Resveratrol inhibits phorbol ester-induced membrane translocation of presynaptic Munc13-1

Abstract

Background Resveratrol (1) is a naturally occurring polyphenol that has been implicated in neuroprotection. One of resveratrol’s several biological targets is Ca2+-sensitive protein kinase C alpha (PKCα). Resveratrol inhibits PKCα by binding to its activator-binding C1 domain. Munc13-1 is a C1 domain-containing Ca2+-sensitive SNARE complex protein essential for vesicle priming and neurotransmitter release. Methods To test if resveratrol could also bind and inhibit Munc13-1, we studied the interaction of resveratrol and its derivatives, (E)-1,3-dimethoxy-5-(4-methoxystyryl)benzene, (E)-5,5’-(ethene-1,2-diyl)bis(benzene-1,2,3-triol), (E)-1,2-bis(3,4,5-trimethoxyphenyl)ethane, and (E)-5-(4-(hexadecyloxy)-3,5-dihydroxystyryl)benzene-1,2,3-triol with Munc13-1 by studying its membrane translocation from cytosol to plasma membrane in HT22 cells and primary hippocampal neurons. Results Resveratrol, but not the derivatives inhibited phorbol ester-induced Munc13-1 translocation from cytosol to membrane in HT22 cells and primary hippocampal neurons, as evidenced by immunoblot analysis and confocal microscopy. Resveratrol did not show any effect on Munc13-1H567K, a mutant which is not sensitive to phorbol ester. Binding studies with Munc13-1 C1 indicated that resveratrol competes with phorbol ester for the binding site. Molecular docking and dynamics studies suggested that hydroxyl groups of resveratrol interact with phorbol-ester binding residues in the binding pocket. Conclusions and significance This study characterizes Munc13-1 as a target of resveratrol and highlights the importance of dietary polyphenol in the management of neurodegenerative diseases.

Probing the Diacylglycerol Binding Site of Presynaptic Munc13-1

“…The docking site is similar to those of our previous docking studies for resveratrol and bryostatin-1. 58,59 However, in PKCδ C1B, the phorbol 13-acetate formed three hydrogen bonds with Thr-242 and Leu-251 that are located deep inside the binding pocket (Figure 2B). The phorbol 13-acetate binding sites of Munc13-1 C1 and PKCδ C1B (PDB entry 1PTQ) are slightly different because of the different orientation of Trp-588.…”

“…The size of the plasma membrane was defined as 300 nm from the outer edge of the cell. The ratios of the mean intensity of the plasma membrane to the whole cells are presented as described previously. , …”

“…The ratios of the mean intensity of the plasma membrane to the whole cells are presented as described previously. 52,53 Expression and Purification of N 15 -Labeled Munc13-1 C1 and Its Mutants for the NMR Study. The pGEX-2TK vector (GE Healthcare Bio-Sciences, Pittsburgh, PA) was used to express the GST-conjugated C1 domain of Munc13-1.…”

Munc13 Is a Molecular Target of Bryostatin 1

“…The mean membrane (membrane size was defined as 300 nm from the outer edge) and whole cell intensities of individual images were measured. The ratios between the mean fluorescence intensity of the membrane and whole cells are presented as described previously. , …”

Protein Kinase C Isozymes and Autophagy during Neurodegenerative Disease Progression

“…Resveratrol binding to the C1 domain of PKCα results in PKC inactivation. As PKCα, Munc13-1, one of the presynaptic proteins regulating neurotransmitter release, has a resveratrol binding region on C1 domain [184]. Both bindings of resveratrol on PKCα and Munc13-1 inhibits neurotoxicity.…”