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Monoclonal non-specific suppressor factor b (MNSFb) is a ubiquitously expressed member of the ubiquitin-like family that is involved in various biological functions. Previous studies have demonstrated that MNSFb covalently binds to intracellular pro-apoptotic protein Bcl-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 MNSFb promotes lipopolysaccharide (LPS)/interferon c (IFNc)-induced apoptosis of Raw264.7 macrophages. In Raw264.7 cells treated with MNSFb small interfering RNA (siRNA), LPS/ IFNc-or NO donor S-nitrosoglutathione-induced apoptosis was inhibited. siRNA-mediated knockdown of MNSFb did not affect inducible nitricoxide synthase (iNOS) expression in LPS/IFNc-stimulated Raw264.7 cells. Conversely, co-transfection with MNSFb and Bcl-G greatly enhanced LPS/IFNc-induced apoptosis in Raw264.7 cells, accompanied by increased expression of p53 and decreased Cox-2 activity. Unlike co-transfection with wild-type MNSFb, co-transfection of a mutant MNSFb (G74A) and Bcl-G did not result in enhancement of LPS/IFNc-induced apoptosis. Co-over-expression of MNSFb and Bcl-G reduced S-nitrosoglutathioneinduced ERK1/2 phosphorylation. Furthermore, electrophoretic mobility shift assay experiments revealed that MNSFb down-regulates the ERK/activator protein 1 (AP-1) signaling cascade which leads to Cox-2 activation. We also observed that MNSFb-Bcl-G promotes LPS/IFNc-induced apoptosis of mouse peritoneal macrophages, together with a decrease in Cox-2 expression. Taken together, our data indicate an apoptosis-enhancing effect of MNSFb-Bcl-G is due in part to down-regulation of Cox-2 activation in macrophages. Structured digital abstractBcl-G physically interacts with MNSFb by anti-bait co-immunoprecipitation (View Interaction: 1,2,3,4) Abbreviations AP-1, activator protein 1; BCL-G, B-cell CLL/lymphoma-G; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; Fau, Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed; GSNO, S-nitrosoglutathione; IFNc, interferon c; IKK, IjB kinase; iNOS, inducible nitric-oxide synthase; LPS, lipopolysaccharide; MEK, mitogen-activated protein kinase/extracellular signalregulated kinase kinase; MAPK, mitogen-activated protein kinase; MNSF, monoclonal non-specific suppressor factor; NFjB, nuclear factor jB; TLR2, Toll-like receptor 2.

MNSFβ, a ubiquitin-like protein, covalently binds to various target proteins including proapoptotic Bcl-G. During the course of isolation of MNSFβ-conjugating enzyme(s), we identified a novel target protein for MNSFβ. MALDI-TOF MS fingerprinting revealed that the MNSFβ-interacting protein is HSPA8 (heat shock 70-kDa protein 8). We observed that MNSFβ noncovalently binds to HSPA8 in the presence of ATP in vitro. Double knockdown of MNSFβ and HSPA8 strongly inhibited RANKL-induced osteoclastogenesis from Raw264.7 macrophage-like cells. The same treatment inhibited RANKL-induced ERK1/2 and p38 phosphorylation and TNFα production, suggesting that the association of MNSFβ with HSPA8 may promote RANKL-induced osteoclastogenesis. This is the first report that MNSFβ binds to a protein substrate via the noncovalent association and exerts biological effects.

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Monoclonal non-specific suppressor factor β (MNSFβ) is a ubiquitously expressed member of the ubiquitin-like family that is involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently binds to intracellular pro-apoptotic protein Bcl-G and regulates apoptosis in macrophages. In this study, we demonstrate that MNSFβ negatively regulates T cell function. In murine T-helper type 2 clone, D10.G4.1 (D10) cells transfected with MNSFβ cDNA, CD3/CD28-induced ERK1/2 phosphorylation leading to IL-4 production was significantly inhibited. The formation of MNSFβ-Bcl-G complex was induced by the CD3/CD28 stimulation. Co-transfection with MNSFβ and Bcl-G greatly enhanced CD3/CD28-induced apoptosis in D10 cells. Similarly, co-over-expression of MNSFβ and Bcl-G caused a marked enhancement of apoptosis in purified splenic T cells. Interestingly, this MNSFβ adduct was also induced in T cells derived from DO11.10 mice stimulated with antigen. Collectively, CD3/CD28-inducible MNSFβ-Bcl-G complex may be involved in the regulation of T cell function and survival.