Tsujita, T. | Search bioRxiv

bioRxiv2026

Cooperative regulation of NF-E2 related factor 1 protein stability and transcriptional activation by endoplasmic reticulum-associated degradation system mediator, Selenoprotein S/K.

NRF1 is a key mediator of the proteasome recovery pathway, yet its regulation by ER-resident factors is not fully elucidated. Here, we demonstrate that selenoproteins SELS and SELK are critical regulators for NRF1 protein dynamics. SELS stabilizes NRF1, while SELK induces its insolubilization. Their deficiency leads to a hyper-accumulation and increased nuclear localization of NRF1 under proteasome inhibition condition. This results in an augmented transcriptional response of proteasome subunits. These results indicate that SELS and SELK cooperatively gate NRF1 activity by controlling its retrotranslocation and solubility, highlighting a novel layer of selenoprotein-mediated quality control in the proteostasis network.

biochemistry↗

bioRxiv2025

High-Throughput Measurement of Oxidative Stress Indicators: d-ROMs and BAP Assays in a 384-Well Plate Format

The bodys oxidative balance is regulated by systems that produce and eliminate reactive oxygen species (ROS), and disruptions in this balance can contribute to the development of lifestyle-related diseases. d-ROMs and BAP tests, which evaluate oxidative balance using just 30 {micro}L of serum per sample in approximately 10 minutes, are widely used for this purpose. However, these methods are not suitable for large-scale studies due to their low-throughput and cost. To overcome these limitations, we established a simultaneous measurement system for multiple samples (144 samples) for measuring d-ROMs and BAP using a pipetting robot and a 384-well plate. The developed system demonstrated good linearity and reproducibility, while significantly reducing reagent and sample consumption. Measurement differences compared to the one of the currently available FREE Carrio Duo system ranged from -5% to 3% for d-ROMs and -12% to 8% for BAP, indicating high consistency with existing methods. Furthermore, the measurement time was substantially shortened from 24 hours (10 minutes x 144 samples) to at longest four hours, although the final color measurements for d-ROMs were performed one week later. This optimized semi-automated system enables the precise and efficient measurement of oxidative stress markers, making it suitable for large-scale studies.

biochemistry↗