The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/β-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic β-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of β-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with β-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune β-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/β-catenin output requires selective modulation of gene expression by transcriptional co-factors.

• MeSH
• algoritmy MeSH
• beta-katenin chemie   metabolismus   MeSH
• buněčná diferenciace MeSH
• chromatin metabolismus   MeSH
• fenotyp MeSH
• genetická transkripce * MeSH
• homeostáza MeSH
• hyperplazie MeSH
• JNK mitogenem aktivované proteinkinasy metabolismus   MeSH
• kmenové buňky metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• mutace genetika   MeSH
• mutantní proteiny metabolismus   MeSH
• myši MeSH
• organoidy metabolismus   MeSH
• proliferace buněk MeSH
• restrukturace chromatinu MeSH
• sekvence nukleotidů MeSH
• signální transdukce MeSH
• střevní sliznice cytologie   MeSH
• transkripční faktory metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.

• MeSH
• krystalografie rentgenová MeSH
• kvantová teorie MeSH
• molekulární modely MeSH
• mutace genetika   MeSH
• mutantní proteiny metabolismus   MeSH
• podjednotky proteinů antagonisté a inhibitory   chemie   metabolismus   MeSH
• proteinové domény MeSH
• pyridiny chemie   farmakologie   MeSH
• pyrimidiny chemie   farmakologie   MeSH
• pyrroly chemie   farmakologie   MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   chemie   metabolismus   MeSH
• termodynamika MeSH
• virová léková rezistence účinky léků   MeSH
• virové proteiny antagonisté a inhibitory   chemie   metabolismus   MeSH
• virus chřipky A účinky léků   enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH

Potato (Solanum tuberosum) mutant (ST) lacking one isoform of manganese-stabilizing protein (MSPI) of photosystem II exhibited besides spontaneous tuberization also growth changes with strongly impaired root system development. Previous studies revealed marked changes in carbohydrate levels and allocation within ST plant body. To verify causal relationship between changed carbohydrate balance and root growth restriction we engaged dark grown sucrose-supplied root organ-cultures of ST plants to exclude/confirm shoot effects. Unexpectedly, in ST root cultures we observed large alterations in growth and architecture as well as saccharide status similar to those found in the intact plant roots. The gene expression analysis, however, proved PsbO1 transcript (coding MSPI protein) neither in ST nor in WT root-organ cultures. Therefore, the results point to indirect effects of PsbO1 allele absence connected possibly with some epigenetic modulations.

• MeSH
• alely MeSH
• fotosyntéza genetika   účinky záření   MeSH
• fotosystém II (proteinový komplex) genetika   metabolismus   MeSH
• hlízy rostlin genetika   růst a vývoj   MeSH
• kořeny rostlin růst a vývoj   metabolismus   MeSH
• kultivované buňky MeSH
• mangan metabolismus   MeSH
• metabolismus sacharidů genetika   MeSH
• mutace MeSH
• mutantní proteiny chemie   genetika   metabolismus   MeSH
• protein - isoformy genetika   metabolismus   MeSH
• regulace genové exprese u rostlin genetika   fyziologie   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sacharosa metabolismus   MeSH
• Solanum tuberosum genetika   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH

Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49-Y51-Q52) and transmembrane domain 2 (G326-K327-F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.

• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• gating iontového kanálu MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• kinetika MeSH
• konzervovaná sekvence MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• luminescentní proteiny chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• mutageneze cílená MeSH
• mutantní proteiny chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• purinergní receptory P2X7 chemie   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• statická elektřina MeSH
• substituce aminokyselin MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Gene dosage is a key defining factor to understand cancer pathogenesis and progression, which requires the development of experimental models that aid better deconstruction of the disease. Here, we model an aggressive form of prostate cancer and show the unconventional association of LKB1 dosage to prostate tumorigenesis. Whereas loss of Lkb1 alone in the murine prostate epithelium was inconsequential for tumorigenesis, its combination with an oncogenic insult, illustrated by Pten heterozygosity, elicited lethal metastatic prostate cancer. Despite the low frequency of LKB1 deletion in patients, this event was significantly enriched in lung metastasis. Modeling the role of LKB1 in cellular systems revealed that the residual activity retained in a reported kinase-dead form, LKB1K78I, was sufficient to hamper tumor aggressiveness and metastatic dissemination. Our data suggest that prostate cells can function normally with low activity of LKB1, whereas its complete absence influences prostate cancer pathogenesis and dissemination.

