RESUMO
BACKGROUND AND AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of symptomatic biliary cysts. Current surgical and pharmacological approaches are ineffective, and liver transplantation represents the only curative option. Ursodeoxycholic acid (UDCA) and histone deacetylase 6 inhibitors (HDAC6is) have arisen as promising therapeutic strategies, but with partial benefits. APPROACH AND RESULTS: Here, we tested an approach based on the design, synthesis, and validation of a family of UDCA synthetic conjugates with selective HDAC6i capacity (UDCA-HDAC6i). Four UDCA-HDAC6i conjugates presented selective HDAC6i activity, UDCA-HDAC6i #1 being the most promising candidate. UDCA orientation within the UDCA-HDAC6i structure was determinant for HDAC6i activity and selectivity. Treatment of polycystic rats with UDCA-HDAC6i #1 reduced their hepatomegaly and cystogenesis, increased UDCA concentration, and inhibited HDAC6 activity in liver. In cystic cholangiocytes UDCA-HDAC6i #1 restored primary cilium length and exhibited potent antiproliferative activity. UDCA-HDAC6i #1 was actively transported into cells through BA and organic cation transporters. CONCLUSIONS: These UDCA-HDAC6i conjugates open a therapeutic avenue for PLDs.
Assuntos
Apoptose , Cistos/tratamento farmacológico , Hepatopatias/tratamento farmacológico , Fígado/patologia , Medicamentos Sintéticos/farmacologia , Ácido Ursodesoxicólico/farmacologia , Animais , Ácidos e Sais Biliares/metabolismo , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Proliferação de Células/efeitos dos fármacos , Cistos/metabolismo , Cistos/patologia , Modelos Animais de Doenças , Desacetilase 6 de Histona/antagonistas & inibidores , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatopatias/metabolismo , Hepatopatias/patologia , Distribuição Aleatória , Ratos , Ácido Ursodesoxicólico/uso terapêuticoRESUMO
BACKGROUND & AIMS: Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple fluid-filled biliary cysts. Most PLD-causative genes participate in protein biogenesis and/or transport. Post-translational modifications (PTMs) are implicated in protein stability, localization and activity, contributing to human pathobiology; however, their role in PLD is unknown. Herein, we aimed to unveil the role of protein SUMOylation in PLD and its potential therapeutic targeting. METHODS: Levels and functional effects of SUMOylation, along with response to S-adenosylmethionine (SAMe, inhibitor of the SUMOylation enzyme UBC9) and/or short-hairpin RNAs (shRNAs) against UBE2I (UBC9), were evaluated in vitro, in vivo and/or in patients with PLD. SUMOylated proteins were determined by immunoprecipitation and proteomic analyses by mass spectrometry. RESULTS: Most SUMOylation-related genes were found overexpressed (mRNA) in polycystic human and rat liver tissue, as well as in cystic cholangiocytes in culture compared to controls. Increased SUMOylated protein levels were also observed in cystic human cholangiocytes in culture, which decreased after SAMe administration. Chronic treatment of polycystic (PCK: Pkdh1-mut) rats with SAMe halted hepatic cystogenesis and fibrosis, and reduced liver/body weight ratio and liver volume. In vitro, both SAMe and shRNA-mediated UBE2I knockdown increased apoptosis and reduced cell proliferation of cystic cholangiocytes. High-throughput proteomic analysis of SUMO1-immunoprecipitated proteins in cystic cholangiocytes identified candidates involved in protein biogenesis, ciliogenesis and proteasome degradation. Accordingly, SAMe hampered proteasome hyperactivity in cystic cholangiocytes, leading to activation of the unfolded protein response and stress-related apoptosis. CONCLUSIONS: Cystic cholangiocytes exhibit increased SUMOylation of proteins involved in cell survival and proliferation, thus promoting hepatic cystogenesis. Inhibition of protein SUMOylation with SAMe halts PLD, representing a novel therapeutic strategy. LAY SUMMARY: Protein SUMOylation is a dynamic post-translational event implicated in numerous cellular processes. This study revealed dysregulated protein SUMOylation in polycystic liver disease, which promotes hepatic cystogenesis. Administration of S-adenosylmethionine (SAMe), a natural UBC9-dependent SUMOylation inhibitor, halted polycystic liver disease in experimental models, thus representing a potential therapeutic agent for patients.
