|Ph.D Student||Kalfon Limor|
|Subject||Molecular Mechanisms and Signaling Pathways Mediating the|
Neuroprotective Action of the Green Tea Polyphenol
(-)-Epigallocatechin 3-Gallate (EGCG)
|Department||Department of Medicine||Supervisor||Professor Emeritus Moussa Youdim|
|Full Thesis text|
The present study attempted to gain a further insight into the early signaling cascades involved in neuronal survival promoted by the major green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG). EGCG caused an acute down-regulation of Bad protein levels and a more pronounced reduction after 24 h in the human neuroblastoma cell line, SH-SY5Y. Co-treatment with EGCG and the protein synthesis inhibitor cycloheximide prominently shortened Bad half-life. Accordingly, the proteasome inhibitors, MG-132 and lactacystin blunted Bad down-regulation by EGCG, independently of ubiquitin conjugation. Also, the general protein kinase C (PKC) inhibitor GF109203X or the down-regulation of conventional and novel, but not atypical PKC isoforms abolished EGCG-induced Bad decline, indicating a PKC and proteasomal-dependent degradation of Bad. The serine 112 phosphorylation site on Bad, a target of PKC, or the PEST motif, identified as a recognition site for proteasomal degradation, did not play a role in the degradation of Bad by EGCG. Pull down assay with recombinant Bad followed by mass spectrometry analysis, revealed a number of Bad-interacting protein candidates whose association could promote its destabilization. The finding that Bad is a signaling mediator of EGCG activity at early stage, prompted us to study its effect on the axis mitochondria- proteasome employing the parkinsonism-inducing neurotoxin, 6-hydroxydopamine (6-OHDA), in the mouse embryonic substantia nigra-derived cell line (SN4741). EGCG attenuated the dissipation of the mitochondrial membrane potential induced by short-term exposure to 6-OHDA. Interestingly, inhibition of the PKC pathway blunted this protective effect, supporting the involvement of this signaling pathway in mitochondrial stabilization by EGCG. The effect of EGCG was even more prominent at the level of ATP, where EGCG almost completely prevented its massive intracellular storage depletion by the neurotoxin. In addition, EGCG significantly attenuated the acute decline in the chemotrypsin-like activity of the proteasome induced by exposure to 6-OHDA.
In order to gain an insight into the global protein expression changes occurring during the signaling phase of EGCG, the iTRAQ proteomics and mass spectrometry were implemented to identify differential protein expression. EGCG significantly affected specific biological classes related to mitochondrial inner membrane, chromatin assembly or disassembly, mRNA binding / processing, protein import into nucleus, calcium-binding EF-hand domains and ribosomal proteins.
In the present study we have identified a novel mechanism of EGCG action involving a rapid PKC and proteasomal-dependent degradation of Bad, accompanied by early elevations in selective components of the cell, probably contributing to its bioenergetic/neuroprotective and neuronal differentiation activity.