Ubiquitin‐like protein MNSF β covalently binds to B cl– G and enhances lipopolysaccharide/interferon γ‐induced apoptosis in macrophages

Monoclonal non‐specific suppressor factor β ( MNSF β) is a ubiquitously expressed member of the ubiquitin‐like family that is involved in various biological functions. Previous studies have demonstrated that MNSF β covalently binds to intracellular pro‐apoptotic protein B cl– G and regulates the extracellular signal‐regulated kinase ( ERK )/mitogen‐activated protein kinase ( MAPK ) cascade in the mouse macrophage cell line Raw264.7. In this study, we demonstrate that MNSF β promotes lipopolysaccharide ( LPS )/interferon γ ( IFN γ)‐induced apoptosis of Raw264.7 macrophages. In Raw264.7 cells treated with MNSF β small interfering RNA (si RNA ), LPS / IFN γ‐ or NO donor S –nitrosoglutathione‐induced apoptosis was inhibited. siRNA‐mediated knockdown of MNSF β did not affect inducible nitric‐oxide synthase ( iNOS ) expression in LPS / IFN γ‐stimulated Raw264.7 cells. Conversely, co‐transfection with MNSF β and Bcl–G greatly enhanced LPS / IFN γ‐ induced apoptosis in Raw264.7 cells, accompanied by increased expression of p53 and decreased Cox–2 activity. Unlike co‐transfection with wild‐type MNSF β, co‐transfection of a mutant MNSF β ( G 74 A ) and Bcl– G did not result in enhancement of LPS / IFN γ‐induced apoptosis. Co‐over‐expression of MNSF β and Bcl–G reduced S –nitrosoglutathione‐induced ERK 1/2 phosphorylation. Furthermore, electrophoretic mobility shift assay experiments revealed that MNSF β down‐regulates the ERK /activator protein 1 (AP–1) signaling cascade which leads to Cox–2 activation. We also observed that MNSF β–Bcl– G promotes LPS / IFN γ‐induced apoptosis of mouse peritoneal macrophages, together with a decrease in Cox–2 expression. Taken together, our data indicate an apoptosis‐enhancing effect of MNSF β–Bcl–G is due in part to down‐regulation of C ox–2 activation in macrophages. Structured digital abstractBcl-G physically interacts with MNSFβ by anti-bait co-immunoprecipitation (View Interaction: 1 , 2 , 3 , 4 )