σ factors are essential parts of bacterial RNA polymerase (RNAP) as they allow to recognize promotor sequences and initiate transcription. Domain 1.1 of vegetative σ factors occupies the primary channel of RNAP and also prevents binding of the σ factor to promoter DNA alone. Here, we show that domain 1.1 of Bacillus subtilis σA exists in more structurally distinct variants in dynamic equilibrium. The major conformation at room temperature is represented by a previously reported well-folded structure solved by nuclear magnetic resonance (NMR), but 4% of the protein molecules are present in a less thermodynamically favorable state. We show that this population increases with temperature and we predict its significant elevation at higher but still biologically relevant temperatures. We characterized the minor state of the domain 1.1 using specialized methods of NMR. We found that, in contrast to the major state, the detected minor state is partially unfolded. Its propensity to form secondary structure elements is especially decreased for the first and third α helices, while the second α helix and β strand close to the C-terminus are more stable. We also analyzed thermal unfolding of the domain 1.1 and performed functional experiments with full length σA and its shortened version lacking domain 1.1 ( σA_Δ1.1 ). The results revealed that while full length σA increases transcription activity of RNAP with increasing temperature, transcription with σA_Δ1.1 remains constant. In summary, this study reveals conformational dynamics of domain 1.1 and provides a basis for studies of its interaction with RNAP and effects on transcription regulation.
- MeSH
- amidy metabolismus MeSH
- Bacillus subtilis * enzymologie MeSH
- DNA řízené RNA-polymerasy * chemie metabolismus MeSH
- molekulární modely MeSH
- proteinové domény MeSH
- protony MeSH
- rozbalení proteinů * MeSH
- sigma faktor * chemie metabolismus MeSH
- teplota * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
P2X receptors (P2X1-7) are trimeric ion channels activated by extracellular ATP. Each P2X subunit contains two transmembrane helices (TM1 and TM2). We substituted all residues in TM1 of rat P2X7 with alanine or leucine one by one, expressed mutants in HEK293T cells, and examined the pore permeability by recording both membrane currents and fluorescent dye uptake in response to agonist application. Alanine substitution of G27, K30, H34, Y40, F43, L45, M46, and D48 inhibited agonist-stimulated membrane current and dye uptake, and all but one substitution, D48A, prevented surface expression. Mutation V41A partially reduced both membrane current and dye uptake, while W31A and A44L showed reduced dye uptake not accompanied by reduced membrane current. Mutations T28A, I29A, and L33A showed small changes in agonist sensitivity, but they had no or small impact on dye uptake function. Replacing charged residues with residues of the same charge (K30R, H34K, and D48E) rescued receptor function, while replacement with residues of opposite charge inhibited (K30E and H34E) or potentiated (D48K) receptor function. Prolonged stimulation with agonist-induced current facilitation and a leftward shift in the dose-response curve in the P2X7 wild-type and most functional mutants, but sensitization was absent in the W31A, L33A, and A44L. Detailed analysis of the decay of responses revealed two kinetically distinct mechanisms of P2X7 deactivation: fast represents agonist unbinding, and slow might represent resetting of the receptor to the resting closed state. These results indicate that conserved and receptor-specific TM1 residues control surface expression of the P2X7 protein, non-polar residues control receptor sensitization, and D48 regulates intrinsic channel properties.
- MeSH
- adenosintrifosfát farmakologie metabolismus MeSH
- biologický transport MeSH
- HEK293 buňky MeSH
- iontové kanály * metabolismus MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- mutace genetika MeSH
- proteinové domény MeSH
- purinergní receptory P2X7 * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Nucleos(t)ide analogues entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are recommended as first-line monotherapies for chronic hepatitis B (CHB). Multiple HBV genotypes/subgenotypes have been described, but their impact on treatment response remains largely elusive. We investigated the effectiveness of ETV/TDF on HBV/D-subgenotypes, D1/D2/D3/D5, studied the structural/functional differences in subgenotype-specific reverse transcriptase (RT) domains of viral polymerase, and identified novel molecules with robust inhibitory activity on various D-subgenotypes. Transfection of Huh7 cells with full-length D1/D2/D3/D5 and in vitro TDF/ETV susceptibility assays demonstrated that D1/D2 had greater susceptibility to TDF/ETV while D3/D5 exhibited poorer response. Additionally, HBV load was substantially reduced in TDF-treated CHB patients carrying D1/D2 but minimally reduced in D3/D5-infected patients. Comparison of RT sequences of D-subgenotypes led to identification of unique subgenotype-specific residues, and molecular modeling/docking/simulation studies depicted differential bindings of TDF/ETV to the active site of their respective RTs. Replacement of signature residues in D3/D5 HBV clones with corresponding amino acids seen in D1/D2 improved their susceptibility to TDF/ETV. Using high throughput virtual screening, we identified N(9)-[3-fluoro-2-(phosphonomethoxy)propyl] (FPMP) derivatives of purine bases, including N6-substituted (S)-FPMP derivative of 2,6-diaminopurine (DAP) (OB-123-VK), as potential binders of RT of different D-subgenotypes. We synthesized (S)-FPMPG prodrugs (FK-381-FEE/FK-381-SEE/FK-382) and tested their effectiveness along with OB-123-VK. Both OB-123-VK and FK-381-FEE exerted similar antiviral activities against all D-subgenotypes, although FK-381-FEE was more potent. Our study highlighted the natural variation in therapeutic response of D1/D2/D3/D5 and emphasized the need for HBV subgenotype determination before treatment. Novel molecules described here could benefit future design/discovery of pan-D-subgenotypic inhibitors. IMPORTANCE Current treatment of chronic hepatitis B relies heavily on nucleotide/nucleoside analogs in particular, tenofovir disoproxil fumarate (TDF) and entecavir (ETV) to keep HBV replication under control and prevent end-stage liver diseases. However, it was unclear whether the therapeutic effects of TDF/ETV differ among patients infected with different HBV genotypes and subgenotypes. HBV genotype D is the most widespread of all HBV genotypes and multiple D-subgenotypes have been described. We here report that different subgenotypes of HBV genotype-D exhibit variable response toward TDF and ETV and this could be attributed to naturally occurring amino acid changes in the reverse transcriptase domain of the subgenotype-specific polymerase. Further, we identified novel molecules and also synthesized prodrugs that are equally effective on different D-subgenotypes and could facilitate management of HBV/D-infected patients irrespective of D-subgenotype.
