Quercetin and HSC70 coregulate the anti-inflammatory action of the ubiquitin-like protein MNSFß.

BACKGROUND: Quercetin is a flavonol that modifies many cellular processes. Monoclonal nonspecific suppressor factor ß is a member of the ubiquitin-like family of proteins that are involved in various biological processes. It has been demonstrated that quercetin regulates the effect of MNSFß on tumor necrosis factor-α secretion in lipopolysaccharide (LPS)-stimulated macrophages. This study found that quercetin and the heat shock protein HSC70 coregulate the action of MNSFß. METHODS AND RESULTS: Quercetin dose-dependently suppressed the LPS/interferon γ-induced nitric oxide production without cytotoxicity in the macrophage-like cell line Raw264.7. SiRNA knockdown experiments showed that quercetin inhibited the MNSFß and HSC70 siRNA-mediated enhancement of TNFα and the production of RANTES, a member of C-C chemokine superfamily, in LPS-stimulated Raw264.7 cells. Western blot analysis showed that quercetin and HSC70 regulated ERK1/2 activation and LPS-stimulated IκBα degradation by affecting the complex formation of MNSFß and the proapoptotic protein Bcl-G. Moreover, MNSFß is implicated in TLR4/MyD88 signaling but not in TLR3 signaling. CONCLUSIONS: HSC70 is an important chaperone that facilitates the stabilization of MNSFß. Quercetin may negatively control the function of MNSFß by regulating the action of the molecular chaperone HSC70. MNSFß mediates TLR4/Myd88 signaling but not TLR3 signaling.

Heat shock protein 60 negatively regulates the biological functions of ubiquitin-like protein MNSFß in macrophages.

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

Ubiquitin-like protein MNSFß noncovalently binds to molecular chaperone HSPA8 and regulates osteoclastogenesis.

MNSFß, a ubiquitin-like protein, covalently binds to various target proteins including proapoptotic Bcl-G. During the course of isolation of MNSFß-conjugating enzyme(s), we identified a novel target protein for MNSFß. MALDI-TOF MS fingerprinting revealed that the MNSFß-interacting protein is HSPA8 (heat shock 70-kDa protein 8). We observed that MNSFß noncovalently binds to HSPA8 in the presence of ATP in vitro. Double knockdown of MNSFß 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 MNSFß with HSPA8 may promote RANKL-induced osteoclastogenesis. This is the first report that MNSFß binds to a protein substrate via the noncovalent association and exerts biological effects.

Ubiquitin-like protein MNSFß covalently binds to cytosolic 10-formyltetrahydrofolate dehydrogenase and regulates thymocyte function.

MNSFß is a ubiquitously expressed member of the ubiquitin-like family that has been involved in various biological functions. Previous studies have demonstrated that MNSFß covalently binds to various target proteins including Bcl-G, a proapoptotic protein. In this study, we purified a 115 kDa MNSFß adduct from murine liver lysates by sequential chromatography on DEAE and anti-MNSFß IgG-conjugated Sepharose in the presence of ATP. MALDI-TOF MS fingerprinting revealed that this MNSFß adduct consists of an 8.5 kDa MNSFß and 10-formyltetrahydrofolate dehydrogenase (FDH), an abundant enzyme of folate metabolism. Interestingly, MNSFß preferably binds to cytosolic but not mitochondrial FDH. Fingerprinting analysis of the MNSFß adduct demonstrate that MNSFß conjugates to cytosolic FDH with a linkage between the C-terminal Gly74 and Lys72. The 115 kDa MNSFß/FDH complex was not expressed in any of the tissues examined, indicating that this adduct formation is not ubiquitous. We found that MNSFß/FDH complex formation was induced by dexamethasone in thymocytes. Double knockdown of MNSFß and FDH strongly reduced dexamethasone-induced apoptosis. Collectively, MNSFß/FDH complex formation may positively regulate apoptosis in thymocytes.