• MeSH
• genetická predispozice k nemoci MeSH
• genetický kód MeSH
• lidé MeSH
• mutace MeSH
• plicní hypertenze genetika   patologie   MeSH
• progrese nemoci MeSH
• Check Tag
• lidé MeSH

BACKGROUND: An emerging metabolic theory of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously demonstrated the existence of hyperproliferative, apoptosis-resistant, proinflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) that exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the tricarboxylic acid cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that in PH-Fibs microRNA-124 (miR-124) regulates PTBP1 (polypyrimidine tract binding protein 1) expression to control alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2, resulting in an increased PKM2/PKM1 ratio, which promotes glycolysis and proliferation even in aerobic environments. METHODS: Pulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects. PTBP1 gene knockdown was achieved via PTBP1-siRNA; restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and shikonin were used to manipulate PKM2 glycolytic function. Histone deacetylase inhibitors were used to treat cells. Metabolic products were determined by mass spectrometry-based metabolomics analyses, and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry. RESULTS: We detected an increased PKM2/PKM1 ratio in PH-Fibs compared with normal subjects. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation, and attenuated macrophage interleukin-1β expression. Furthermore, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, ie, lactate), rescued mitochondrial reprogramming, and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and shikonin or treatment with histone deacetylase inhibitors produced similar results. CONCLUSIONS: In PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative, and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH.

• MeSH
• alternativní sestřih MeSH
• antagomiry metabolismus   MeSH
• cévní endotel cytologie   MeSH
• fibroblasty cytologie   účinky léků   metabolismus   MeSH
• glykolýza MeSH
• heterogenní jaderné ribonukleoproteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• inhibitory histondeacetylas farmakologie   MeSH
• interleukin-1beta metabolismus   MeSH
• lidé MeSH
• makrofágy cytologie   imunologie   metabolismus   MeSH
• mikro RNA antagonisté a inhibitory   genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• naftochinony farmakologie   MeSH
• plicní hypertenze metabolismus   patologie   MeSH
• proliferace buněk MeSH
• protein - isoformy antagonisté a inhibitory   genetika   metabolismus   MeSH
• protein vázající polypyrimidinové úseky RNA antagonisté a inhibitory   genetika   metabolismus   MeSH
• pyruvátkinasa antagonisté a inhibitory   genetika   metabolismus   MeSH
• RNA interference MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Targeting ubiquitin E3 ligases is therapeutically attractive; however, the absence of an active-site pocket impedes computational approaches for identifying inhibitors. In a large, unbiased biochemical screen, we discover inhibitors that bind a cryptic cavity distant from the catalytic cysteine of the homologous to E6-associated protein C terminus domain (HECT) E3 ligase, SMAD ubiquitin regulatory factor 1 (SMURF1). Structural and biochemical analyses and engineered escape mutants revealed that these inhibitors restrict an essential catalytic motion by extending an α helix over a conserved glycine hinge. SMURF1 levels are increased in pulmonary arterial hypertension (PAH), a disease caused by mutation of bone morphogenetic protein receptor-2 (BMPR2). We demonstrated that SMURF1 inhibition prevented BMPR2 ubiquitylation, normalized bone morphogenetic protein (BMP) signaling, restored pulmonary vascular cell homeostasis, and reversed pathology in established experimental PAH. Leveraging this deep mechanistic understanding, we undertook an in silico machine-learning-based screen to identify inhibitors of the prototypic HECT E6AP and confirmed glycine-hinge-dependent allosteric activity in vitro. Inhibiting HECTs and other glycine-hinge proteins opens a new druggable space.

• MeSH
• alosterická regulace účinky léků   MeSH
• lidé MeSH
• myši MeSH
• plicní arteriální hypertenze farmakoterapie   MeSH
• receptory morfogenetických kostních proteinů typu II MeSH
• signální transdukce účinky léků   MeSH
• ubikvitinace účinky léků   MeSH
• ubikvitinligasy * antagonisté a inhibitory   metabolismus   chemie   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• vnitřní lékařství MeSH
• Publikační typ
• učebnice MeSH

• Konspekt
• Lékařské vědy. Lékařství
• NLK Obory
• vnitřní lékařství
• NLK Publikační typ
• kolektivní monografie