The nonsense-mediated mRNA decay (NMD) pathway rapidly detects and degrades mRNA containing premature termination codons (PTCs). UP-frameshift 1 (UPF1), the master regulator of the NMD process, has two alternatively-spliced isoforms; one carries 353-GNEDLVIIWLR-363 insertion in the 'regulatory loop (involved in mRNA binding)'. Such insertion can induce catalytic and/or ATPase activity, as determined experimentally; however, the kinetics and molecular level information are not fully understood. Herein, applying all-atom molecular dynamics, we probe the binding specificity of UPF1 with different GC- and AU-rich mRNA motifs and the influence of insertion to the viable control over UPF1 catalytic activity. Our results indicate two distinct conformations between 1B and RecA2 domains of UPF1: 'open (isoform_2; without insertion)' and 'closed (isoform_1; with insertion)'. These structural movements correspond to an important stacking pattern in mRNA motifs, i.e., absence of stack formation in mRNA, with UPF1 isoform_2 results in the 'open conformation'. Particularly, for UPF1 isoform_1, the increased distance between 1B and RecA2 domains has resulted in reducing the mRNA-UPF1 interactions. Lower fluctuating GC-rich mRNA motifs have better binding with UPF1, compared with AU-rich sequences. Except CCUGGGG, all other GC-rich motifs formed a 4-stack pattern with UPF1. High occupancy R363, D364, T627, and G862 residues were common binding GC-rich motifs, as were R363, N535, and T627 for the AU-rich motifs. The GC-rich motifs behave distinctly when bound to either of the isoforms; lower stability was observed with UPF1 isoform_2. The cancer-associated UPF1 variants (P533L/T and A839T) resulted in decreased protein-mRNA binding efficiency. Lack of mRNA stacking poses in the UPF1P533T system significantly decreased UPF1-mRNA binding efficiency and increased distance between 1B-RecA2. These novel findings can serve to further inform NMD-associated mechanistic and kinetic studies.

• Klíčová slova
• AU-rich, GC-rich, NMD, PTC, UPF1, alternatively spliced, degradation, isoform, mRNA, molecular dynamics, motifs, regulatory loop, stability,
• MeSH
• alternativní sestřih * MeSH
• fosforylace MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• nonsense mediated mRNA decay * MeSH
• protein - isoformy MeSH
• regulace genové exprese * MeSH
• RNA-helikasy genetika   metabolismus   MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• messenger RNA MeSH
• protein - isoformy MeSH
• RNA-helikasy MeSH
• trans-aktivátory MeSH
• UPF1 protein, human MeSH Prohlížeč

G-quadruplexes have important regulatory roles in the nuclear genome but their distribution and potential roles in mitochondrial DNA (mtDNA) are poorly understood. We analysed 11883 mtDNA sequences from 18 taxonomic sub-groups and identified their frequency and location within mtDNA. Large differences in both the frequency and number of putative quadruplex-forming sequences (PQS) were observed amongst all the organisms and PQS frequency was negatively correlated with an increase in evolutionary age. PQS were over-represented in the 3'UTRs, D-loops, replication origins, and stem loops, indicating regulatory roles for quadruplexes in mtDNA. Variations of the G-quadruplex-forming sequence in the conserved sequence block II (CSBII) region of the human D-loop were conserved amongst other mammals, amphibians, birds, reptiles, and fishes. This D-loop PQS was conserved in the duplicated control regions of some birds and reptiles, indicating its importance to mitochondrial function. The guanine tracts in these PQS also displayed significant length heterogeneity and the length of these guanine tracts were generally longest in bird mtDNA. This information provides further insights into how G4s may contribute to the regulation and function of mtDNA and acts as a database of information for future studies investigating mitochondrial G4s in organisms other than humans.

• Klíčová slova
• D-loop, Evolution, G-quadruplex, Genome, Mitochondria,
• MeSH
• G-kvadruplexy * MeSH
• genom MeSH
• lidé MeSH
• mitochondriální DNA genetika   MeSH
• mitochondrie MeSH
• regulační oblasti nukleových kyselin genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mitochondriální DNA MeSH

IL-17 mediates immune protection from fungi and bacteria, as well as it promotes autoimmune pathologies. However, the regulation of the signal transduction from the IL-17 receptor (IL-17R) remained elusive. We developed a novel mass spectrometry-based approach to identify components of the IL-17R complex followed by analysis of their roles using reverse genetics. Besides the identification of linear ubiquitin chain assembly complex (LUBAC) as an important signal transducing component of IL-17R, we established that IL-17 signaling is regulated by a robust negative feedback loop mediated by TBK1 and IKKε. These kinases terminate IL-17 signaling by phosphorylating the adaptor ACT1 leading to the release of the essential ubiquitin ligase TRAF6 from the complex. NEMO recruits both kinases to the IL-17R complex, documenting that NEMO has an unprecedented negative function in IL-17 signaling, distinct from its role in NF-κB activation. Our study provides a comprehensive view of the molecular events of the IL-17 signal transduction and its regulation.

