Polycomb repressive complex 2 (PRC2) is involved in maintaining transcriptionally silent chromatin states through methylating lysine 27 of histone H3 by the catalytic subunit enhancer of zeste [E(z)]. Here, we report the diversity of PRC2 core subunit proteins in different eukaryotic supergroups with emphasis on the early-diverged lineages and explore the molecular evolution of PRC2 subunits by phylogenetics. For the first time, we identify the putative ortholog of E(z) in Discoba, a lineage hypothetically proximal to the eukaryotic root, strongly supporting emergence of PRC2 before the diversification of eukaryotes. Analyzing 283 species, we robustly detect a common presence of E(z) and ESC, indicating a conserved functional core. Full-length Su(z)12 orthologs were identified in some lineages and species only, indicating, nonexclusively, high divergence of VEFS-Box-containing Su(z)12-like proteins, functional convergence of sequence-unrelated proteins, or Su(z)12 dispensability. Our results trace E(z) evolution within the SET-domain protein family, proposing a substrate specificity shift during E(z) evolution based on SET-domain and H3 histone interaction prediction.

• MeSH
• fylogeneze MeSH
• histony genetika   metabolismus   MeSH
• lysin metabolismus   MeSH
• PRC2 * genetika   metabolismus   MeSH
• proteiny Drosophily * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The chromodomain (CD) of HP1 proteins is an established H3K9me3 reader that also binds H1, EHMT2 and H3K23 lysine-methylated targets. Structural experiments have provided atomistic pictures of its recognition of the conserved ARKme3S/T motif, but structural dynamics' contribution to the recognition may have been masked by ensemble averaging. METHODS: We acquired ~350 μs of explicit solvent molecular dynamics (MD) simulations of the CD domain interacting with several peptides using the latest AMBER force fields. RESULTS: The simulations reproduced the experimentally observed static binding patterns well but also revealed visible structural dynamics at the interfaces. While the buried K0me3 and A-2 target residues are tightly bound, several flanking sidechains sample diverse sites on the CD surface. Different amino acid positions of the targets can substitute for each other by forming mutually replaceable interactions with CD, thereby explaining the lack of strict requirement for cationic H3 target residues at the -3 position. The Q-4 residue of H3 targets further stabilizes the binding. The recognition pattern of the H3K23 ATKme3A motif, for which no structure is available, is predicted. CONCLUSIONS: The CD reads a longer target segment than previously thought, ranging from positions -7 to +3. The CD anionic clamp can be neutralized not only by the -3 and -1 residues, but also by -7, -6, -5 and +3 residues. GENERAL SIGNIFICANCE: Structural dynamics, not immediately apparent from the structural data, contribute to molecular recognition between the HP1 CD domain and its targets. Mutual replaceability of target residues increases target sequence flexibility.

• MeSH
• chromozomální proteiny, nehistonové chemie   metabolismus   MeSH
• histokompatibilita - antigeny metabolismus   MeSH
• histonlysin-N-methyltransferasa metabolismus   MeSH
• histony metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• lysin metabolismus   MeSH
• metylace MeSH
• posttranslační úpravy proteinů MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.

• MeSH
• astrocyty metabolismus   patologie   MeSH
• biologické modely MeSH
• buněčný rodokmen MeSH
• chromatin metabolismus   MeSH
• embryo savčí metabolismus   MeSH
• epigeneze genetická MeSH
• genetická transkripce MeSH
• gliom genetika   patologie   MeSH
• histony genetika   metabolismus   MeSH
• interneurony metabolismus   MeSH
• karcinogeneze genetika   patologie   MeSH
• lysin metabolismus   MeSH
• mutace genetika   MeSH
• myši inbrední C57BL MeSH
• nádory mozku genetika   patologie   MeSH
• nervové kmenové buňky metabolismus   MeSH
• oligodendroglie metabolismus   MeSH
• přední mozek embryologie   MeSH
• přeprogramování buněk genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• regulace genové exprese u nádorů MeSH
• růstový faktor odvozený z trombocytů - receptor alfa genetika   metabolismus   MeSH
• stupeň nádoru MeSH
• transkriptom genetika   MeSH
• umlčování genů MeSH
• zvířata MeSH
• Check Tag
• 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

