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Excessive generation and accumulation of highly reactive oxidizing molecules causes oxidative stress and oxidative damage to cellular components. Accumulating evidence indicates that autophagy diminishes oxidative damage in cells and maintains redox homeostasis by degrading and recycling intracellular damaged components. Here, we show that TRAF6 E3 ubiquitin ligase and A20 deubiquitinase coordinate to regulate ATG9A ubiquitination and autophagy activation in cells responding to oxidative stress. The ROS-dependent TRAF6-mediated non-proteolytic, K48/63-linked ubiquitination of ATG9A enhances its association with Beclin 1 and the assembly of VPS34-UVRAG complex, thereby stimulating autophagy. Notably, expression of the ATG9A ubiquitination mutants impairs ROS-induced VPS34 activation and autophagy. We further find that lipopolysaccharide (LPS)-induced ROS production also stimulates TRAF6-mediated ATG9A ubiquitination. Ablation of ATG9A causes aberrant TLR4 endosomal trafficking and decreases IRF-3 phosphorylation in LPS-stimulated macrophages. Our findings provide important insights into how K48/K63-linked ubiquitination of ATG9A contributes to the regulation of oxidative stress-induced autophagy.

This paper investigates using an optical measurement method to determine the concentration ratio in a liquid mixture of methanol and ethanol. To better understand the relative mixture solution concentration, the dilution method is used to determine slight variations in the refractive index of the variable mixture concentration. The test concentration range of both methanol and ethanol is between 0–90%. The results of our analysis indicate that the refractive index of a mixture of methanol and ethanol lies in the interval between the refractive index of pure ethanol and methanol. The optical property of the mixture is similar to that of the solution material with the highest quantity. Regression analysis using a quadratic function resulted in an error of 25%. Regression analysis using a cubic function reduced the error to 6.25%. Therefore, the regression analysis function used in this study is the cubic function, as the mixture solution includes the methanol liquid. Combining the optical measurement and dilution methods can yield the solution concentration of the liquid mixture.