Structural characterization and anti-aging activity of a novel extracellular polysaccharide from fungus Phellinus sp. in a mammalian system.

Little is known about the chemical structure of purified extracellular polysaccharides from Phellinus sp., a fungal species with known medicinal properties. A combination of IR spectroscopy, methylation analysis and NMR were performed for the structural analysis of a purified extracellular polysaccharide derived from Phellinus sp. culture, denoted as SHP-1, along with an evaluation of the anti-aging effect in vivo of the polysaccharide supplementation. The structure of SHP-1 was established, with a backbone composed of →2,4)-α-d-glucopyranose-(1→ and →2)-ß-d-mannopyranose-(1→ and two terminal glucopyranose branches. Biochemical analysis from mammalian animal experiments demonstrated that SHP-1 possesses the ability to enhance antioxidant enzyme activities, such as catalase (CAT) and superoxide dismutase (SOD) activities, Trolox equivalent antioxidant capacity (TEAC) in serum of d-galactose-aged mice, while reducing lipofuscin levels, another indicator of cell aging, indicating a potential association with anti-aging activities in a dose dependent manner. This compound had a favourable influence on immune organ indices, and a marked amelioration ability of histopathological hepatic lesions such as necrosis, karyolysis and reduced inflammation and apoptosis in mouse hepatocytes. These results suggest that SHP-1 has strong antioxidant activities and a significant protective effect against oxidative stress or hepatotoxicity induced by d-galactose in mice and it could be developed as a food ingredient or a pharmaceutical to prevent many age-associated diseases such as major depressive disorder and hepatotoxicity. To our knowledge, this is the first report on the antioxidant effects of a novel purified exopolysaccharide derived from Phellinus sp.

Regulation of hERG and hEAG channels by Src and by SHP-1 tyrosine phosphatase via an ITIM region in the cyclic nucleotide binding domain.

Members of the EAG K(+) channel superfamily (EAG/Kv10.x, ERG/Kv11.x, ELK/Kv12.x subfamilies) are expressed in many cells and tissues. In particular, two prototypes, EAG1/Kv10.1/KCNH1 and ERG1/Kv11.1/KCNH2 contribute to both normal and pathological functions. Proliferation of numerous cancer cells depends on hEAG1, and in some cases, hERG. hERG is best known for contributing to the cardiac action potential, and for numerous channel mutations that underlie 'long-QT syndrome'. Many cells, particularly cancer cells, express Src-family tyrosine kinases and SHP tyrosine phosphatases; and an imbalance in tyrosine phosphorylation can lead to malignancies, autoimmune diseases, and inflammatory disorders. Ion channel contributions to cell functions are governed, to a large degree, by post-translational modulation, especially phosphorylation. However, almost nothing is known about roles of specific tyrosine kinases and phosphatases in regulating K(+) channels in the EAG superfamily. First, we show that tyrosine kinase inhibitor, PP1, and the selective Src inhibitory peptide, Src40-58, reduce the hERG current amplitude, without altering its voltage dependence or kinetics. PP1 similarly reduces the hEAG1 current. Surprisingly, an 'immuno-receptor tyrosine inhibitory motif' (ITIM) is present within the cyclic nucleotide binding domain of all EAG-superfamily members, and is conserved in the human, rat and mouse sequences. When tyrosine phosphorylated, this ITIM directly bound to and activated SHP-1 tyrosine phosphatase (PTP-1C/PTPN6/HCP); the first report that a portion of an ion channel is a binding site and activator of a tyrosine phosphatase. Both hERG and hEAG1 currents were decreased by applying active recombinant SHP-1, and increased by the inhibitory substrate-trapping SHP-1 mutant. Thus, hERG and hEAG1 currents are regulated by activated SHP-1, in a manner opposite to their regulation by Src. Given the widespread distribution of these channels, Src and SHP-1, this work has broad implications in cell signaling that controls survival, proliferation, differentiation, and other ERG1 and EAG1 functions in many cell types.

High NaCl-induced inhibition of PTG contributes to activation of NFAT5 through attenuation of the negative effect of SHP-1.

Activation of the transcription factor NFAT5 by high NaCl involves changes in phosphorylation. By siRNA screening, we previously found that protein targeting to glycogen (PTG), a regulatory subunit of protein phosphatase1 (PP1), contributes to regulation of high NaCl-induced NFAT5 transcriptional activity. The present study addresses the mechanism involved. We find that high NaCl-induced inhibition of PTG elevates NFAT5 activity by increasing NFAT5 transactivating activity, protein abundance, and nuclear localization. PTG acts via a catalytic subunit PP1γ. PTG associates physically with PP1γ, and NaCl reduces both this association and remaining PTG-associated PP1γ activity. High NaCl-induced phosphorylation of p38, ERK, and SHP-1 contributes to activation of NFAT5. Knockdown of PTG does not affect phosphorylation of p38 or ERK. However, PTG and PP1γ bind to SHP-1, and knockdown of either PTG or PP1γ increases high NaCl-induced phosphorylation of SHP-1-S591, which inhibits SHP-1. Mutation of SHP-1-S591 to alanine, which cannot be phosphorylated, increases inhibition of NFAT5 by SHP-1. Thus high NaCl reduces the stimulatory effect of PTG and PP1γ on SHP-1, which in turn reduces the inhibitory effect of SHP-1 on NFAT5. Our findings add to the known functions of PTG, which was previously recognized only for its glycogenic activity.