In silico and in vitro studies suggest epigallocatechin gallate (EGCG), a polyphenol in green tea, can bind and modulate the aggregation and cytotoxicity of the full-length TDP-43 protein implicated in TDP-43 proteinopathies.

Abstract

Misfolding and aggregation of TDP-43 protein are implicated in several proteinopathies like ALS and FTLD. Extracellular TDP-43 is also proposed to propagate in a prion-like pathogenic manner to the neighbouring cells. Here, using turbidity and sedimentation assay, we show that a polyphenol in green tea, epigallocatechin gallate (EGCG), can inhibit the in vitro aggregation of the full-length TDP-43 protein. Furthermore, Alexa-Fluor-labelled TDP-43 protein failed to show aggregates in the presence of EGCG in fluorescence microscopy. Also, AFM imaging revealed that EGCG co-incubation with TDP-43 allows formation of only small oligomers in contrast to the larger TDP-43 aggregates formed otherwise. A physical binding of EGCG with TDP-43 was observed using triphenyl tetrazolium chloride (TTC) staining and isothermal titration calorimetry (ITC). ITC also revealed a high-affinity binding site for EGCG on TDP-43 with a Kd value of 7.8 {micro}M and a binding free energy of -6.9 kcal/mol. Furthermore, in silico molecular docking and molecular dynamic simulation (MDS) studies using different available structures of the N-terminal, RRM1-2 and C-terminal domains of TDP-43, predicted a preferable and stable binding of EGCG to the structure of the aggregation prone C-terminal domain (CTD) (PDB ID:7KWZ). Also, EGCG complexed with CTD of TDP-43 yielded a negative {Delta}G value of -20.29 kcal/mol using MM-PBSA analysis of the MDS data thereby further suggesting a stable complex formation. Also, in MDS, EGCG interacted with the amino acids Phe-313 and Ala-341 of TDP-43, which were previously projected to be important for the recruitment of monomers for the amyloid formation by CTD, thereby suggesting a possible mechanism of EGCGs inhibition of the TDP-43 aggregation. Notably, while the in vitro-made aggregates of full-length TDP-43 caused mild cytotoxicity to the HEK293 cells, the small oligomers of TDP-43 formed in presence of EGCG did not. In totality, EGCG can in vitro interact with TDP-43 and inhibit its aggregation, possibly via interaction with the amyloidogenic domain, thereby preventing it from assuming cytotoxic conformations. As EGCG is a natural molecule, it could be relevant to the therapeutic quest against the TDP-43 proteinopathies.