• Klíčová slova
• LUBAC, NEMO, IKKε, IL-17, TBK1,
• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• kinasa I-kappa B genetika   metabolismus   MeSH
• lidé MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• receptory interleukinu-17 genetika   metabolismus   MeSH
• signální transdukce * MeSH
• zpětná vazba fyziologická * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• IKBKE protein, human MeSH Prohlížeč
• IKBKG protein, human MeSH Prohlížeč
• IL17RA protein, human MeSH Prohlížeč
• kinasa I-kappa B MeSH
• protein-serin-threoninkinasy MeSH
• receptory interleukinu-17 MeSH
• TBK1 protein, human MeSH Prohlížeč
• TRAF3IP2 protein, human MeSH Prohlížeč

Epithelial-mesenchymal transition (EMT) is a process involved not only in morphogenesis and embryonic development, but also in cancer progression, whereby tumor cells obtain a more aggressive metastatic phenotype. Anterior gradient protein 2 (AGR2) maintains the epithelial phenotype and blocks the induction of EMT, thus playing an undeniable role in tumor progression. However, the mechanism through which AGR2 expression is regulated, not only during EMT, but also in the early stages of cancer development, remains to be elucidated. In the present study, we show an inverse correlation of AGR2 with ZEB1 (zinc finger enhancer binding protein, δEF1) that was verified by analysis of several independent clinical data sets of lung adenocarcinomas. We also identified the ZEB1 binding site within the AGR2 promoter region and confirmed AGR2 as a novel molecular target of ZEB1. The overexpression of ZEB1 decreased the promoter activity of the AGR2 gene, which resulted in reduced AGR2 protein level and the acquisition of a more invasive phenotype of these lung cancer cells. Conversely, silencing of ZEB1 led not only to increased levels of AGR2 protein, but also attenuated the invasiveness of tumor cells. The AGR2 knockout, vice versa, increased ZEB1 expression, indicating that the ZEB1/AGR2 regulatory axis may function in a double negative feedback loop. In conclusion, we revealed for the first time that ZEB1 regulates AGR2 at the transcriptional level, while AGR2 presence contributes to ZEB1 mRNA degradation. Thus, our data identify a new regulatory mechanism between AGR2 and ZEB1, two rivals in the EMT process, tightly associated with the development of metastasis.

• Klíčová slova
• AGR2, EMT, ZEB1, cancer,
• Publikační typ
• časopisecké články MeSH

Chronic myeloid leukemia (CML) is caused by constituve activity of BCR-ABL tyrosine kinase. Despite of high efficiency of imatinib, selective BCR-ABL inhibitor, about 30% of patients develop resistance. Novel markers and targets for therapy are thus necessary. MicroRNAs are small intereference RNAs whose role in physiological and malignant hematopoiesis has been shown. This study is focused on miR-451 in CML. Following our observation of miR-451 downregulation in CML, we further show its relation to BCR-ABL activity. Our data together with current literature indicate a more complex relationship of miR-451 and BCR-ABL in CML.

• MeSH
• bcr-abl fúzní proteiny antagonisté a inhibitory   MeSH
• chronická myeloidní leukemie genetika   MeSH
• geny abl fyziologie   MeSH
• lidé MeSH
• mikro RNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bcr-abl fúzní proteiny MeSH
• mikro RNA MeSH
• MIRN451 microRNA, human MeSH Prohlížeč

Homologous recombination (HR) is essential for maintaining genomic integrity, which is challenged by a wide variety of potentially lethal DNA lesions. Regardless of the damage type, recombination is known to proceed by RAD51-mediated D-loop formation, followed by DNA repair synthesis. Nevertheless, the participating polymerases and extension mechanism are not well characterized. Here, we present a reconstitution of this step using purified human proteins. In addition to Pol δ, TLS polymerases, including Pol η and Pol κ, also can extend D-loops. In vivo characterization reveals that Pol η and Pol κ are involved in redundant pathways for HR. In addition, the presence of PCNA on the D-loop regulates the length of the extension tracks by recruiting various polymerases and might present a regulatory point for the various recombination outcomes.