• MeSH
• epitel enzymologie   patologie   MeSH
• fosfohydroláza PTEN metabolismus   MeSH
• HEK293 buňky MeSH
• heterozygot MeSH
• lidé MeSH
• metastázy nádorů MeSH
• mutantní proteiny metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši nahé MeSH
• nádorové buněčné linie MeSH
• nádory prostaty enzymologie   MeSH
• progrese nemoci MeSH
• prostata enzymologie   patologie   MeSH
• protein-serin-threoninkinasy nedostatek   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

β-N-Acetylhexosaminidases (EC 3.2.1.52) are a unique family of glycoside hydrolases with dual substrate specificity and a particular reaction mechanism. Though hydrolytic enzymes per se, their good stability, easy recombinant production, absolute stereoselectivity, and a broad substrate specificity predestine these enzymes for challenging applications in carbohydrate synthesis. This mini-review aims to demonstrate the catalytic potential of β-N-acetylhexosaminidases in a range of unusual reactions, processing of unnatural substrates, formation of unexpected products, and demanding reaction designs. The use of unconventional media can considerably alter the progress of transglycosylation reactions. By means of site-directed mutagenesis, novel catalytic machineries can be constructed. Glycosylation of difficult substrates such as sugar nucleotides was accomplished, and the range of afforded glycosidic bonds comprises unique non-reducing sugars. Specific functional groups may be tolerated in the substrate molecule, which makes β-N-acetylhexosaminidases invaluable allies in difficult synthetic problems.

• MeSH
• beta-N-acetylhexosaminidasy genetika   metabolismus   MeSH
• biokatalýza * MeSH
• glykosylace MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Tail-anchored (TA) proteins are membrane proteins that are found in all domains of life. They consist of an N-terminal domain that performs various functions and a single transmembrane domain (TMD) near the C-terminus. In eukaryotes, TA proteins are targeted to the membranes of mitochondria, the endoplasmic reticulum (ER), peroxisomes and in plants, chloroplasts. The targeting of these proteins to their specific destinations correlates with the properties of the C-terminal domain, mainly the TMD hydrophobicity and the net charge of the flanking regions. Trichomonas vaginalis is a human parasite that has adapted to oxygen-poor environment. This adaptation is reflected by the presence of highly modified mitochondria (hydrogenosomes) and the absence of peroxisomes. The proteome of hydrogenosomes is considerably reduced; however, our bioinformatic analysis predicted 120 putative hydrogenosomal TA proteins. Seven proteins were selected to prove their localization. The elimination of the net positive charge in the C-tail of the hydrogenosomal TA4 protein resulted in its dual localization to hydrogenosomes and the ER, causing changes in ER morphology. Domain mutation and swap experiments with hydrogenosomal (TA4) and ER (TAPDI) proteins indicated that the general principles for specific targeting are conserved across eukaryotic lineages, including T. vaginalis; however, there are also significant lineage-specific differences.

• MeSH
• multienzymové komplexy metabolismus   MeSH
• mutační analýza DNA MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• organely metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• transport proteinů MeSH
• Trichomonas vaginalis enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Studies have suggested a role for the mammalian (or mechanistic) target of rapamycin (mTOR) in skeletal development and homeostasis, yet there is no evidence connecting mTOR with the key signaling pathways that regulate skeletogenesis. We identified a parathyroid hormone (PTH)/PTH-related peptide (PTHrP)-salt-inducible kinase 3 (SIK3)-mTOR signaling cascade essential for skeletogenesis. While investigating a new skeletal dysplasia caused by a homozygous mutation in the catalytic domain of SIK3, we observed decreased activity of mTOR complex 1 (mTORC1) and mTORC2 due to accumulation of DEPTOR, a negative regulator of both mTOR complexes. This SIK3 syndrome shared skeletal features with Jansen metaphyseal chondrodysplasia (JMC), a disorder caused by constitutive activation of the PTH/PTHrP receptor. JMC-derived chondrocytes showed reduced SIK3 activity, elevated DEPTOR, and decreased mTORC1 and mTORC2 activity, indicating a common mechanism of disease. The data demonstrate that SIK3 is an essential positive regulator of mTOR signaling that functions by triggering DEPTOR degradation in response to PTH/PTHrP signaling during skeletogenesis.

