Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil.
Departamento de Bioquímica, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil; Departamento de Ciências Farmacêuticas, Universidade Federal de São Paulo, Diadema, São Paulo, Brazil.
Biochimie. 2023 Sep;212:21-30. doi: 10.1016/j.biochi.2023.03.013. Epub 2023 Mar 28.
Thimet oligopeptidase (THOP) is a cytosolic metallopeptidase known to regulate the fate of post-proteasomal peptides, protein turnover and peptide selection in the antigen presentation machinery (APM) system. Oxidative stress influences THOP expression and regulates its proteolytic activity, generating variable cytosolic peptide levels, possibly affecting the immune evasion of tumor cells. In the present work, we examined the association between THOP expression/activity and stress oxidative resistance in human leukemia cells using the K562 cell line, a chronic myeloid leukemia (CML), and the multidrug-resistant (MDR) Lucena 1 (K562-derived MDR cell line) as model. The Lucena 1 phenotype was validated under vincristine treatment and the relative THOP1 mRNA levels and protein expression compared to K562 cell line. Our data demonstrated increased THOP1 gene and protein levels in K562 cells in contrast to the oxidative-resistant Lucena 1, even after HO treatment, suggesting an oxidative stress dependence in THOP regulation. Further, it was observed higher basal levels of reactive oxygen species (ROS) in K562 compared to Lucena 1 cell line using DHE fluorescent probe. Since THOP activity is dependent on its oligomeric state, we also compared its proteolytic activity under reducing agent treatment, which demonstrated that its function modulation with respect to changes in redox state. Finally, the mRNA expression and FACS analyses demonstrated a reduced expression of MHC I only in K562 cell line. In conclusion, our results highlight THOP redox modulation, which could influence antigen presentation in multidrug resistant leukemia cells.
硫醇内切酶（THOP）是一种细胞溶质金属肽酶，已知可调节蛋白酶体后肽的命运、蛋白质周转和抗原呈递机制（APM）系统中的肽选择。氧化应激会影响 THOP 的表达并调节其蛋白水解活性，从而产生不同的细胞溶质肽水平，可能会影响肿瘤细胞的免疫逃逸。在本工作中，我们使用 K562 细胞系（慢性髓细胞白血病（CML））和多药耐药（MDR）Lucena 1（K562 衍生的 MDR 细胞系）作为模型，研究了 THOP 表达/活性与人类白血病细胞中氧化应激抵抗之间的相关性。Lucena 1 表型在长春新碱处理下得到验证，并与 K562 细胞系的相对 THOP1 mRNA 水平和蛋白表达进行了比较。我们的数据表明，与氧化应激抵抗的 Lucena 1 相比，K562 细胞中的 THOP1 基因和蛋白水平增加，即使在 HO 处理后也是如此，这表明 THOP 调节依赖于氧化应激。此外，使用 DHE 荧光探针观察到 K562 细胞中基础水平的活性氧（ROS）高于 Lucena 1 细胞系。由于 THOP 活性依赖于其寡聚状态，我们还比较了还原剂处理下的其蛋白水解活性，这表明其功能调节与氧化还原状态的变化有关。最后，mRNA 表达和 FACS 分析表明，只有在 K562 细胞系中 MHC I 的表达降低。总之，我们的结果强调了 THOP 的氧化还原调节，这可能会影响多药耐药白血病细胞中的抗原呈递。
