Identification of Selenoprotein H Isoforms and Impact of Selenoprotein H Overexpression on Protein But Not mRNA Levels of 2 Other Selenoproteins in 293T Cells

Selenoprotein H (SELONOH), a member of the thioredoxin-like family proteins, is prioritized to degradation in selenium (Se) insufficiency. Recent studies implicate protective roles of SELENOH in oxidative stress, cellular senescence, and intestinal tumorigenesis. Although the nonselenoprotein H0YE28 is suggested as shortened SELENOH according to genomic and proteomic data repositories, this variant has not been verified biochemically. We sought to identify SELENOH isoforms and explore the impact of Se flux on selenoprotein expression in SELENOH-overexpressing cells. A vector expressing a FLAG (the DYKDDDDK sequence) tag on the N-terminal end of wild-type SELENOH was constructed and transiently transfected into 293T cells incubated with graded concentrations of Na2SeO3 (0–200 nM). Cells were subjected to immunoprecipitation, LC-MS/MS protein analysis, immunoblotting, qRT-PCR, and senescence assays. Data were analyzed by 1-way or 2-way ANOVA. Results of anti-FLAG immunoblotting showed that FLAG-SELENOH transfection increased (3.7-fold; P