• MeSH
• HEK293 buňky MeSH
• homozygot MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• lidé MeSH
• missense mutace genetika   MeSH
• mTORC1 metabolismus   MeSH
• mTORC2 metabolismus   MeSH
• mutantní proteiny chemie   metabolismus   MeSH
• osteogeneze * MeSH
• parathormon metabolismus   MeSH
• protein podobný parathormonu metabolismus   MeSH
• proteinkinasy chemie   nedostatek   genetika   metabolismus   MeSH
• proteolýza MeSH
• růstová ploténka metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce * MeSH
• TOR serin-threoninkinasy metabolismus   MeSH
• typy dědičnosti genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

The spontaneous host-range mutants 812F1 and K1/420 are derived from polyvalent phage 812 that is almost identical to phage K, belonging to family Myoviridae and genus Kayvirus. Phage K1/420 is used for the phage therapy of staphylococcal infections. Endolysin of these mutants designated LysF1, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase (CHAP) domain and C-terminal SH3b cell wall-binding domain, has deleted middle amidase domain compared to wild-type endolysin. In this work, LysF1 and both its domains were prepared as recombinant proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus species of the 43 tested. SH3b domain influenced antimicrobial activity of LysF1, since the lytic activity of the truncated variant containing the CHAP domain alone was decreased. The results of a co-sedimentation assay of SH3b domain showed that it was able to bind to three types of purified staphylococcal peptidoglycan 11.2, 11.3, and 11.8 that differ in their peptide bridge, but also to the peptidoglycan type 11.5 of Streptococcus uberis, and this capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy. Using several different approaches, including NMR, we have not confirmed the previously proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell wall. The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic spectrum, and therefore is an appropriate enzyme for practical use. The binding spectrum of SH3b domain covering all known staphylococcal peptidoglycan types is a promising feature for creating new chimeolysins by combining it with more effective catalytic domains.

• MeSH
• endopeptidasy genetika   izolace a purifikace   metabolismus   MeSH
• hostitelská specificita * MeSH
• mutantní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• Myoviridae enzymologie   genetika   fyziologie   MeSH
• peptidoglykan metabolismus   MeSH
• proteinové domény MeSH
• sekvenční delece * MeSH
• Staphylococcus virologie   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a genetic expansion of the CAG repeat region in the huntingtin (HTT) gene. Studies in HD mouse models have shown that artificial miRNAs can reduce mutant HTT, but evidence for their effectiveness and safety in larger animals is lacking. HD transgenic sheep express the full-length human HTT with 73 CAG repeats. AAV9 was used to deliver unilaterally to HD sheep striatum an artificial miRNA targeting exon 48 of the human HTT mRNA under control of two alternative promoters: U6 or CβA. The treatment reduced human mutant (m) HTT mRNA and protein 50-80% in the striatum at 1 and 6 months post injection. Silencing was detectable in both the caudate and putamen. Levels of endogenous sheep HTT protein were not affected. There was no significant loss of neurons labeled by DARPP32 or NeuN at 6 months after treatment, and Iba1-positive microglia were detected at control levels. It is concluded that safe and effective silencing of human mHTT protein can be achieved and sustained in a large-animal brain by direct delivery of an AAV carrying an artificial miRNA.

• MeSH
• Dependovirus genetika   MeSH
• elektrolyty metabolismus   MeSH
• genetické vektory metabolismus   MeSH
• geneticky modifikovaná zvířata MeSH
• genom virový MeSH
• Huntingtonova nemoc genetika   patologie   MeSH
• imunoanalýza MeSH
• injekce MeSH
• játra patofyziologie   MeSH
• ledviny patofyziologie   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• mikro RNA genetika   metabolismus   MeSH
• mikroglie metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• mutantní proteiny metabolismus   MeSH
• neostriatum metabolismus   MeSH
• neurony metabolismus   MeSH
• ovce MeSH
• protein huntingtin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH