Decoding the Cellular Response to Methylthioadenosine Uncovers Novel Targets for Chronic Liver Disease

Progressive familial intrahepatic cholestasis type 3 (PFIC3), caused by MDR3 deficiency (MDR2 in mice), is a lethal pediatric liver disease characterized by early-onset persistent cholestasis, progressive fibrosis, cirrhosis, and eventual liver failure. MDR3 is a phosphatidylcholine transporter located in the canalicular membrane of hepatocytes, and its deficiency leads to the accumulation of free bile acids, which damage epithelial cells due to their detergent-like effect. Therapeutic options remain limited, highlighting the need to identify molecular drivers of disease progression and novel pharmacological strategies. To investigate the molecular alterations associated with this deficiency, our laboratory performed functional proteomic analyses in patient samples and murine hepatic organoids, revealing consistent reprogramming of one-carbon metabolism (OCM). Based on these findings, we explored the therapeutic potential and molecular mechanisms of 5'-methylthioadenosine (MTA), a metabolite involved in this pathway. Using hepatocellular carcinoma cells (PLCs), we combined quantitative shotgun proteomics with the proteome integral solubility alteration (PISA) assay to characterize functional responses and identify direct protein targets of MTA. To reinforce the physiological relevance of our findings, we extended the PISA analysis to liver protein extracts from the Mdr2^-/- mouse model of chronic cholestatic injury, enabling validation of candidate target interactions observed in cellular extracts. Proteomic profiling revealed that MTA induces significant alterations in cell division processes, epigenetic regulatory networks, and energy metabolism, thereby promoting an adaptive phenotype characterized by metabolic plasticity, microenvironmental reorganization, and immunomodulation. These findings provide a robust molecular framework for understanding the hepatic response to MTA and shed new light on the mechanisms that may underlie its beneficial effects. In addition, they provide a valuable platform for further investigation of the therapeutic potential of this compound and its prospective translation into clinical practice.