Computational Insights Into the Mechanism of EGCG's Binding and Inhibition of the TDP-43 Aggregation.

Misfolding and aggregation of TAR DNA-binding protein, TDP-43, is linked to devastating proteinopathies such as ALS. Therefore, targeting TDP-43's aggregation is significant for therapeutics. Recently, green tea polyphenol, EGCG, was observed to promote non-toxic TDP-43 oligomer formation disallowing TDP-43 aggregation. Here, we investigated if the anti-aggregation effect of EGCG is mediated via EGCG's binding to TDP-43. In silico molecular docking and molecular dynamics (MD) simulation suggest a strong binding of EGCG with TDP-43's aggregation-prone C-terminal domain (CTD). Three replicas, each having 800 ns MD simulation of the EGCG-TDP-43-CTD complex, yielded a high negative binding free energy (ΔG) inferring a stable complex formation. Simulation snapshots show that EGCG forms close and long-lasting contacts with TDP-43's Phe-313 and Ala-341 residues, which were previously identified for monomer recruitment in CTD's aggregation. Notably, stable physical interactions between TDP-43 and EGCG were also detected in vitro using TTC staining and isothermal titration calorimetry which revealed a high-affinity binding site of EGCG on TDP-43 (Kd, 7.8 µM; ΔG, -6.9 kcal/mol). Additionally, TDP-43 co-incubated with EGCG was non-cytotoxic when added to HEK293 cells. In summary, EGCG's binding to TDP-43 and blocking of residues important for aggregation can be a possible mechanism of its anti-aggregation effects on TDP-43.

In Vitro Interaction of a C-Terminal Fragment of TDP-43 Protein with Human Serum Albumin Modulates Its Aggregation.

An increased level of naturally occurring anti-TDP-43 antibodies was observed in the serum and cerebrospinal fluid (CSF) of amyotrophic lateral sclerosis patients. Human serum albumin (HSA), the most abundant protein in blood plasma and CSF, is found to interact with pathological proteins like Aß and α-synuclein. Therefore, we examined the effect on the in vitro aggregation of a C-terminal fragment of TDP-43 in the presence of HSA. We found that the lag phase in TDP-432C aggregation is abrogated in the presence of HSA, but there is an overall decreased aggregation as examined by thioflavin-T fluorescence spectroscopy and microscopy. An early onset of TDP-432C oligomer formation in the presence of HSA was observed using atomic force microscopy and transmission electron microscopy. Also, a known chemical inhibitor of TDP-432Caggregation, AIM4, abolishes the HSA-induced early formation of TDP-432C oligomers. Notably, the aggregates of TDP-432C formed in the presence of HSA are more stable against sarkosyl detergent. Using affinity copurification, we observed that HSA can bind to TDP-432C, and biolayer interferometry further supported their physical interaction and suggested the binding affinity to be in sub-micromolar range. Taken together, the data support that HSA can interact with TDP-432C in vitro and affect its aggregation.