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Monoclonal nonspecific suppressor factor β (MNSFβ) is a ubiquitously expressed ubiquitin-like protein known to be involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently modify its target proteins including Bcl-G, a proapoptotic protein. In this study, we purified a 65 kDa MNSFβ adduct from mouse liver lysates by sequential chromatography on DEAE and glutathione S-transferase (GST)-fusioned MNSFβ immobilized on glutathione-Sepharose beads in the presence of ATP. MALDI-TOF mass spectrometry fingerprinting revealed that this MNSFβ adduct consists of an 8.5 kDa MNSFβ and heat shock protein 60 (HSP60), a mitochondrial protein involved in protein folding. Fingerprinting analysis of the MNSFβ adduct demonstrates that MNSFβ conjugates to HSP60 with a linkage between the C-terminal Gly74 and Lys481. HSP60 siRNA neutralized the inhibition of apoptosis by MNSFβ siRNA in LPS/IFNγ-stimulated Raw264.7, a murine macrophage cell line. HSP60 siRNA also down-regulated the enhancement of TNFα production by MNSFβ siRNA in LPS-stimulated Raw264.7 cells. Here, we firstly report that MNSFβ activity is negatively regulated by molecular chaperone.

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.

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 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 MNSFβ promotes lipopolysaccharide (LPS)/interferon γ (IFNγ)-induced apoptosis of Raw264.7 macrophages. In Raw264.7 cells treated with MNSFβ small interfering RNA (siRNA), LPS/IFNγ- or NO donor S–nitrosoglutathione-induced apoptosis was inhibited. siRNA-mediated knockdown of MNSFβ did not affect inducible nitric-oxide synthase (iNOS) expression in LPS/IFNγ-stimulated Raw264.7 cells. Conversely, co-transfection with MNSFβ and Bcl–G greatly enhanced LPS/IFNγ- induced apoptosis in Raw264.7 cells, accompanied by increased expression of p53 and decreased Cox–2 activity. Unlike co-transfection with wild-type MNSFβ, co-transfection of a mutant MNSFβ (G74A) and Bcl–G did not result in enhancement of LPS/IFNγ-induced apoptosis. Co-over-expression of MNSFβ and Bcl–G reduced S–nitrosoglutathione-induced ERK1/2 phosphorylation. Furthermore, electrophoretic mobility shift assay experiments revealed that MNSFβ down-regulates the ERK/activator protein 1 (AP–1) signaling cascade which leads to Cox–2 activation. We also observed that MNSFβ–Bcl–G promotes LPS/IFNγ-induced apoptosis of mouse peritoneal macrophages, together with a decrease in Cox–2 expression. Taken together, our data indicate an apoptosis-enhancing effect of MNSFβ–Bcl–G is due in part to down-regulation of Cox–2 activation in macrophages. Structured digital abstract Bcl-G physically interacts with MNSFβ by anti-bait co-immunoprecipitation (View Interaction: 1, 2, 3, 4)

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.

The ordered Ll 0 -FePt phase with a size of 20-200 nm was directly formed by rapidly quenching the melt in the compositional range of 2-5 at.% Zr and 17-20 at.% B, and the alloys exhibit coercivity (H c ) higher than 200 kA/m in an as-quenched state. In particular, the melt-spun (Fe 0.50 Pt 0.50 ) 78 Zr 4 B 18 alloy exhibits high H c of 684 kA/m. The melting temperature (T m ) remarkably decreases by the addition of Zr and B, e.g., from 1837 K for FePt to 1345 K for (Feo.55Pto.45) 78 Zr 4 B 18 . The Ll 0 phase with Zr and B directly formed by the rapidly quenching shows high thermal stability and maintains up to T m . The simultaneous addition of Zr and B facilitates the direct formation of the ordered Ll 0 phase with a grain size of 20-200 nm by rapidly quenching the melt through the effect of the decreasing T m and the increasing the stability of the Ll 0 phase by the solution of Zr and B into the phase. The substitution of Fe by Co monotonously decreases T m from 1345 K of (Fe 0.50 Pt 0.50 ) 78 Zr 4 B 18 to 1083 K of (Co 0.50 Pt 0.50 ) 78 Zr 4 B 18 . However, H c drastically decreases from 684 kA/m of (Fe 0.50 Pt 0.50 ) 78 Zr 4 B 18 to 12 kA/m of (Co 0.50 Pt 0.50 ) 78 Zr 4 B 18 , which has a single fcc structure in an as-quenched state. The decrease in H c with increasing Co content up to Fe:Co = 1:3 is caused by the decreasing of the magnetocrystalline anisotropy, which originates from the decreasing the long-range order parameter of the Llo phase.

