RESUMO
The ubiquitin-proteasome system (UPS) and autophagy are two major intracellular degradative mechanisms that mediate the turnover of complementary repertoires of intracellular proteomes. Simultaneously activating both UPS and autophagy might provide a powerful strategy for the clearance of misfolded proteins. However, it is not clear whether UPS and autophagy can be controlled by a common regulatory mechanism. K48 deubiquitination by USP14 is known to inhibit UPS. Here we show that USP14 regulates autophagy by negatively controlling K63 ubiquitination of Beclin 1. Furthermore, we show that activation of USP14 by Akt-mediated phosphorylation provides a mechanism for Akt to negatively regulate autophagy by promoting K63 deubiquitination. Our study suggests that Akt-regulated USP14 activity modulates both proteasomal degradation and autophagy through controlling K48 and K63 ubiquitination, respectively. Therefore, regulation of USP14 provides a mechanism for Akt to control both proteasomal and autophagic degradation. We propose that inhibition of USP14 may provide a strategy to promote both UPS and autophagy for developing novel therapeutics targeting neurodegenerative diseases.
Assuntos
Autofagia/fisiologia , Proteína Beclina-1/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Proteína Oncogênica v-akt/metabolismo , Fosforilação , Ubiquitina Tiolesterase/genéticaRESUMO
The generation of functional hepatocytes independent of donor liver organs is of great therapeutic interest with regard to regenerative medicine and possible cures for liver disease. Induced hepatic differentiation has been achieved previously using embryonic stem cells or induced pluripotent stem cells. Particularly, hepatocytes generated from a patient's own induced pluripotent stem cells could theoretically avoid immunological rejection. However, the induction of hepatocytes from induced pluripotent stem cells is a complicated process that would probably be replaced with the arrival of improved technology. Overexpression of lineage-specific transcription factors directly converts terminally differentiated cells into some other lineages, including neurons, cardiomyocytes and blood progenitors; however, it remains unclear whether these lineage-converted cells could repair damaged tissues in vivo. Here we demonstrate the direct induction of functional hepatocyte-like (iHep) cells from mouse tail-tip fibroblasts by transduction of Gata4, Hnf1α and Foxa3, and inactivation of p19(Arf). iHep cells show typical epithelial morphology, express hepatic genes and acquire hepatocyte functions. Notably, transplanted iHep cells repopulate the livers of fumarylacetoacetate-hydrolase-deficient (Fah(-/-)) mice and rescue almost half of recipients from death by restoring liver functions. Our study provides a novel strategy to generate functional hepatocyte-like cells for the purpose of liver engineering and regenerative medicine.
Assuntos
Diferenciação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Proteínas de Ligação a DNA/deficiência , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Perfilação da Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 3-gama Nuclear de Hepatócito/genética , Fator 3-gama Nuclear de Hepatócito/metabolismo , Hepatócitos/fisiologia , Hepatócitos/transplante , Hidrolases/deficiência , Hidrolases/genética , Fígado/citologia , Fígado/enzimologia , Fígado/fisiologia , Fígado/fisiopatologia , Hepatopatias/enzimologia , Hepatopatias/patologia , Hepatopatias/fisiopatologia , Hepatopatias/terapia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Medicina Regenerativa/métodos , Taxa de Sobrevida , Cauda/citologia , Engenharia Tecidual/métodos , Transdução GenéticaRESUMO
Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina/metabolismo , Linhagem Celular , Humanos , FosforilaçãoRESUMO
Metabonomics has emerged as an important technology for exploring the underlying mechanisms of diseases and screening for biomarkers. In this investigation, to comprehensively assess metabolite changes in D-galactosamine (GalN)-induced liver injury in Chinese miniature pigs and to increase our understanding of physiological changes in normal and pathological states, we used ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) to analyze metabolites and identify biomarkers in serum. Blood samples were collected both from 18 h after GalN treatment group and control group pigs. We performed multivariate analyses on the metabolite profiles to identify potential biomarkers of acute liver injury, which were then confirmed by tandem MS. Based on "variable of importance in the project" (VIP) values and S-plots, four groups of biomarkers were identified--namely conjugated bile acids, lysophosphatidylcholines (LPCs), phosphatidylcholines (PCs) and fatty acid amides (FAAs)--that were present at significantly different levels in the control and GalN-induced groups. LPCs, PCs, and FAAs showed marked decreases in the GalN-treated group, whereas conjugated bile acids in the treated group showed considerable increases. Taken together, our results suggested that obvious metabolic disturbances occur during acute liver injury, which provided novel insights into the molecular mechanism(s) of D-galactosamine (GalN)-induced liver injury, and will facilitate future research and management of liver injury.
Assuntos
Ácidos e Sais Biliares/sangue , Doença Hepática Induzida por Substâncias e Drogas/sangue , Ácidos Graxos/sangue , Lisofosfatidilcolinas/sangue , Fosfatidilcolinas/sangue , Animais , Biomarcadores/sangue , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Masculino , Metabolômica/métodos , Análise Multivariada , Distribuição Aleatória , Espectrometria de Massas por Ionização por Electrospray/métodos , Suínos , Porco Miniatura , Espectrometria de Massas em Tandem/métodosRESUMO
Niacin has been demonstrated to activate a PI3K/Akt signaling cascade to prevent brain damage after stroke and UV-induced skin damage; however, the underlying molecular mechanisms for HCA2-induced Akt activation remain to be elucidated. Using CHO-K1 cells stably expressing HCA2 and A431 cells, a human epidermoid cell line with high levels of endogenous expression of functional HCA2 receptors, we first demonstrated that niacin induced a robust Akt phosphorylation at both Thr308 and Ser473 in a time-dependent fashion, with a maximal activation at 5 min and a subsequent reduction to baseline by 30 min through HCA2, and that the activation was significantly blocked by pertussis toxin. The HCA2-mediated activation of Akt was also significantly inhibited by the PKC inhibitors GF109203x and Go6983 in both cell lines, by the PDGFR-selective inhibitor tyrphostin A9 in CHO-HCA2 cells and by the MMP inhibitor GM6001 and EGFR-specific inhibitor AG1478 in A431 cells. These results suggest that the PKC pathway and PDGFR/EGFR transactivation pathway play important roles in HCA2-mediated Akt activation. Further investigation indicated that PI3K and the Gßγ subunit were likely to play an essential role in HCA2-induced Akt activation. Moreover, Immunobloting analyses using an antibody that recognizes p70S6K1 phosphorylated at Thr389 showed that niacin evoked p70S6K1 activation via the PI3K/Akt pathway. The results of our study provide new insight into the signaling pathways involved in HCA2 activation.