Radicalization of Glyceraldehyde-3-Phosphate Dehydrogenase by HOCl in Living Cells

SANDRA E GOMEZ-MEJIBA, DARIO C RAMIREZ; MARCOS D MUÑOZ , CECILIA DELLA VEDOVA

Enzime Engineering

Enzime Engineering

Año: 2015 vol. 4 p. 1 - 9

2329-6674

A number of post-translational oxidative modifications of the enzyme ?cell-redox sensor? glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been reported. These modifications affect GAPDH structure, function,and cell fate; however no free-radical mechanisms have been reported in these processes. Herein we used thenitrone 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-based spin trapping techniques to examine a novel free radicalmechanism that causes GAPDH inactivation and aggregation in RAW264.7 cells primed with lipopolysaccharide(LPS). In these primed cells, GAPDH is oxidized by myeloperoxidase (MPO)-derived hypochlorous acid (HOCl)resulting in loss of enzyme activity and aggregation, accumulation of lactate and cell death. Due to the close spatialand physical proximity between MPO and GAPDH, and the oxidizing potential of HOCl, it may be the main speciesthat triggers radicalization of GAPDH that ultimately results in enzyme aggregation and inactivation in LPS-primedmacrophages. Lysine residues are the primary radicalization sites formed upon reaction of HOCl with the enzyme.Our data highlight the important relationship between radicalization of GAPDH and fate of stressed cells, which mighthelp teasing out the cell response to stress at sites of inflammation.