Resumen de Study of the molecular mechanisms underlying liver diseases by mass spectrometry-based proteomics
Liver diseases cause approximately 2 million deaths per year worldwide and their incidence has increased over the last decade. Risk factors include excessive alcohol consumption, obesity, diabetes, ingestion of hepatotoxic substances such as aflatoxin, viral infections (viral hepatitis), and genetic determinants. Liver cancer is the sixth most prevalent and the third deadliest (second in men). The low survival rate (less than 20% after 5 years) is partly explained by late diagnosis, highlighting the need for new molecular biomarkers for early diagnosis. In this work, proteomic techniques based on mass spectrometry have been applied to the study of liver diseases such as cholestasis, liver cirrhosis, and hepatocellular carcinoma with the aim of understanding the underlying molecular mechanisms of these pathological processes, as well as discovering potential biomarkers for the diagnosis and prognosis of patients. Cholestasis is a disease characterized by the interruption of bile flow from hepatocytes to the small intestine. Its treatment, depending on the cause and severity of the disease, may include the administration of ursodeoxycholic acid, removal of gallstones obstructing the duct in cases of physical obstruction, or liver transplantation in severe cases. It is precisely in these latter cases where rapid and adequate intervention is essential for a favorable patient outcome. With the aim of developing tools for a more effective patient management, we conducted a study of the livers of patients with cholestasis of various etiologies, combining mass spectrometry and machine learning tools. In addition to identifying some of the cellular processes involved in the molecular pathogenesis of cholestasis, we have defined a panel of proteins using linear discriminant analysis (LDA) that allows for the stratification of patients according to the etiology of cholestasis with high precision (91%). This mathematical approach, based on the establishment of an importance criterion for the measured proteins in relation to their ability to discriminate the studied cases, represents an increasingly used analysis method to objectively define panels of biomolecules for patient stratification. Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a serious and rare liver disease that affects between 1 in 50,000 and 1 in 100,000 children. It is caused by mutations in the phosphatidylcholine transporter ABCB4 (MDR3), leading to intrahepatic accumulation of free bile acids and resulting in chronic liver damage. PFIC3 typically manifests at an early age, progresses rapidly, and has a poor prognosis. Currently, besides the palliative use of ursodeoxycholic acid, the only available treatment for this disease is liver transplantation, which poses a challenge, especially in young patients. To uncover the molecular basis of PFIC3 progression and propose new strategies for its treatment, we conducted a proteomic and phosphoproteomic analysis comparing liver samples from PFIC3 patients and controls. The results were validated in an MDR2-deficient PFIC3 mouse model. The functional interpretation of the proteomic analysis results indicates that essential cellular processes, such as inflammation, cell proliferation, cytoskeletal organization, and significant reconfiguration of intermediary metabolism, particularly glucose metabolism and onecarbon metabolism (OCM), are affected in PFIC3. OCM is a highly relevant metabolic cycle that connects intermediary metabolism with epigenetic regulation. This cycle has a tissue-specific configuration, and in the liver, some of the participating enzymes show a specific expression pattern, which is essential for maintaining the differentiated and quiescent state of hepatocytes. Considering its critical role in hepatocyte biology, we have developed a targeted proteomic method to monitor specifically OCM enzymes. Our results indicate that systematic determination of the levels of these proteins reveals a reprogramming of OCM in chronic liver damage processes such as cholestasis, which is further accentuated in situations of severe liver damage, losing metabolic capacity in hepatocellular carcinoma (HCC) and adopting a nonspecific profile similar to that of other tissues. These results point to OCM as an indicator of liver function and differentiation that could help to monitor patients with chronic liver diseases, facilitating early prognoses that could enable more effective interventions. According to these results, we believe that the metabolic alteration of OCM occurring in liver damage processes is a relevant factor in disease progression, and the administration of metabolites to modulate this pathway could represent a potential therapeutic option. Therefore, we have studied the effect of methylthioadenosine (MTA), an intermediate metabolite of this cycle, on an established hepatocellular carcinoma cell line, evaluating the response through the analysis of the changes induced in the proteome. Our results show that treatment with MTA may have a hepatoprotective role by reversing several tumour phenotypic characteristics, such as proliferation and reconfiguration of OCM. In conclusion, the application of state-of-the-art proteomic techniques has allowed us to establish some of the mechanisms involved in the progression of chronic liver diseases, including cholestasis, cirrhosis, and HCC. Based on this knowledge, we have demonstrated the relevance of OCM and developed an analytical method to monitor OCM enzymes, enabling assessment of liver function and hepatocyte differentiation status. Therefore, it can be highly useful in monitoring patients with chronic liver diseases. Finally, we have developed an LDA method that allows the definition of models to stratify patients with similar pathologies, such as different etiologies of cholestasis. Our results suggest the great potential of proteomics in the development of precision medicine
