示出与外泌体掺入不同的特性。野生型 USP8 的再表达，但催化失活或定位受损的 ΔMIT 结构域突变体都不能挽救 CTLA4 的延迟降解或抵消其在成簇内体中的积累。CTLA4 相关泛素链键的 UbiCRest 分析确定了常规 Lys63 链和更不常见的 Lys27 和 Lys29 连接聚泛素链的复杂混合物，这可能是蛋白质周转快速的原因。