• Klíčová slova
• D-loop, DNA repair synthesis, Homologous recombination, Reconstitution, TLS polymerases,
• MeSH
• DNA-dependentní DNA-polymerasy chemie   fyziologie   MeSH
• DNA-polymerasa III chemie   fyziologie   MeSH
• DNA-polymerasa iota MeSH
• HeLa buňky MeSH
• homologní rekombinace * MeSH
• jednovláknová DNA biosyntéza   MeSH
• lidé MeSH
• osmolární koncentrace MeSH
• poškození DNA MeSH
• proliferační antigen buněčného jádra chemie   fyziologie   MeSH
• protein FUS vázající RNA chemie   fyziologie   MeSH
• rekombinasa Rad51 chemie   MeSH
• replikace DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-dependentní DNA-polymerasy MeSH
• DNA-polymerasa III MeSH
• DNA-polymerasa iota MeSH
• jednovláknová DNA MeSH
• POLI protein, human MeSH Prohlížeč
• POLK protein, human MeSH Prohlížeč
• proliferační antigen buněčného jádra MeSH
• protein FUS vázající RNA MeSH
• Rad30 protein MeSH Prohlížeč
• RAD51 protein, human MeSH Prohlížeč
• rekombinasa Rad51 MeSH

Cisplatin is the most widely used chemotherapeutic drug for the treatment of various types of cancer; however, its administration brings also numerous side effects. It was demonstrated that cisplatin can inhibit the Na+/K+-ATPase (NKA), which can explain a large part of the adverse effects. In this study, we have identified five cysteinyl residues (C452, C456, C457, C577, and C656) as the cisplatin binding sites on the cytoplasmic loop connecting transmembrane helices 4 and 5 (C45), using site-directed mutagenesis and mass spectrometry experiments. The identified residues are known to be susceptible to glutathionylation indicating their involvement in a common regulatory mechanism.

• Klíčová slova
• C45 loop, Na+/K+-ATPase, binding site, cisplatin, cysteine mutants, sodium pump,
• MeSH
• cisplatina chemie   farmakologie   MeSH
• cystein antagonisté a inhibitory   metabolismus   MeSH
• cytoplazma účinky léků   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• mutageneze cílená MeSH
• myši MeSH
• protinádorové látky chemie   farmakologie   MeSH
• simulace molekulární dynamiky MeSH
• sodíko-draslíková ATPasa antagonisté a inhibitory   genetika   metabolismus   MeSH
• vazebná místa účinky léků   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cisplatina MeSH
• cystein MeSH
• protinádorové látky MeSH
• sodíko-draslíková ATPasa MeSH

Heptahelical receptor coupling selectivity to G-proteins is controlled by a large contact area that involves several portions of the receptor and each subunit of the G-protein. In the G-protein alpha subunit, the C-terminal 5 residues, the N terminus, and the alpha N-beta 1 and alpha 4-alpha 5 loops play important roles. On the receptor side, both the second and third (i2 and i3) intracellular loops as well as the C-terminal tail probably contact these different regions of the G-protein. It is now accepted that the C terminus of the alpha subunit binds in a cavity formed by the i2 and i3 loops. Among the various G-protein-coupled receptors (GPCRs), class III receptors that include metabotropic glutamate (mGlu) receptors greatly differ from the rhodopsin-like GPCRs, but the contact zone between these receptors and the G-protein is less understood. The C terminus of the alpha subunit has been shown to play a pivotal role in the selective recognition of class III GPCRs. Indeed, the mGlu2 and mGlu4 and -8 receptors can discriminate between alpha subunits that differ at the level of their C-terminal end only (such as Gqo and Gqz). Here, we examine the role of the i2 loop of mGluRs in the selective recognition of this region of the alpha subunit. To that aim, we analyzed the coupling properties of mGlu2 and mGlu4 or -8 receptors and chimeras containing the i2 loop of the converse receptor to G-protein alpha subunits that only differ by their C termini (Gqo,Gqz, and their point mutants). Our data demonstrate that the central portion of the i2 loop is responsible for the selective recognition of the C-terminal end of the alpha subunit, especially the residue on position -4. These data are consistent with the proposal that the C-terminal end of the G-protein alpha subunit interacts with residues in a cavity formed by the i2 and i3 loops in class III GPCRs, as reported for class I GPCRs.

