Inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS) is a dominant negative transcription factor that represses hypoxia-inducible factor 1 (HIF-1) activity. In this study, we show that IPAS also functions as a pro-apoptotic protein through binding to pro-survival Bcl-2 family members. In a previous paper, we reported that NF-jB-dependent IPAS induction by cobalt chloride repressed the hypoxic response in PC12 cells. We found that prolonged incubation under the same conditions caused apoptosis in PC12 cells. Repression of IPAS induction protected cells from apoptosis. Furthermore, knockdown of IPAS recovered cell viability. EGFP-IPAS protein was localized in both the nucleus and the cytoplasm, with a large fraction associated with mitochondria. Mitochondrial IPAS induced mitochondria depolarization and caspase-3 activation. Immunoprecipitation assays revealed that IPAS is associated with Bcl-x L , Bcl-w and Mcl-1. The association of IPAS with Bcl-x L was also observed in living cells by the FLIM-based FRET analysis, indicating direct binding between the two proteins. IPAS contributed to dysfunction of Bcl-x L by inhibiting the interaction of Bcl-x L with Bax. These results demonstrate that IPAS functions as a dual function protein involved in transcription repression and apoptosis.
Tricholoma matsutake, a high-class edible mushroom in Japan, has been reported to have excellent biological activities, but difficulty in cultivating the fruit bodies and limited bulk availability have restricted detailed studies. We have developed a method of culturing in tanks, enabling the bulk supply of the mycelia. The preparation (CM6271) exerts modulative effects on the immune competence of mice and rats. In this study, a sodium hydroxide extract of CM6271 was defatted followed by fractionation with a combination of ion exchange chromatography and gel filtration in order to identify the components involved in the expression of the activity, and a single peak fraction (MPG-1) was obtained with reversed phase chromatography. MPG-1 was a glycoprotein (sugar:protein ratio, 94.3:5.7) with a relative molecular mass of 360 kDa, and the sugar moiety contained about 90% glucose. NMR spectra and methylation analysis revealed that the alpha-1,4-linkage was the predominant glucan linkage with alpha-1,6- and alpha-1,2-linkages in the minority. The amino acid composition in the protein moiety was rich in glutamine, alanine, asparagine, leucine, glycine, valine, serine, threonine, isoleucine, and proline. MPG-1 was resistant to degradation with amylase or protease. The oral administration of MPG-1 promoted, in a dose-dependent manner, the recovery of the mouse natural killer cell activity and serum IL-12 level that had been reduced by the loading of restraint stress. The dose of MPG-1 (25 mg/kg) required for the expression of the effect decreases to 1/12 of that of CM6271 (300 mg/kg). Furthermore, MPG-1 formed a complex with TGF-beta1 in vitro, modulating the biological activity of TGF-beta1 by binding to its active form. These results indicate that the mycelium of T. matsutake contains a novel alpha-glucan-protein complex with immunomodulatory activities.
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