Pulmonary hypertension (PH) is a heterogeneous and life-threatening cardiopulmonary disorder in which mitochondrial dysfunction is believed to drive pathogenesis, although the underlying mechanisms remain unclear. To determine if abnormal SIRT3 (sirtuin 3) activity is related to mitochondrial dysfunction in adventitial fibroblasts from patients with idiopathic pulmonary arterial hypertension (IPAH) and hypoxic PH calves (PH-Fibs) and whether SIRT3 could be a potential therapeutic target to improve mitochondrial function, SIRT3 concentrations in control fibroblasts, PH-Fibs, and lung tissues were determined using quantitative real-time PCR and western blot. SIRT3 deacetylase activity in cells and lung tissues was determined using western blot, immunohistochemistry staining, and immunoprecipitation. Glycolysis and mitochondrial function in fibroblasts were measured using respiratory analysis and fluorescence-lifetime imaging microscopy. The effects of restoring SIRT3 activity (by overexpression of SIRT3 with plasmid, activation SIRT3 with honokiol, and supplementation with the SIRT3 cofactor nicotinamide adenine dinucleotide [NAD+]) on mitochondrial protein acetylation, mitochondrial function, cell proliferation, and gene expression in PH-Fibs were also investigated. We found that SIRT3 concentrations were decreased in PH-Fibs and PH lung tissues, and its cofactor, NAD+, was also decreased in PH-Fibs. Increased acetylation in overall mitochondrial proteins and SIRT3-specific targets (MPC1 [mitochondrial pyruvate carrier 1] and MnSOD2 [mitochondrial superoxide dismutase]), as well as decreased MnSOD2 activity, was identified in PH-Fibs and PH lung tissues. Normalization of SIRT3 activity, by increasing its expression with plasmid or with honokiol and supplementation with its cofactor NAD+, reduced mitochondrial protein acetylation, improved mitochondrial function, inhibited proliferation, and induced apoptosis in PH-Fibs. Thus, our study demonstrated that restoration of SIRT3 activity in PH-Fibs can reduce mitochondrial protein acetylation and restore mitochondrial function and PH-Fib phenotype in PH.

• Klíčová slova
• SIRT3, honokiol, mitochondria, nicotinamide adenine dinucleotide, pulmonary hypertension,
• MeSH
• fibroblasty metabolismus   MeSH
• lidé MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• NAD metabolismus   MeSH
• plicní hypertenze * patologie   MeSH
• sirtuin 3 * genetika   metabolismus   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• honokiol MeSH Prohlížeč
• mitochondriální proteiny MeSH
• NAD MeSH
• SIRT3 protein, human MeSH Prohlížeč
• sirtuin 3 * MeSH

Wild type mitochondrial isocitrate dehydrogenase (IDH2) was previously reported to produce oncometabolite 2-hydroxyglutarate (2HG). Besides, mitochondrial deacetylase SIRT3 has been shown to regulate the oxidative function of IDH2. However, regulation of 2HG formation by SIRT3-mediated deacetylation was not investigated yet. We aimed to study mitochondrial IDH2 function in response to acetylation and deacetylation, and focus specifically on 2HG production by IDH2. We used acetylation surrogate mutant of IDH2 K413Q and assayed enzyme kinetics of oxidative decarboxylation of isocitrate, 2HG production by the enzyme, and 2HG production in cells. The purified IDH2 K413Q exhibited lower oxidative reaction rates than IDH2 WT. 2HG production by IDH2 K413Q was largely diminished at the enzymatic and cellular level, and knockdown of SIRT3 also inhibited 2HG production by IDH2. Contrary, the expression of putative mitochondrial acetylase GCN5L likely does not target IDH2. Using mass spectroscopy, we further identified lysine residues within IDH2, which are the substrates of SIRT3. In summary, we demonstrate that 2HG levels arise from non-mutant IDH2 reductive function and decrease with increasing acetylation level. The newly identified lysine residues might apply in regulation of IDH2 function in response to metabolic perturbations occurring in cancer cells, such as glucose-free conditions.

