Weiss Konstantin, Agarkova Yelizaveta, Zwosta Alexandra, Hoevel Sarah, Himmelreich Ann-Kathrin, Shumanska Magdalena, Etich Julia, Poschmann Gereon, Brachvogel Bent, Bogeski Ivan, Mielenz Dirk, Riemer Jan
Redox Metabolism Group, Institute for Biochemistry, University of Cologne, Cologne, Germany.
Division of Molecular Immunology, Department of Internal Medicine 3, Friedrich-Alexander-Universität Erlangen-Nürnberg and Universitätsklinikum Erlangen, Erlangen, Germany.
Life Sci Alliance. 2025 May 12;8(8). doi: 10.26508/lsa.202403076. Print 2025 Aug.
Dipeptidyl peptidases 8 and 9 (DPP8/9) are critical for the quality control of mitochondrial and endoplasmic reticulum protein import, immune regulation, cell adhesion, and cell migration. Dysregulation of DPP8/9 is associated with pathologies including tumorigenesis and inflammation. Commonly, DPP8/9 activity is analysed by in vitro assays using artificial substrates, which allow neither continuously monitoring DPP8/9 activity in individual, living cells nor detecting effects from endogenous interactors and posttranslational modifications. Here, we developed DiPAK (for DPP8/9 activity sensor based on AK2), a ratiometric genetically encoded fluorescent sensor, which enables studying DPP8/9 activity in living cells. Using DiPAK, we determined the dynamic range of DPP8/9 activity in cells overexpressing or lacking DPP9. We identified distinct activity levels among melanoma cell lines and found that LPS-induced primary B-cell activation depends on DPP8/9 as the absence of DPP8/9 activity results in apoptotic but not pyroptotic cell death. Consistently, we observed increasing DPP8/9 activity during B-cell maturation. Overall, DiPAK is a versatile tool for real-time single-cell monitoring of DPP8/9 activity in a broad range of cells and organisms.
二肽基肽酶8和9（DPP8/9）对于线粒体和内质网蛋白质导入的质量控制、免疫调节、细胞黏附和细胞迁移至关重要。DPP8/9的失调与包括肿瘤发生和炎症在内的多种病理状况相关。通常，DPP8/9的活性通过使用人工底物的体外测定来分析，这种方法既不能连续监测单个活细胞中的DPP8/9活性，也无法检测内源性相互作用分子和翻译后修饰的影响。在此，我们开发了DiPAK（基于AK2的DPP8/9活性传感器），这是一种比率型基因编码荧光传感器，能够在活细胞中研究DPP8/9的活性。利用DiPAK，我们确定了过表达或缺乏DPP9的细胞中DPP8/9活性的动态范围。我们在黑色素瘤细胞系中鉴定出不同的活性水平，并发现脂多糖诱导的原代B细胞活化依赖于DPP8/9，因为缺乏DPP8/9活性会导致细胞凋亡而非焦亡。一致地，我们观察到在B细胞成熟过程中DPP8/9活性增加。总体而言，DiPAK是一种通用工具，可用于在广泛的细胞和生物体中实时单细胞监测DPP8/9的活性。