• MeSH
• bodová mutace MeSH
• buněčné linie MeSH
• inositolfosfáty metabolismus   MeSH
• kinetika MeSH
• ledviny MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• proteiny vázající GTP chemie   genetika   metabolismus   MeSH
• receptory metabotropního glutamátu chemie   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   metabolismus   MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• transfekce MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inositolfosfáty MeSH
• podjednotky proteinů MeSH
• proteiny vázající GTP MeSH
• receptory metabotropního glutamátu MeSH
• rekombinantní fúzní proteiny MeSH
• rekombinantní proteiny MeSH

The paired domain, DNA-binding domain of Pax6 and other Pax transcription factors, is composed of two subdomains (PAI and RED), each recognizing distinct half-sites of the bipartite binding site in adjacent major grooves of the DNA helix. The alternatively spliced Pax6(5a) isoform containing 14 extra amino acids within the PAI domain recognizes the 5aCON sequence consisting of four interdigitated 5' half-sites of the bipartite consensus sequence. A genome database search for similar tetrameric Pax6(A) recognition sequences led to the identification of a Pax6-binding site in the lens-specific enhancer of the mouse E- and F-crystallin genes. This binding site combines the properties of bipartite and tetrameric recognition sequences and, by mutational analysis, is shown to mediate Pax6-dependent regulation of the E- and F-crystallin promoter constructs both in primary chicken lens cells and in chicken embryo fibroblasts. The Pax6-binding site is adjacent to a previously identified retinoic acid response element and is itself required for retinoic acid induction of the F- and E-crystallin genes, suggesting that Pax proteins and retinoic acid receptors cooperate in transcriptional regulation. In summary, our protein-DNA binding and transactivation studies suggest that -crystallin genes are under the control of a multifunctional enhancer element that mediates Pax6 regulation as well as retinoic acid-mediated induction.

• MeSH
• DNA vazebné proteiny genetika   fyziologie   MeSH
• down regulace MeSH
• epitelové buňky metabolismus   MeSH
• homeodoménové proteiny genetika   fyziologie   MeSH
• krystaliny genetika   MeSH
• kultivované buňky MeSH
• kuřecí embryo MeSH
• luciferasy genetika   metabolismus   MeSH
• oční čočka cytologie   metabolismus   MeSH
• oční proteiny MeSH
• plazmidy genetika   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• receptory kyseliny retinové genetika   fyziologie   MeSH
• regulace genové exprese účinky léků   MeSH
• regulační oblasti nukleových kyselin genetika   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• represorové proteiny genetika   fyziologie   MeSH
• retinoidní X receptory MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie nukleových kyselin MeSH
• transfekce MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory bHLH MeSH
• transkripční faktory paired box MeSH
• transkripční faktory genetika   fyziologie   MeSH
• tretinoin farmakologie   MeSH
• vazebná místa genetika   MeSH
• zesilovače transkripce genetika   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• gro protein, Drosophila MeSH Prohlížeč
• homeodoménové proteiny MeSH
• krystaliny MeSH
• luciferasy MeSH
• oční proteiny MeSH
• Pax6 protein, mouse MeSH Prohlížeč
• receptory kyseliny retinové MeSH
• rekombinantní fúzní proteiny MeSH
• represorové proteiny MeSH
• retinoidní X receptory MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory bHLH MeSH
• transkripční faktory paired box MeSH
• transkripční faktory MeSH
• tretinoin MeSH

G-quadruplexes are unusual DNA and RNA secondary structures ubiquitous in a variety of organisms including vertebrates, plants, viruses and bacteria. The folding topology and stability of intramolecular G-quadruplexes are determined to a large extent by their loops. Loop permutation is defined as swapping two or three of these regions so that intramolecular G-quadruplexes only differ in the sequential order of their loops. Over the past two decades, both length and base composition of loops have been studied extensively, but a systematic study on the effect of loop permutation has been missing. In the present work, 99 sequences from 21 groups with different loop permutations were tested. To our surprise, both conformation and thermal stability are greatly dependent on loop permutation. Loop permutation actually matters as much as loop length and base composition on G-quadruplex folding, with effects on Tm as high as 17°C. Sequences containing a longer central loop have a high propensity to adopt a stable non-parallel topology. Conversely, sequences containing a short central loop tend to form a parallel topology of lower stability. In addition, over half of interrogated sequences were found in the genomes of diverse organisms, implicating their potential regulatory roles in the genome or as therapeutic targets. This study illustrates the structural roles of loops in G-quadruplex folding and should help to establish rules to predict the folding pattern and stability of G-quadruplexes.

• MeSH
• algoritmy MeSH
• cirkulární dichroismus MeSH
• DNA chemie   MeSH
• G-kvadruplexy * MeSH
• konformace nukleové kyseliny * MeSH
• sekvence nukleotidů MeSH
• termodynamika * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH