Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Qingdao, 266237, People's Republic of China.
Hubei Key Laboratory of Edible Wild Plants Conservation & Utilization, Hubei Engineering Research Center of Typical Wild Vegetable Breeding and Comprehensive Utilization Technology, Hubei Normal University, Huangshi, 435002, People's Republic of China.
Cell Mol Biol Lett. 2021 Dec 7;26(1):50. doi: 10.1186/s11658-021-00297-2.
Esterase D (ESD) is a nonspecific esterase that detoxifies formaldehyde. Many reports have stated that ESD activity is associated with a variety of physiological and pathological processes. However, the detailed signaling pathway of ESD remains poorly understood.
Considering the advantages of the small chemical molecule, our recent work demonstrated that 4-chloro-2-(5-phenyl-1-(pyridin-2-yl)-4,5-dihydro-1H-pyrazol-3-yl) phenol (FPD5) activates ESD, and will be a good tool for studying ESD further. Firstly, we determined the interaction between ESD and FK506 binding protein 25 (FKBP25) by yeast two-hybrid assay and co-immunoprecipitation (CO-IP) and analyzed the phosphorylation levels of mTORC1, P70S6K and 4EBP1 by western blot. Furthermore, we used the sulforhodamine B (SRB) and chick chorioallantoic membrane (CAM) assay to analyze cell viability in vitro and in vivo after treatment with ESD activator FPD5.
We screened FKBP25 as a candidate protein to interact with ESD by yeast two-hybrid assay. Then we verified the interaction between ESD and endogenous FKBP25 or ectopically expressed GFP-FKBP25 by CO-IP. Moreover, the N-terminus (1-90 aa) domain of FKBP25 served as the crucial element for their interaction. More importantly, ESD reduced the K48-linked poly-ubiquitin chains of FKBP25 and thus stabilized cytoplasmic FKBP25. ESD also promoted FKBP25 to bind more mTORC1, suppressing the activity of mTORC1. In addition, ESD suppressed tumor cell growth in vitro and in vivo through autophagy.
These findings provide novel evidence for elucidating the molecular mechanism of ESD and ubiquitination of FKBP25 to regulate autophagy and cancer cell growth. The ESD/FKBP25/mTORC1 signaling pathway is involved in inhibiting tumor cell growth via regulating autophagy.
酯酶 D（ESD）是一种非特异性酯酶，可解毒甲醛。许多报道指出，ESD 活性与多种生理和病理过程有关。然而，ESD 的详细信号通路仍知之甚少。
考虑到小分子化学物质的优势，我们最近的工作表明 4-氯-2-（5-苯基-1-（2-吡啶基）-4,5-二氢-1H-吡唑-3-基）苯酚（FPD5）激活 ESD，并且将成为进一步研究 ESD 的良好工具。首先，我们通过酵母双杂交测定和免疫共沉淀（CO-IP）确定了 ESD 与 FK506 结合蛋白 25（FKBP25）之间的相互作用，并通过 Western blot 分析了 mTORC1、P70S6K 和 4EBP1 的磷酸化水平。此外，我们使用磺基罗丹明 B（SRB）和鸡胚绒毛尿囊膜（CAM）测定法分析了 ESD 激活剂 FPD5 处理后体外和体内细胞活力。
我们通过酵母双杂交测定筛选 FKBP25 作为与 ESD 相互作用的候选蛋白。然后，我们通过 CO-IP 验证了 ESD 与内源性 FKBP25 或异位表达的 GFP-FKBP25 之间的相互作用。此外，FKBP25 的 N 端（1-90 aa）结构域是它们相互作用的关键元素。更重要的是，ESD 减少了 FKBP25 的 K48 连接多泛素链，从而稳定了细胞质 FKBP25。ESD 还促进 FKBP25 与更多 mTORC1 结合，从而抑制 mTORC1 的活性。此外，ESD 通过自噬抑制体外和体内肿瘤细胞的生长。
这些发现为阐明 ESD 的分子机制以及 FKBP25 的泛素化在调节自噬和肿瘤细胞生长中的作用提供了新的证据。ESD/FKBP25/mTORC1 信号通路通过调节自噬参与抑制肿瘤细胞生长。
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