A precisely balanced activity of canonical Wnt signaling is essential for a number of biological processes and its perturbation leads to developmental defects or diseases. Here, we demonstrate that alternative isoforms of the KDM2A and KDM2B lysine demethylases have the ability to negatively regulate canonical Wnt signaling. These KDM2A and KDM2B isoforms (KDM2A-SF and KDM2B-SF) lack the N-terminal demethylase domain, but they still have the ability to bind to CpG islands in promoters and to interact with their protein partners via their other functional domains. We have observed that KDM2A-SF and KDM2B-SF bind to the promoters of axin 2 and cyclin D1, two canonical Wnt signaling target genes, and repress their activity. Moreover, KDM2A-SF and KDM2B-SF are both able to strongly repress a Wnt-responsive luciferase reporter. The transcriptional repression mediated by KDM2A-SF and KDM2B-SF, but also by KDM2A-LF, is dependent on their DNA binding domain, while the N-terminal demethylase domain is dispensable for this process. Surprisingly, KDM2B-LF is unable to repress both the endogenous promoters and the luciferase reporter. Finally, we show that both KDM2A-SF and KDM2B-SF are able to interact with TCF7L1, one of the transcriptional mediators of canonical Wnt signaling. KDM2A-SF and KDM2B-SF are thus likely to negatively affect the transcription of canonical Wnt signaling target genes by binding to their promoters and by interacting with TCF7L1 and other co-repressors.

• MeSH
• CpG ostrůvky MeSH
• cyklin D1 genetika   metabolismus   MeSH
• F-box proteiny genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• Jumonjiho doména s histondemethylasami genetika   metabolismus   MeSH
• lidé MeSH
• lysin genetika   metabolismus   MeSH
• promotorové oblasti (genetika) * MeSH
• protein - isoformy MeSH
• protein 1 podobný transkripčnímu faktoru 7 genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• signální dráha Wnt * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A feared adverse effect of dyslipidaemia therapy by fibrates is myopathy. We examined the effect of fenofibrate (FF) on protein and amino acid metabolism. Rats received a low (50 mg/kg, LFFD) or high (300 mg/kg, HFFD) dose of FF or vehicle daily by oral gavage. Blood plasma, liver, and soleus and extensor digitorum longus muscles were analysed after 10 days. The FF-treated rats developed hepatomegaly associated with increased hepatic carnitine and ATP and AMP concentrations, decreased protein breakdown, and decreased concentrations of DNA and triglycerides. HFFD increased plasma ALT and AST activities. The weight and protein content of muscles in the HFFD group were lower compared with controls. In muscles of the LFFD group there were increased ATP and decreased AMP concentrations; in the HFFD group AMP was increased. In both FF-treated groups there were increased glycine, phenylalanine, and citrulline and decreased arginine and branched-chain keto acids (BCKA) in blood plasma. After HFFD there were decreased levels of branched-chain amino acids (BCAA; valine, leucine and isoleucine), methionine, and lysine and increased homocysteine. Decreased arginine and increased glycine concentrations were found in both muscles in FF-treated animals; in HFFD-treated animals lysine, methionine, and BCAA were decreased. We conclude that FF exerts protein-anabolic effects on the liver and catabolic effects on muscles. HFFD causes signs of hepatotoxicity, impairs energy and protein balance in muscles, and decreases BCAA, methionine, and lysine. It is suggested that increased glycine and decreased lysine and methionine levels are due to activated carnitine synthesis; decreased BCAA and BCKA levels are due to increased BCAA oxidation.

