Non-alcoholic fatty liver disease (NAFLD), encompassing fatty liver and its progression into nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), is one of the rapidly rising health concerns worldwide. SIRT6 is an essential nuclear sirtuin that regulates numerous pathological processes including insulin resistance and inflammation, and recently it has been implicated in the amelioration of NAFLD progression. SIRT6 overexpression protects from formation of fibrotic lesions. However, the underlying molecular mechanisms are not fully delineated. Moreover, new allelic variants of SIRT6 (N308K/A313S) were recently associated with the longevity in Ashkenazi Jews by improving genome maintenance and DNA repair, suppressing transposons and killing cancer cells. Whether these new SIRT6 variants play different or enhanced roles in liver diseases is currently unknown. In this study, we aimed to clarify how these new centenarian-associated SIRT6 genetic variants affect liver metabolism and associated diseases. We present evidence that overexpression of centenarian-associated SIRT6 variants dramatically altered the metabolomic and secretomic profiles of unchallenged immortalized human hepatocytes (IHH). Most amino acids were increased in the SIRT6 N308K/A313S overexpressing IHH when compared to IHH transfected with the SIRT6 wild-type sequence. Several unsaturated fatty acids and glycerophospholipids were increased, and ceramide tended to be decreased upon SIRT6 N308K/A313S overexpression. Furthermore, we found that overexpression of SIRT6 N308K/A313S in a 3D hepatic spheroid model formed by the co-culture of human immortalized hepatocytes (IHH) and hepatic stellate cells (LX2) inhibited collagen deposition and fibrotic gene expression in absence of metabolic or dietary challenges. Hence, our findings suggest that novel longevity associated SIRT6 N308K/A313S variants could favor the prevention of NASH by altering hepatocyte proteome and lipidome.
- MeSH
- hepatocelulární karcinom * metabolismus patologie MeSH
- hepatocyty metabolismus patologie MeSH
- kolagen metabolismus MeSH
- lidé MeSH
- nádory jater * metabolismus patologie MeSH
- nealkoholová steatóza jater * genetika metabolismus patologie MeSH
- senioři nad 80 let MeSH
- sirtuiny * genetika metabolismus MeSH
- století lidé MeSH
- Check Tag
- lidé MeSH
- senioři nad 80 let MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lysine deacetylases, like histone deacetylases (HDACs) and sirtuins (SIRTs), are involved in many regulatory processes such as control of metabolic pathways, DNA repair, and stress responses. Besides robust deacetylase activity, sirtuin isoforms SIRT2 and SIRT3 also show demyristoylase activity. Interestingly, most of the inhibitors described so far for SIRT2 are not active if myristoylated substrates are used. Activity assays with myristoylated substrates are either complex because of coupling to enzymatic reactions or time-consuming because of discontinuous assay formats. Here we describe sirtuin substrates enabling direct recording of fluorescence changes in a continuous format. Fluorescence of the fatty acylated substrate is different when compared to the deacylated peptide product. Additionally, the dynamic range of the assay could be improved by the addition of bovine serum albumin, which binds the fatty acylated substrate and quenches its fluorescence. The main advantage of the developed activity assay is the native myristoyl residue at the lysine side chain avoiding artifacts resulting from the modified fatty acyl residues used so far for direct fluorescence-based assays. Due to the extraordinary kinetic constants of the new substrates (KM values in the low nM range, specificity constants between 175,000 and 697,000 M-1s-1) it was possible to reliably determine the IC50 and Ki values for different inhibitors in the presence of only 50 pM of SIRT2 using different microtiter plate formats.
Histone deacylase 11 and human sirtuins are able to remove fatty acid-derived acyl moieties from the ε-amino group of lysine residues. Specific substrates are needed for investigating the biological functions of these enzymes. Additionally, appropriate screening systems are required for identification of modulators of enzymatic activities of HDAC11 and sirtuins. We designed and synthesized a set of activity probes by incorporation of a thioamide quencher unit into the fatty acid-derived acyl chain and a fluorophore in the peptide sequence. Systematic variation of both fluorophore and quencher position resulted "super-substrates" with catalytic constants of up to 15,000,000 M-1s-1 for human sirtuin 2 (Sirt2) enabling measurements using enzyme concentrations down to 100 pM in microtiter plate-based screening formats. It could be demonstrated that the stalled intermediate formed by the reaction of Sirt2-bound thiomyristoylated peptide and NAD+ has IC50 values below 200 pM.
