Liying Zhang,Zhiheng Deng,Yunxiang Du,Ziyu Xu,Tianyi Zhang,Zebin Tong,Huasong Ai,Lu-Jun Liang,Lei Liu
The 2-((2-chloroethyl)amino)ethane-1-thiol (CAET)-based chemical trapping strategy is a practical tool for mechanistic studies of E3-catalysed ubiquitination. However, the construction of ubiquitination intermediate mimics (E2-Ub-substrate conjugates) via CAET has been limited to peptides, while its application to folded protein substrates remains unexplored. Here, we report that disulfide bond formation between E2-Ub (RAD6A-Ub) and the folded protein substrate PCNA (proliferating cell nuclear antigen) occurs upon the addition of the PCNA-associated E3 ligase RAD18. Leveraging this finding, we employed intein splicing technology to generate a stable, covalently linked RAD18-RAD6A-Ub-PCNA complex, enabling chemical crosslinking mass spectrometry (CX–MS) analysis to study the structure of this complex. This work showcases use of a substrate-associated E3 ligase to promote disulfide bond formation between an E2-Ub conjugate and a folded substrate for CAET-based trapping, thereby expanding the scope of this technique.
Background Chemotherapy resistance of osteosarcoma (OS) is still the crux of poor clinical curative effect.E3 ubiquitin-protein ligase Rad18 (Rad18) contributed to doxorubicin resistance in OS, which ultimately mediated DNA damage tolerance and led to a poor prognosis and chemotherapy response in patients. Methods In this study, doxorubicin was loaded in the process of Fe 2+ and siRad18 forming nanoparticles(FSD) through coordination, chitosan modified with cell penetrating peptide (H6R6) was synthesized and coated on the surface of the NPs(FSD-CHR). FSD-CHR was then dispersed in thermosensitive hydrogel(PPP) for peritumoral injection of osteosarcoma in situ. Subsequently, the physicochemical properties and molecular biological characteristics of the drug delivery system were characterized. Finally, an osteosarcoma model was established to study the anti-tumor effects of multifunctional nanoparticles and the immunotherapy effect combined with αPD-L1. Results FSD-CHR has enhanced tumor tissue permeability, siRad18 can significantly reduce Dox-mediated DNA damage tolerance and enhance anti-tumor effects, and iron-based NPs show enhanced ROS upregulation. FSD-CHR@PPP showed significant inhibition of osteosarcoma growth in vivo and a reduced incidence of lung metastasis. In addition, siRad18 was unexpectedly found to enhance Dox-mediated immunogenic cell death (ICD).FSD-CHR@PPP combined with PD-L1 blocking significantly enhanced anti-tumor effects due to decreased PD-L1 enrichment. Conclusion Hydrogel encapsulation of permeable nanoparticles provides an effective strategy for doxorubicin-resistant OS, showing that gene therapy blocking DNA damage tolerance can enhance treatment response to chemotherapy and appears to enhance the effect of ICD inducers to activate the immune system.
危险品化学品经营许可证（带存储）