Probing Oxidant Effects on Superoxide Dismutase 1 Oligomeric States in Live Cells Using Single-Molecule Fluorescence Anisotropy  

作　　者：Huanhuan Chen Tai-Yen Chen 

机构地区：[1]Department of Chemistry,University of Houston,Houston,Texas 77204,United States

出　　处：《Chemical & Biomedical Imaging》2023年第1期49-57,共9页化学与生物医学影像（英文）

基　　金：supported by the National Institutes of Health(Grant No.R35GM133505)and the University of Houston.

摘　　要：The protein Cu/Zn superoxide dismutase (SOD1) isknown to function as a dimer, but its concentration in cells (∼50μM) and the dimerization constant (K_(d) of 500 μM) results suggestthat it exists in a monomer-dimer equilibrium. It is unclear how theoligomeric state of SOD1 changes when cells are initially exposed tohigh levels of extracellular oxidative stress. To address this problem,we introduced the single-molecule ffuorescence anisotropy (smFA)assay to explore SOD1 oligomeric states in live COS7 cells. smFAspeciffcally probes the ffuorescence polarization changes caused bymolecular rotations where the fast-rotating molecules (either due tosmaller hydrodynamic volume or less viscous environments)deteriorate the emission polarization and thus lower the anisotropy. After validating that smFA is effective in distinguishingmonomeric and dimeric ffuorescence proteins, we overexpressed SOD1 in live COS7 cells and investigated how its oligomeric statechanges under basal, 2 h, and 24 h 100 μM H_(2)O_(2) treatments. We found that treating cells with H_(2)O_(2) promotes SOD1 dimerizationand decreases cellular viscosity in 2 h. Interestingly, prolonged H_(2)O_(2) treatments show similar results as the basal conditions,indicating that cells return to a steady state similar to the basal state after 24 h, despite the presence of H_(2)O_(2). Our resultsdemonstrate that SOD1 changes its oligomeric state equilibrium in response to extracellular oxidative stresses. smFA will open newopportunities to explore the relationship between the SOD1 oligomer state and its H_(2)O_(2)-based signaling and transcription regulationroles.

关 键 词：single-molecule ffuorescence anisotropy protein rotation hydrogen peroxide H_(2)O_(2) superoxide dismutase 1 SOD1 protein oligomerization cellular viscosity 

分 类 号：Q25[生物学—细胞生物学]