Screening, identification and functional validation of Microcystin-LR direct binding target proteins based on thermal...

Microcystin-LR (MC-LR) is one of the most common harmful cyanobacterial toxin and poses a serious threat to human health and ecosystems. The accepted toxic effect of MC-LR is to inhibit its enzymatic activity by covalently binding to protein phosphatase 2A (PP2A). However, numerous researches have revealed that the toxic effects of MC-LR are not solely dependent on PP2A. To date, there have been no relevant reports of MC-LR binding to other exact targets to produce toxic effects, and there is an urgent need to decipher the potential direct targets of MC-LR. Thermal proteome profiling (TPP) is a novel technique for the identification of active small molecule target proteins based on the principle that protein-ligand binding can increase the thermal stability of proteins. For this purpose, we used the TPP technique in combination with SWATH-DIA mass spectrometry to systematically assess the changes in the thermal stability of the proteins, thus searching for potential direct-acting target proteins of MC-LR. The results showed that 129 proteins, including PP2A, were potential binding targets of MC-LR. Bioinformatics analysis of 129 proteins enriched for response to dopamine, proteasome complex, and NF-kappaB binding was consistent with previous MC-LR toxicity studies. MC-LR could directly bind to target proteins such as PSMD4, PSMB9, HDAC2, and MAPK1 by CETSA-Western blot and MST assay. It was further confirmed by functional validation that MC-LR may lead to inhibition of proteasome activity through binding to PSMD4/PSMB9, suggesting that the proteasome is one of the toxic targets of MC-LR. This study reveals the existence of multiple targets of MC-LR after entering the organism, which broadens the horizon and provides a valuable reference for the study of the toxicity mechanism of MC-LR.