Assuntos
Cistos , Hepatopatias , RNA Interferente Pequeno/farmacologia , S-Adenosilmetionina/farmacologia , Sumoilação/efeitos dos fármacos , Enzimas de Conjugação de Ubiquitina , Animais , Apoptose/efeitos dos fármacos , Ductos Biliares/efeitos dos fármacos , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Proliferação de Células/efeitos dos fármacos , Cistos/metabolismo , Cistos/patologia , Cistos/terapia , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes/métodos , Humanos , Hepatopatias/metabolismo , Hepatopatias/patologia , Hepatopatias/terapia , Modelos Teóricos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
PURPOSE OF REVIEW: This review provides an outline of the most recent insights and significant discoveries regarding the genetic mechanisms involved in polycystic liver disease. RECENT FINDINGS: Polycystic liver disease includes a heterogeneous group of genetic disorders characterized by multiple hepatic cysts. Isolated liver cysts are caused by mutations in Protein Kinase C Substrate 80K-H (PRKCSH), SEC63, and LDL Receptor Related Protein 5 (LRP5), whereas Polycystic Kidney Disease (PKD)1, PKD2, and PKHD1 mutations cause kidney cysts often accompanied by liver cysts. Glucosidase II Alpha Subunit (GANAB) has been reported to cause both phenotypes. These mutations, together with the newly identified ones in SEC61B and Alpha-1,3-Glucosyltransferase (ALG8), can be found in â¼50% of patients with isolated polycystic liver disease. Somatic second hit-mutations are hypothesized as driving force leading to cystogenesis. Subsequently, loss of heterozygosity in the cystic tissue aggravates disease progression. All genetic mutations lead to reduced levels of functional polycystin-1. This ciliary protein is therefore considered to be the central factor in the development and severity of liver cysts. SUMMARY: Recent advances of the genetic complexity leading to hepatic cystogenesis provide novel candidate genes and important mechanistic insights with polycystin-1 as a common denominator.
Assuntos
Cistos/genética , Hepatopatias/genética , Animais , Humanos , Mutação , Peptídeos/metabolismoRESUMO
BACKGROUND: Polycystic liver diseases (PLDs) are genetic inherited disorders characterized by the progressive growth of numerous intrahepatic biliary cysts, which are the main cause of morbidity. Previous studies revealed that cystic cholangiocytes are characterized by endoplasmic reticulum stress and aberrant posttranslational modification (PTM) of proteins, in particular hyper-SUMOylation, that promote PLD pathobiology. Protein NEDDylation is a newly characterized PTM that modulates a plethora of biological processes and its dysregulation is associated with the development and progression of several human diseases. However, the role of NEDDylation in PLD remains elusive. OBJECTIVE: To explore the role of protein NEDDylation in PLD and its potential therapeutic regulatory value. METHODS: Levels and functional effects of NEDDylation, including response to Pevonedistat (first-in-class selective inhibitor of the NEDDylation E1 enzyme NAE), were assessed in vitro, in vivo, and/or in patients with PLD. NEDDylated protein levels in normal and cystic human cholangiocytes were assessed by immunoprecipitation, and the proteomic profile was further analyzed by mass spectrometry. RESULTS AND CONCLUSION: The genes involved in the NEDDylation pathway were found overexpressed (mRNA) in polycystic human and rat liver tissue, as well as in cystic cholangiocytes in culture, compared to controls. Elevated levels of NEDDylated proteins were further confirmed in cystic cholangiocytes in vitro, which diminished under Pevonedistat incubation. Pevonedistat promoted apoptotic cell death and reduced proliferation in cystic cholangiocytes in vitro. Comparative proteomic profiling of NEDD8-immunoprecipitated proteins between normal and cystic cholangiocytes in culture reported candidate proteins involved in cystogenesis, mostly associated with protein biogenesis and quality control. All these data indicate that cystic cholangiocytes display increased protein NEDDylation, contributing to cell survival and proliferation, ultimately supporting hepatic cystogenesis. Targeting of protein hyper-NEDDylation in cystic cholangiocytes inhibits cystogenesis in experimental models, representing a novel therapeutic opportunity in PLD.