• MeSH
• aminokyseliny účinky léků   metabolismus   MeSH
• energetický metabolismus účinky léků   MeSH
• fenofibrát aplikace a dávkování   MeSH
• glycin metabolismus   MeSH
• hepatomegalie chemicky indukované   metabolismus   MeSH
• hypolipidemika aplikace a dávkování   MeSH
• játra účinky léků   metabolismus   MeSH
• karnitin krev   MeSH
• kosterní svaly účinky léků   metabolismus   MeSH
• krysa rodu rattus MeSH
• leucin metabolismus   MeSH
• lidé MeSH
• lysin metabolismus   MeSH
• methionin metabolismus   MeSH
• oxidace-redukce MeSH
• potkani Wistar MeSH
• proteiny účinky léků   metabolismus   MeSH
• větvené aminokyseliny krev   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Humoral immunity in mammals relies on the function of two developmentally and functionally distinct B-cell subsets-B1 and B2 cells. While B2 cells are responsible for the adaptive response to environmental antigens, B1 cells regulate the production of polyreactive and low-affinity antibodies for innate humoral immunity. The molecular mechanism of B-cell specification into different subsets is understudied. In this study, we identified lysine methyltransferase NSD2 (MMSET/WHSC1) as a critical regulator of B1 cell development. In contrast to its minor impact on B2 cells, deletion of the catalytic domain of NSD2 in primary B cells impairs the generation of B1 lineage. Thus, NSD2, a histone H3 K36 dimethylase, is the first-in-class epigenetic regulator of a B-cell lineage in mice.

• MeSH
• analýza přežití MeSH
• B-lymfocyty metabolismus   MeSH
• histonlysin-N-methyltransferasa chemie   metabolismus   MeSH
• histony metabolismus   MeSH
• humorální imunita MeSH
• katalytická doména * MeSH
• lysin metabolismus   MeSH
• metylace MeSH
• myši inbrední C57BL MeSH
• novorozená zvířata MeSH
• přesmyk imunoglobulinových tříd MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zárodečné centrum lymfatické uzliny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Post-translational modifications of proteins enable swift physiological adaptation of cells to altered growth conditions and stress. Aside from protein phosphorylation, acetylation on ε-amino groups of lysine residues (N-ε-lysine acetylation) represents another important post-translational modification of proteins. For many bacterial pathogens, including the whooping cough agent Bordetella pertussis, the role and extent of protein acetylation remain to be defined. We expressed in Escherichia coli the BP0960 and BP3063 genes encoding two putative deacetylases of B. pertussis and show that BP0960 encodes a lysine deacetylase enzyme, named Bkd1, that regulates acetylation of a range of B. pertussis proteins. Comparison of the proteome and acetylome of a Δbkd1 mutant with the proteome and acetylome of wild-type B. pertussis (PRIDE ID. PXD016384) revealed that acetylation on lysine residues may modulate activities or stabilities of proteins involved in bacterial metabolism and histone-like proteins. However, increased acetylation of the BvgA response regulator protein of the B. pertussis master virulence-regulating BvgAS two-component system affected neither the total levels of produced BvgA nor its phosphorylation status. Indeed, the Δbkd1 mutant was not impaired in the production of key virulence factors and its survival within human macrophages in vitro was not affected. The Δbkd1 mutant exhibited an increased growth rate under carbon source-limiting conditions and its virulence in the in vivo mouse lung infection model was somewhat affected. These results indicate that the lysine deacetylase Bkd1 and N-ε-lysine acetylation primarily modulate the general metabolism rather than the virulence of B. pertussis.

