Alteration of the selenoproteome by cellular hypomethylation promotes endothelial cell activation

TY - JOUR

T1 - Alteration of the selenoproteome by cellular hypomethylation promotes endothelial cell activation

AU - Brigham, Cardiovascular Department of

PY - 2018/3/27

Y1 - 2018/3/27

N2 - The mechanisms by which hyperhomocysteinemia (HHcy) contributes to cardiovascular disease are unclear. We have identified suppression of the antioxidant selenoprotein GPx-1 as a link between HHcy and endothelial dysfunction. Selenoprotein translation is dependent on a unique system that recognizes a UGA codon as a site for selenocysteine (Sec) incorporation rather than a stop codon. Translation of a subset of selenoproteins, including GPx-1, is dependent on methylation of the tRNASec to the Um34 form. Accumulation of the methylation inhibitor, S-adenosylhomocysteine (SAH), occurs during HHcy. We postulated that SAH accumulation may impair tRNASec methylation, affecting the selenoproteome, which is involved in cell detoxification and antioxidant systems. We decreased SAH hydrolase activity, with a siRNA or a pharmacological inhibitor, to increase SAH in endothelial cells (EC). The selenoproteome was studied after [75Se] labeling. GPx-1 and thioredoxin reductase (TR1, TR2) expression was confirmed by western blot (WB). tRNASec methylation status was evaluated by RPC-5 chromatography after aminoacylation with [3H]-serine, the Sec-precursor that is attached to tRNASec. H2O2 release was measured with Amplex Red. ICAM-1 expression was evaluated by WB and FACS, and leukocyte adhesion studies were performed. An increase in intracellular SAH decreased the expression of selenoproteins dependent on the Um34-containing tRNASec, with a 37 ± 2.7% decrease of GPx-1 (p<0.005). Under these conditions, Um34-tRNASec was undetectable, although total tRNASec was increased by 1.64 ± 0.03 fold. In contrast, TR expression, which is independent of the Um34 modification, was upregulated 36.7% (TR1) and 23.1% (TR2) (p<0.05). The decrease in stress-related selenoproteins (e.g., GPx1) may account for the 2.9 ± 0.3 fold (p<0.001) increase of H2O2 release and the increase in total (2.1 ± 0.2 fold, p<0.001) and surface (2.1 ± 0.3 fold, p<0.02) expression of ICAM-1, as this phenomenon was attenuated with antioxidants. Leukocyte adhesion was also enhanced (41 ± 13%, p<0.05) in SAH-activated EC. These results suggest that SAH accumulation in EC can induce tRNASec hypomethylation which alters the selenoproteome to contribute to a pro-atherogenic endothelial phenotype.

AB - The mechanisms by which hyperhomocysteinemia (HHcy) contributes to cardiovascular disease are unclear. We have identified suppression of the antioxidant selenoprotein GPx-1 as a link between HHcy and endothelial dysfunction. Selenoprotein translation is dependent on a unique system that recognizes a UGA codon as a site for selenocysteine (Sec) incorporation rather than a stop codon. Translation of a subset of selenoproteins, including GPx-1, is dependent on methylation of the tRNASec to the Um34 form. Accumulation of the methylation inhibitor, S-adenosylhomocysteine (SAH), occurs during HHcy. We postulated that SAH accumulation may impair tRNASec methylation, affecting the selenoproteome, which is involved in cell detoxification and antioxidant systems. We decreased SAH hydrolase activity, with a siRNA or a pharmacological inhibitor, to increase SAH in endothelial cells (EC). The selenoproteome was studied after [75Se] labeling. GPx-1 and thioredoxin reductase (TR1, TR2) expression was confirmed by western blot (WB). tRNASec methylation status was evaluated by RPC-5 chromatography after aminoacylation with [3H]-serine, the Sec-precursor that is attached to tRNASec. H2O2 release was measured with Amplex Red. ICAM-1 expression was evaluated by WB and FACS, and leukocyte adhesion studies were performed. An increase in intracellular SAH decreased the expression of selenoproteins dependent on the Um34-containing tRNASec, with a 37 ± 2.7% decrease of GPx-1 (p<0.005). Under these conditions, Um34-tRNASec was undetectable, although total tRNASec was increased by 1.64 ± 0.03 fold. In contrast, TR expression, which is independent of the Um34 modification, was upregulated 36.7% (TR1) and 23.1% (TR2) (p<0.05). The decrease in stress-related selenoproteins (e.g., GPx1) may account for the 2.9 ± 0.3 fold (p<0.001) increase of H2O2 release and the increase in total (2.1 ± 0.2 fold, p<0.001) and surface (2.1 ± 0.3 fold, p<0.02) expression of ICAM-1, as this phenomenon was attenuated with antioxidants. Leukocyte adhesion was also enhanced (41 ± 13%, p<0.05) in SAH-activated EC. These results suggest that SAH accumulation in EC can induce tRNASec hypomethylation which alters the selenoproteome to contribute to a pro-atherogenic endothelial phenotype.

UR - https://www.ahajournals.org/doi/10.1161/circ.128.suppl_22.A10272

U2 - 10.1161/circ.128.suppl_22.A10272

DO - 10.1161/circ.128.suppl_22.A10272

M3 - Conference abstract in journal

VL - 128

JO - CIRCULATION

JF - CIRCULATION

SN - 0009-7322

IS - suppl_22

ER -