• MeSH
• acetylace MeSH
• glutaráty metabolismus   MeSH
• isocitrátdehydrogenasa genetika   metabolismus   MeSH
• isocitráty chemie   MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• nádorové buněčné linie MeSH
• NADP metabolismus   MeSH
• oxidace-redukce MeSH
• proteiny nervové tkáně metabolismus   MeSH
• sirtuin 3 metabolismus   MeSH
• umlčování genů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alpha-hydroxyglutarate MeSH Prohlížeč
• BLOC1S1 protein, human MeSH Prohlížeč
• glutaráty MeSH
• IDH2 protein, human MeSH Prohlížeč
• isocitrátdehydrogenasa MeSH
• isocitráty MeSH
• isocitric acid MeSH Prohlížeč
• NADP MeSH
• proteiny nervové tkáně MeSH
• SIRT3 protein, human MeSH Prohlížeč
• sirtuin 3 MeSH

Nicotinamide phosphoribosyltransferase (NAMPT) is located in both the nucleus and cytoplasm and has multiple biological functions including catalyzing the rate-limiting step in NAD synthesis. Moreover, up-regulated NAMPT expression has been observed in many cancers. However, the determinants and regulation of NAMPT's nuclear transport are not known. Here, we constructed a GFP-NAMPT fusion protein to study NAMPT's subcellular trafficking. We observed that in unsynchronized 3T3-L1 preadipocytes, 25% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 62% had higher GFP-NAMPT fluorescence in the nucleus. In HepG2 hepatocytes, 6% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 84% had higher GFP-NAMPT fluorescence in the nucleus. In both 3T3-L1 and HepG2 cells, GFP-NAMPT was excluded from the nucleus immediately after mitosis and migrated back into it as the cell cycle progressed. In HepG2 cells, endogenous, untagged NAMPT displayed similar changes with the cell cycle, and in nonmitotic cells, GFP-NAMPT accumulated in the nucleus. Similarly, genotoxic, oxidative, or dicarbonyl stress also caused nuclear NAMPT localization. These interventions also increased poly(ADP-ribosyl) polymerase and sirtuin activity, suggesting an increased cellular demand for NAD. We identified a nuclear localization signal in NAMPT and amino acid substitution in this sequence (424RSKK to ASGA), which did not affect its enzymatic activity, blocked nuclear NAMPT transport, slowed cell growth, and increased histone H3 acetylation. These results suggest that NAMPT is transported into the nucleus where it presumably increases NAD synthesis required for cell proliferation. We conclude that specific inhibition of NAMPT transport into the nucleus might be a potential avenue for managing cancer.

• Klíčová slova
• GFP fusion, NAMPT, cancer, epigenetics, nicotinamide adenine dinucleotide (NAD), nuclear localization, pre–B cell colony enhancing factor (PBEF), sirtuin, visfatin,
• MeSH
• akrylamidy farmakologie   MeSH
• aktivní transport - buněčné jádro MeSH
• buněčné jádro metabolismus   MeSH
• buňky 3T3-L1 MeSH
• buňky Hep G2 MeSH
• cytoplazma metabolismus   MeSH
• histony metabolismus   MeSH
• kontrolní body buněčného cyklu MeSH
• lidé MeSH
• mutageneze cílená MeSH
• myši MeSH
• NAD metabolismus   MeSH
• nikotinamidfosforibosyltransferasa chemie   genetika   metabolismus   MeSH
• oxidační stres MeSH
• piperidiny farmakologie   MeSH
• poly(ADP-ribosa)polymerasy metabolismus   MeSH
• proliferace buněk MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• sirtuiny metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akrylamidy MeSH
• histony MeSH
• N-(4-(1-benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide MeSH Prohlížeč
• NAD MeSH
• nikotinamidfosforibosyltransferasa MeSH
• piperidiny MeSH
• poly(ADP-ribosa)polymerasy MeSH
• rekombinantní fúzní proteiny MeSH
• sirtuiny MeSH