The L 1 0 -FePt nanocrystalline phase is directly formed by rapidly quenching the (Fe 1− x Pt x ) 77−78 Zr 2−5 B 17−20 ( x = 0.45–0.50) alloy melt. The as-quenched alloys exhibit coercivity higher than 200 kA/m. The in situ X-ray diffractometry at high temperatures reveals that the quenched L 1 0 -FePt phase in the alloys is stable up to the melting temperature. The L 1 0 -FePt phase forms directly without allowing the formation of the disordered face-centered cubic phase during rapid solidification.

The hard magnetic properties and the structure of the melt-spun (Fe 0.55 Pt 0.45 )-(Nb, Ti)-B alloys have been investigated. The almost single L1 0 -FePt structure was directly formed in the wide compositional range of 2–10 at% Nb and 15–24 at% B, and 2–6 at% Ti and 3–20 at% B, respectively. The highest H c values are 588 kA/m for (Fe 0.55 Pt 0.45 ) 72 Nb 8 B 20 and 357 kA/m for (Fe 0.55 Pt 0.45 ) 83 Ti 2 B 15 , respectively. The rapidly quenching the melt is useful to realize the good hard magnetic properties through the grain size refinement of the near equi-atomic FePt alloys.

For studying molecular mechanisms regulating the fate of ethanol-treated hepatocytes, involvement of Fas in ethanol-induced apoptosis was examined in human liver adenocarcinoma (SK-Hep1) cells in which the function of Fas-associated death domain (FADD) protein was knocked down by transfection. In FADD-knocked down cells, while ethanol-induced increase in generation of reactive oxygen species (ROS) was unaffected, apoptosis was significantly suppressed, demonstrating the involvement of Fas in ethanol-induced hepatocyte apoptosis more directly than in the past reports. On the other hand, effects of mitogen-activated protein kinase (MAPK), which is well known to determine the fate of various cells, on ethanol-induced apoptosis have not been examined in SK-Hep1 cells. Of three major MAPKs, only p38 MAPK and JNK were found activated by 200 mM ethanol treatment. When cells were incubated with inhibitors of p38 MAPK and JNK, ethanol-induced apoptosis was decreased while ROS generation was unaffected, and examination of pro-apoptotic Bax and anti-apoptotic Bcl-2 levels showed decrease of the former and increase of the latter. We concluded that oxidative stress inflicted by ROS triggered Fas-mediated and mitochondria-mediated apoptotic pathways in ethanol-treated SK-Hep1 cells, and that p38 MAPK and JNK were promoting mitochondrial pathway, suggesting interaction between apoptosis and MAPK signaling systems.

The microstructure and the hard magnetic properties of the melt-spun (Fe1−xPtx)78Zr4B18 (x=0.385, 0.45, 0.50, and 0.55) nanocrystalline alloys have been investigated. The ordered L10-FePt phase with a grain size smaller than the critical single domain size is directly formed by rapidly quenching of the alloy melt with x=0.45 and 0.50. The alloys have a mixed microstructure composed of the L10-FePt and fcc phases, and exhibit high coercivity (Hc) of 649 and 688kA∕m, respectively, in an as-quenched state. On the other hand, the alloys with x=0.385 and 0.55 are found to composed of two fcc phases and fcc+L12-FePt3+L10-FePt, respectively, and exhibit Hc values lower than 70kA∕m in an as-quenched state. The rapidly quenching of the melt of the (Fe1−xPtx)78Zr4B18 alloys with x=0.45–0.50 is useful to realize the good hard magnetic properties through the grain size refinement of the near equiatomic FePt alloys.