regulatory loop Dotaz Zobrazit nápovědu
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.
- 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
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.
- 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.
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.
- 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
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.
- 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
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.
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.
- MeSH
- DNA-dependentní DNA-polymerasy chemie fyziologie MeSH
- DNA-polymerasa III chemie fyziologie 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
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.
- 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
We examine the modular structure of the metabolic network when combined with the regulatory network representing direct regulation of enzymes by small metabolites in E. coli. We introduce novel clustering algorithm and compare it with mainstream module detection method based on simulated annealing. Both methods identify the similar modular core. Slight but significant increase in modularity is observed after regulatory interactions addition. We also identify new functional modules in the combined network, which cannot be detected in the metabolic network only. Regulatory loops in the modules are the source of their autonomy, and allow us to hypothesise about module function.
