Binding Interface and Electron Transfer Between Nicotine Oxidoreductase and Its Cytochrome c Electron Acceptor

The enzyme nicotine oxidoreductase (NicA2) is a member of the flavoprotein amine oxidase family that uses a cytochrome c protein (CycN) as its oxidant instead of dioxygen, which is the oxidant used by most other members of this enzyme family. We recently identified a potential binding site for CycN on the surface of NicA2 through rigid body docking [J. Biol. Chem. 2022, 298 (8), 102251]. However, this potential binding interface has not been experimentally validated. In this paper, we used unnatural amino acid incorporation to probe the binding interface between NicA2 and CycN. Our results are consistent with a structural model of the NicA2-CycN complex predicted by protein–protein docking and AlphaFold, suggesting that this is the binding site for CycN on NicA2’s surface. Based on additional mutagenesis of potentially redox active residues in NicA2, we propose that electron transfer from NicA2’s flavin to CycN’s heme occurs without the assistance of a protein-derived wire

Pre-validation of an in vitro skin irritation test for medical devices using the reconstructed human tissue model EpiDerm™.

Assessment of dermal irritation is an essential component of the safety evaluation of medical devices. Reconstructed human epidermis (RhE) models have replaced rabbit skin irritation testing for neat chemicals and their mixtures (OECD Test Guideline 439). However, this guideline cannot be directly applied to the area of medical devices (MD) since their non-toxicity assessment is largely based on the testing of MD extracts that may have very low irritation potential. Therefore, the RhE-methods previously validated with neat chemicals needed to be modified to reflect the needs for detection of low levels of potential irritants. A protocol employing RhE EpiDerm was optimized in 2013 using known irritants and spiked polymers (Casas et al., 2013, TIV). In 2014 and 2015 MatTek In Vitro Life Science Laboratories (IVLSL) and RIVM assessed the transferability of the assay. After the successful transfer and standardization of the protocol, 17 laboratories were trained in the use of the protocol in the preparation for the validation. Laboratories produced data with 98% agreement of predictions for the selected references and controls. We conclude that a modified RhE skin irritation test has the potential to address the skin irritation potential of the medical devices. Standardization and focus on the technical issues is essential for accurate prediction

Artemisinin Blocks Prostate Cancer Growth and Cell Cycle Progression by Disrupting Sp1 Interactions with the Cyclin-dependent Kinase-4 (CDK4) Promoter and Inhibiting CDK4 Gene Expression*

Artemisinin, a naturally occurring component of Artemisia annua, or sweet wormwood, is a potent anti-malaria compound that has recently been shown to have anti-proliferative effects on a number of human cancer cell types, although little is know about the molecular mechanisms of this response. We have observed that artemisinin treatment triggers a stringent G1 cell cycle arrest of LNCaP (lymph node carcinoma of the prostate) human prostate cancer cells that is accompanied by a rapid down-regulation of CDK2 and CDK4 protein and transcript levels. Transient transfection with promoter-linked luciferase reporter plasmids revealed that artemisinin strongly inhibits CDK2 and CDK4 promoter activity. Deletion analysis of the CDK4 promoter revealed a 231-bp artemisinin-responsive region between -1737 and -1506. Site-specific mutations revealed that the Sp1 site at -1531 was necessary for artemisinin responsiveness in the context of the CDK4 promoter. DNA binding assays as well as chromatin immunoprecipitation assays demonstrated that this Sp1-binding site in the CDK4 promoter forms a specific artemisinin-responsive DNA-protein complex that contains the Sp1 transcription factor. Artemisinin reduced phosphorylation of Sp1, and when dephosphorylation of Sp1 was inhibited by treatment of cells with the phosphatase inhibitor okadaic acid, the ability of artemisinin to down-regulate Sp1 interactions with the CDK4 promoter was ablated, rendering the CDK4 promoter unresponsive to artemisinin. Finally, overexpression of Sp1 mostly reversed the artemisinin down-regulation of CDK4 promoter activity and partially reversed the cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin anti-proliferative effects in prostate cancer cells is the transcriptional down-regulation of CDK4 expression by disruption of Sp1 interactions with the CDK4 promoter