Abstract
Several ellagitannins inhibited the activity of protein phosphatase-1 (PP1) and -2 A (PP2A) catalytic subunits (PP1c and PP2Ac) with preferential suppression of PP1c over PP2Ac. The inhibitory potency for PP1c followed the order of tellimagrandin I > mahtabin A > praecoxin B > 1.2-Di-O-galloyl-4.6-(S)-HHDP-β-D-glucopyranose > pedunculagin with IC50 values ranging from 0.20 µM to 2.47 µM. The interaction of PP1c and tellimagrandin I was assessed by NMR saturation transfer difference, surface plasmon resonance, isothermal titration calorimetry, and microscale thermophoresis based binding techniques. Tellimagrandin I suppressed viability and phosphatase activity of HeLa cells, while mahtabin A was without effect. Conversely, mahtabin A increased the phosphorylation level of SNAP-25Thr138 and suppressed exocytosis of cortical synaptosomes, whereas tellimagrandin I was without influence. Our results establish ellagitannins as partially selective inhibitors of PP1 and indicate that these polyphenols may act distinctly in cellular systems depending on their membrane permeability and/or their actions on cell membranes.
Highlights
It is well accepted that the phosphorylation of proteins is an important regulatory device in many cellular processes and it is regulated by the phosphorylating protein kinases but the dephosphorylating protein phosphatases as well[1]
The ellagitannins (Figure 1) assayed on the activity of PP1c or PP2Ac in this study are similar to PGG in composition, but they differ structurally in two important aspects: (i) all of them include hexahydroxydiphenoyl unit linked in various positions, except for pedunculagin which includes two of these linkages; (ii) tellimagrandin I, pedunculagin, and praecoxin B have free glycosidic hydroxyls, while GHG has an unmodified hydroxyl at position 3 of the glucopyranose ring
We determined the activity of PP1 and PP2A in the lysates using I2 to inhibit PP1, and low concentration of okadaic acid (OA) (2 nM) to suppress PP2A in order to determine the contribution of PP1 and PP2A to the phosphatase activity of lysates (Figure 2(E))
Summary
It is well accepted that the phosphorylation of proteins is an important regulatory device in many cellular processes and it is regulated by the phosphorylating protein kinases but the dephosphorylating protein phosphatases as well[1]. Serious efforts have been made to distinguish between PP1 and PP2A in biochemical and even more importantly in cellular assays in order to establish their physiological roles by targeting the catalytic centre and the substrate binding regions of PP1c or PP2Ac with inhibitory compounds. In this regard, a number of naturally occurring toxins (okadaic acid, calyculin A, tautomycin, microcystin, etc) have been identified as potent inhibitors of PP1 and PP2A5. A recent publication[10] has shown that conversion of microcystin by chemical methods leads to a series of analogues, which have higher selectivity toward PP2A than PP1
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