• MeSH
• acetylace MeSH
• bakteriální proteiny * genetika   metabolismus   MeSH
• Bordetella pertussis genetika   MeSH
• lysin * metabolismus   MeSH
• myši MeSH
• regulace genové exprese u bakterií MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Histone deacetylase 6 (HDAC6) is a multidomain cytosolic hydrolase acting mostly on nonhistone protein substrates. Investigations of the substrate specificity of HDAC6 are confounded by the presence of 2 catalytically active deacetylase domains (DD1 and DD2). In this study, acetylome peptide microarrays and peptide libraries were used to map the substrate specificity of DD1 and DD2 of human HDAC6. The results show that DD1 is solely responsible for the deacetylation of substrates harboring the acetyllysine at their C terminus, whereas DD2 exclusively deacetylates peptides with an internal acetyllysine residue. Also, statistical analysis of the deacetylation data revealed amino acid preferences at individual positions flanking the acetyllysine, where glycine and arginine residues are favored at positions N-terminal to the central acetyllysine; negatively charged glutamate is strongly disfavored throughout the sequence. Finally, the deacylation activity of HDAC6 was profiled by using a panel of acyl derivatives of the optimized peptide substrate and showed that HDAC6 acts as a proficient deformylase. Our data thus offer a detailed insight into the substrate preferences of the individual HDAC6 domains at the peptide level, and these findings can in turn help in elucidating the biologic roles of the enzyme and facilitate the development of new domain-specific inhibitors as research tools or therapeutic agents.-Kutil, Z., Skultetyova, L., Rauh, D., Meleshin, M., Snajdr, I., Novakova, Z., Mikesova, J., Pavlicek, J., Hadzima, M., Baranova, P., Havlinova, B., Majer, P., Schutkowski, M., Barinka, C. The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries.

• MeSH
• HEK293 buňky MeSH
• histondeacetylasa 6 chemie   metabolismus   MeSH
• katalytická doména * MeSH
• lidé MeSH
• lysin chemie   metabolismus   MeSH
• peptidové fragmenty chemie   metabolismus   MeSH
• statická elektřina MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We developed a one-step direct assay for the determination of histone deacylase (HDAC) activity by substituting the carbonyl oxygen of the acyl moiety with sulfur, resulting in thioacylated lysine side chains. This modification is recognized by class I HDACs with different efficiencies ranging from not accepted for HDAC1 to kinetic constants similar to that of the parent oxo substrate for HDAC8. Class II HDACs can hydrolyze thioacylated substrates with approximately 5-10-fold reduced kcat values, which resembles the effect of thioamide substitution in metallo-protease substrates. Class IV HDAC11 accepts thiomyristoyl modification less efficiently with an ∼5-fold reduced specificity constant. On the basis of the unique spectroscopic properties of thioamide bonds (strong absorption in spectral range of 260-280 nm and efficient fluorescence quenching), HDAC-mediated cleavage of thioamides could be followed by ultraviolet-visible and fluorescence spectroscopy in a continuous manner. The HDAC activity assay is compatible with microtiter plate-based screening formats up to 1536-well plates with Z' factors of >0.75 and signal-to-noise ratios of >50. Using thioacylated lysine residues in p53-derived peptides, we optimized substrates for HDAC8 with a catalytic efficiency of >250000 M-1 s-1, which are more than 100-fold more effective than most of the known substrates. We determined inhibition constants of several inhibitors for human HDACs using thioacylated peptidic substrates and found good correlation with the values from the literature. On the other hand, we could introduce N-methylated, N-acylated lysine residues as inhibitors for HDACs with an IC50 value of 1 μM for an N-methylated, N-myristoylated peptide derivative and human HDAC11.

• MeSH
• biokatalýza MeSH
• histondeacetylasy chemie   genetika   metabolismus   MeSH
• inhibitory histondeacetylas chemie   metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• lysin chemie   metabolismus   MeSH
• thioamidy chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3'-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling.

• MeSH
• ABC transportéry chemie   genetika   metabolismus   MeSH
• arginin metabolismus   MeSH
• delece genu MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu MeSH
• genetické vektory chemie   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• klonování DNA MeSH
• kodon chemie   metabolismus   MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• lysin metabolismus   MeSH
• molekulární modely MeSH
• priony chemie   genetika   metabolismus   MeSH
• regulace genové exprese u hub * MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• ribozomy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• sekvenční seřazení MeSH
• terminace translace peptidového řetězce * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH