Endothelial dysfunction induced by hyperhomocysteinemia (HHcy) plays a critical role in vascular pathology. However, little is known about the role of endoplasmic reticulum (ER) redox homeostasis in HHcy-induced endothelial dysfunction. Here, we show that Hcy induces ER oxidoreductin-1α (Ero1α) expression with ER stress and inflammation in human umbilical vein endothelial cells and in the arteries of HHcy mice. Hcy upregulates Ero1α expression by promoting binding of hypoxia-inducible factor 1α to the ERO1A promoter. Notably, Hcy rather than other thiol agents markedly increases the GSH/GSSG ratio in the ER, therefore allosterically activating Ero1α to produce H2O2 and trigger ER oxidative stress. By contrast, the antioxidant pathway mediated by ER glutathione peroxidase 7 (GPx7) is downregulated in HHcy mice. Ero1α knockdown and GPx7 overexpression protect the endothelium from HHcy-induced ER oxidative stress and inflammation. Our work suggests that targeting ER redox homeostasis could be used as an intervention for HHcy-related vascular diseases.

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Shieldin, including SHLD1, SHLD2, SHLD3 and REV7, functions as a bridge linking 53BP1-RIF1 and single-strand DNA to suppress the DNA termini nucleolytic resection during nonhomologous end joining (NHEJ). However, the mechanism of shieldin assembly remains unclear. Here we present the crystal structure of the SHLD3-REV7-SHLD2 ternary complex and reveal an unexpected C (closed)-REV7-O (open)-REV7 conformational dimer mediated by SHLD3. We show that SHLD2 interacts with O-REV7 and the N-terminus of SHLD3 by forming β sheet sandwich. Disruption of the REV7 conformational dimer abolishes the assembly of shieldin and impairs NHEJ efficiency. The conserved FXPWFP motif of SHLD3 binds to C-REV7 and blocks its binding to REV1, which excludes shieldin from the REV1/Pol ζ translesion synthesis (TLS) complex. Our study reveals the molecular architecture of shieldin assembly, elucidates the structural basis of the REV7 conformational dimer, and provides mechanistic insight into orchestration between TLS and NHEJ.

Heteronemin, a marine sesterterpenoid-type natural product, possesses diverse bioactivities, especially antitumor effect. Accumulating evidence shows that heteronemin may act as a potent anticancer agent in clinical therapy. To fully understand the antitumor mechanism of heteronemin, we further explored the precise molecular targets in prostate cancer cells. Initially, heteronemin exhibited potent cytotoxic effect against LNcap and PC3 prostate cancer cells with IC50 1.4 and 2.7 μM after 24 h, respectively. In the xenograft animal model, the tumor size was significantly suppressed to about 51.9% in the heteronemin-treated group in comparison with the control group with no significant difference in the mice body weights. In addition, the results of a cell-free system assay indicated that heteronemin could act as topoisomerase II (topo II) catalytic inhibitor through the elimination of essential enzymatic activity of topoisomerase IIα expression. We found that the use of heteronemin-triggered apoptosis by 20.1–68.3%, caused disruption of mitochondrial membrane potential (MMP) by 66.9–99.1% and promoted calcium release by 1.8-, 2.0-, and 2.1-fold compared with the control group in a dose-dependent manner, as demonstrated by annexin-V/PI, rhodamine 123 and Fluo-3 staining assays, respectively. Moreover, our findings indicated that the pretreatment of LNcap cells with an inhibitor of protein tyrosine phosphatase (PTPi) diminished growth inhibition, oxidative and Endoplasmic Reticulum (ER) stress, as well as activation of Chop/Hsp70 induced by heteronemin, suggesting PTP activation plays a crucial rule in the cytotoxic activity of heteronemin. Using molecular docking analysis, heteronemin exhibited more binding affinity to the N-terminal ATP-binding pocket of Hsp90 protein than 17-AAG, a standard Hsp90 inhibitor. Finally, heteronemin promoted autophagy and apoptosis through the inhibition of Hsp 90 and topo II as well as PTP activation in prostate cancer cells. Taken together, these multiple targets present heteronemin as an interesting candidate for its future development as an antiprostatic agent.