- MeSH
- fluorescenční barviva chemie farmakologie MeSH
- fotochemické procesy MeSH
- histondeacetylasy chemie genetika metabolismus MeSH
- lidé MeSH
- molekulární struktura MeSH
- pozitronová emisní tomografie * MeSH
- sirtuiny antagonisté a inhibitory chemie metabolismus MeSH
- thioamidy chemie farmakologie MeSH
- transport elektronů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Epigenetics is the study of heritable alterations in phenotypes that are not caused by changes in DNA sequence. In the present study, we characterized the genetic and phenotypic alterations of the bacterial plant pathogen Xanthomonas campestris pv. campestris (Xcc) under different treatments with several epigenetic modulating chemicals. The use of DNA demethylating chemicals unambiguously caused a durable decrease in Xcc bacterial virulence, even after its reisolation from infected plants. The first-time use of chemicals to modify the activity of sirtuins also showed some noticeable results in terms of increasing bacterial virulence, but this effect was not typically stable. Changes in treated strains were also confirmed by using methylation sensitive amplification (MSAP), but with respect to registered SNPs induction, it was necessary to consider their contribution to the observed polymorphism. The molecular basis of the altered virulence was deciphered by using dualRNA-seq analysis of treated Xcc strains infecting Brassica rapa plants. The results of the present study should promote more intensive research in the generally understudied field of bacterial epigenetics, where artificially induced modification by epigenetic modulating chemicals can significantly increase the diversity of bacterial properties and potentially contribute to the further development of the fields, such as bacterial ecology and adaptation.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- Brassica rapa mikrobiologie MeSH
- epigeneze genetická účinky léků MeSH
- inhibitory enzymů farmakologie MeSH
- jednonukleotidový polymorfismus MeSH
- metylace DNA MeSH
- puriny farmakologie MeSH
- sirtuiny antagonisté a inhibitory genetika metabolismus MeSH
- virulence genetika MeSH
- Xanthomonas campestris účinky léků genetika patogenita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Sirtuin 6 (SIRT6), a member of sirtuin family (SIRT1-7), regulates a variety of cellular processes involved in aging, metabolism, and cancer. Dysregulation of SIRT6 is widely observed in different breast cancer subtypes; however, the role and function of SIRT6 in cancer development remain largely unexplored. The aim of this study was to identify novel compounds targeting SIRT6 which may provide a new approach in development of anti-cancer therapy for breast cancer. Virtual screening was utilized to discover potential compounds targeting SIRT6 for in vitro screening. In addition, novel 1,4-dihydropyridine derivatives were synthetized and further subjected for the screening. The impact of the compounds on the deacetylation activity of SIRT6 was determined with HPLC method. The anti-cancer activities were screened for a panel of breast cancer cells. A set of 1,4-dihydropyridine derivatives was identified as SIRT6 inhibitors. A SIRT6 activating compound, (2,4-dihydroxy-phenyl)-2-oxoethyl 2-(3-methyl-4-oxo-2-phenyl-4H-chromen-8-yl)acetate (later called as 4H-chromen), was discovered and it provided 30-40-fold maximal activation. 4H-chromen was proposed to bind similarly to quercetin and place to previously reported SIRT6 activator sites. 4H-chromen was investigated in various breast cancer cells, and it decreased cell proliferation in all cells as well as arrested cell cycle in triple negative cells. Overall, this study describes a highly potent SIRT6 activator and new inhibitors that represent a novel tool to study the mechanism of SIRT6 function.
- MeSH
- antitumorózní látky chemie farmakologie terapeutické užití MeSH
- časná detekce nádoru metody MeSH
- léky antitumorózní - screeningové testy metody MeSH
- lidé MeSH
- nádory prsu farmakoterapie metabolismus MeSH
- sekundární struktura proteinů MeSH
- simulace molekulového dockingu metody MeSH
- sirtuiny antagonisté a inhibitory chemie metabolismus MeSH
- terciární struktura proteinů MeSH
- viabilita buněk účinky léků fyziologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články 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.
- 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
- MeSH
- antioxidancia MeSH
- chromatin MeSH
- genetická transkripce * MeSH
- heterochromatin MeSH
- histondeacetylasy * MeSH
- histony MeSH
- kalorická restrikce MeSH
- lidé MeSH
- NAD+ nukleosidasa MeSH
- posttranslační úpravy proteinů * MeSH
- sirtuiny * fyziologie klasifikace metabolismus MeSH
- stárnutí MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
A vast collection of data obtained during the last decade supports the view on sirtuins as sensors of actual cellular metabolic state being involved in cell cycle progression, apoptosis/survival decision making, longevity, inflammation etc. Moreover, sirtuins themselves can control metabolism through their ability to consume NAD(+). In turn, cellular NAD parameters may affect the generation of ATP, a main cellular currency of energy. Therefore, sirtuins became recognized as critical affectors of cellular metabolism which participate in fat mobilization, gluconeogenesis, caloric restriction etc. Cellular senescence is viewed as a mechanism to restrict excessive cell growth when it is unnecessary or harmful. It is therefore necessary to understand the mechanism of senescence to design new approaches to combat cancer. Growth in turn depends on metabolism as it requires energy. Therefore, in this review, we address the connection of sirtuins to senescence through their participation in the regulation of metabolic and biochemical parameters and related signaling.
- MeSH
- antiflogistika nesteroidní farmakologie MeSH
- lidé MeSH
- NAD metabolismus MeSH
- signální transdukce MeSH
- sirtuiny účinky léků metabolismus MeSH
- stárnutí účinky léků MeSH
- stilbeny farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- MeSH
- geriatrická psychiatrie metody trendy MeSH
- geriatrie metody trendy MeSH
- křehký senior MeSH
- neurokognitivní poruchy epidemiologie MeSH
- potravinová alergie imunologie MeSH
- preventivní lékařství metody trendy MeSH
- senioři MeSH
- sirtuiny metabolismus MeSH
- stárnutí kůže MeSH
- zdravé chování MeSH
- Check Tag
- senioři MeSH
- Publikační typ
- kongresy MeSH
