Exosomes are small vesicles released by cells, which have crucial functions in intercellular communication. Exosomes originated from cell membrane invagination and are released followed by multivesicular bodies (MVBs) fused with the cell membrane. It is known that Polymerase I and Transcript Release Factor (PTRF, also known as Caveolin-associated Protein-1, CAVIN1) plays an important role in caveolae formation and exosome secretion. And PTRF in exosomes has been identified as a potential biomarker in multiple malignancies such as glioma and renal cell carcinoma. However, the mechanisms of how to regulate the secretion of exosome-related PTRF remain unknown. We performed exogenous and endogenous immunoprecipitation assays to investigate the interaction between ubiquitin-conjugating enzyme E2O (UBE2O) and PTRF. We identified UBE2O ubiquitinated PTRF using ubiquitination assays. Then, exosomes were isolated by ultracentrifugation and identified by transmission electronic microscopy, western blot and nanoparticle tracking analysis. The effect of UBE2O on the secretion of exosome-related PTRF was analyzed by western blot, and the effect of UBE2O on exosome secretion was evaluated by exosome markers and the total protein content of exosomes. Here, we showed that UBE2O interacts with PTRF directly and ubiquitinates PTRF. Functionally, we found that UBE2O inhibited the effects of PTRF on exosome secretion via decreasing caveolae formation. Importantly, UBE2O decreased exosome secretion, resulting in downregulating PTRF secretion via exosomes. Our study also identified Serum Deprivation Protein Response (SDPR, also known as Caveolin-associated Protein-2, CAVIN2) interacted with both UBE2O and PTRF. Furthermore, we found that SDPR promotes PTRF expression in exosomes. Interestingly, even in the presence of SDPR, UBE2O still inhibited the secretion of exosome-related PTRF. Our study demonstrated that UBE2O downregulated exosome release and controlled the secretion of exosome-related PTRF through ubiquitinating PTRF. Since exosomes play an important role in malignant tumor growth and PTRF included in exosomes is a biomarker for several malignant tumors, increasing UBE2O expression in cells has the potential to be developed as a novel approach for cancer treatment.

中文翻译：

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UBE2O 泛素化 PTRF/CAVIN1 并抑制外泌体相关 PTRF/CAVIN1 的分泌

外泌体是由细胞释放的小囊泡，在细胞间通讯中起着至关重要的作用。外泌体起源于细胞膜内陷，随后释放出与细胞膜融合的多泡体 (MVB)。众所周知，聚合酶 I 和转录物释放因子（PTRF，也称为小窝蛋白相关蛋白 1，CAVIN1）在小窝形成和外泌体分泌中起重要作用。外泌体中的 PTRF 已被确定为多种恶性肿瘤（如神经胶质瘤和肾细胞癌）的潜在生物标志物。然而，如何调节外泌体相关 PTRF 分泌的机制仍然未知。我们进行了外源性和内源性免疫沉淀测定，以研究泛素结合酶 E2O (UBE2O) 和 PTRF 之间的相互作用。我们使用泛素化分析鉴定了 UBE2O 泛素化 PTRF。然后，通过超速离心分离外泌体，并通过透射电子显微镜、蛋白质印迹和纳米粒子追踪分析进行鉴定。通过western blot分析UBE2O对外泌体相关PTRF分泌的影响，通过外泌体标志物和外泌体总蛋白含量评价UBE2O对外泌体分泌的影响。在这里，我们表明 UBE2O 直接与 PTRF 相互作用并泛素化 PTRF。在功能上，我们发现 UBE2O 通过减少细胞膜穴样内陷的形成来抑制 PTRF 对外泌体分泌的影响。重要的是，UBE2O 减少了外泌体分泌，导致通过外泌体下调 PTRF 分泌。我们的研究还确定了血清剥夺蛋白反应（SDPR，也称为 Caveolin 相关蛋白 2，CAVIN2) 与 UBE2O 和 PTRF 相互作用。此外，我们发现 SDPR 促进外泌体中的 PTRF 表达。有趣的是，即使在 SDPR 存在的情况下，UBE2O 仍然抑制外泌体相关 PTRF 的分泌。我们的研究表明，UBE2O 下调外泌体释放并通过泛素化 PTRF 控制外泌体相关 PTRF 的分泌。由于外泌体在恶性肿瘤生长中起着重要作用，外泌体中包含的 PTRF 是几种恶性肿瘤的生物标志物，因此增加细胞中 UBE2O 的表达有可能被开发为一种新的癌症治疗方法。我们的研究表明，UBE2O 下调外泌体释放并通过泛素化 PTRF 控制外泌体相关 PTRF 的分泌。由于外泌体在恶性肿瘤生长中起着重要作用，外泌体中包含的 PTRF 是几种恶性肿瘤的生物标志物，因此增加细胞中 UBE2O 的表达有可能被开发为一种新的癌症治疗方法。我们的研究表明，UBE2O 下调外泌体释放并通过泛素化 PTRF 控制外泌体相关 PTRF 的分泌。由于外泌体在恶性肿瘤生长中起着重要作用，外泌体中包含的 PTRF 是几种恶性肿瘤的生物标志物，因此增加细胞中 UBE2O 的表达有可能被开发为一种新的癌症治疗方法。