- MeSH
- antivirové látky chemie farmakologie terapeutické užití MeSH
- chronická hepatitida B farmakoterapie virologie MeSH
- genotyp MeSH
- guanin analogy a deriváty chemie farmakologie terapeutické užití MeSH
- inhibitory reverzní transkriptasy chemie farmakologie terapeutické užití MeSH
- lidé MeSH
- mutace MeSH
- organofosfonáty chemie farmakologie MeSH
- prekurzory léčiv MeSH
- proteinové domény MeSH
- racionální návrh léčiv * MeSH
- reverzní transkriptasa chemie genetika MeSH
- tenofovir chemie farmakologie terapeutické užití MeSH
- virová léková rezistence účinky léků genetika MeSH
- virová nálož účinky léků MeSH
- virus hepatitidy B účinky léků enzymologie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cilia formation is essential for human life. One of the earliest events in the ciliogenesis program is the recruitment of tau-tubulin kinase 2 (TTBK2) by the centriole distal appendage component CEP164. Due to the lack of high-resolution structural information on this complex, it is unclear how it is affected in human ciliopathies such as nephronophthisis. Furthermore, it is poorly understood if binding to CEP164 influences TTBK2 activities. Here, we present a detailed biochemical, structural, and functional analysis of the CEP164-TTBK2 complex and demonstrate how it is compromised by two ciliopathic mutations in CEP164. Moreover, we also provide insights into how binding to CEP164 is coordinated with TTBK2 activities. Together, our data deepen our understanding of a crucial step in cilia formation and will inform future studies aimed at restoring CEP164 functionality in a debilitating human ciliopathy.
- MeSH
- ciliopatie genetika MeSH
- cirkulární dichroismus MeSH
- HEK293 buňky MeSH
- konformace proteinů MeSH
- lidé MeSH
- mikrotubulární proteiny chemie genetika metabolismus MeSH
- molekulární modely MeSH
- mutace * MeSH
- protein-serin-threoninkinasy chemie metabolismus MeSH
- proteinové domény MeSH
- proteiny asociované s mikrotubuly metabolismus MeSH
- stabilita proteinů MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Proteins are naturally formed by domains edging their functional and structural properties. A domain out of the context of an entire protein can retain its structure and to some extent also function on its own. These properties rationalize construction of artificial fusion multidomain proteins with unique combination of various functions. Information on the specific functional and structural characteristics of individual domains in the context of new artificial fusion proteins is inevitably encoded in sequential order of composing domains defining their mutual spatial positions. So the challenges in designing new proteins with new domain combinations lie dominantly in structure/function prediction and its context dependency. Despite the enormous body of publications on artificial fusion proteins, the task of their structure/function prediction is complex and nontrivial. The degree of spatial freedom facilitated by a linker between domains and their mutual orientation driven by noncovalent interactions is beyond a simple and straightforward methodology to predict their structure with reasonable accuracy. In the presented manuscript, we tested methodology using available modeling tools and computational methods. We show that the process and methodology of such prediction are not straightforward and must be done with care even when recently introduced AlphaFold II is used. We also addressed a question of benchmarking standards for prediction of multidomain protein structures-x-ray or Nuclear Magnetic Resonance experiments. On the study of six two-domain protein chimeras as well as their composing domains and their x-ray structures selected from PDB, we conclude that the major obstacle for justified prediction is inappropriate sampling of the conformational space by the explored methods. On the other hands, we can still address particular steps of the methodology and improve the process of chimera proteins prediction.
Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility." Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular "spindle and thread" mechanism unblocks protein translation in vitro.
- MeSH
- lidé MeSH
- nádorový supresorový protein p53 * metabolismus MeSH
- nádory * MeSH
- proteinové domény MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Due to the fast global spreading of the Severe Acute Respiratory Syndrome Coronavirus - 2 (SARS-CoV-2), prevention and treatment options are direly needed in order to control infection-related morbidity, mortality, and economic losses. Although drug and inactivated and attenuated virus vaccine development can require significant amounts of time and resources, DNA and RNA vaccines offer a quick, simple, and cheap treatment alternative, even when produced on a large scale. The spike protein, which has been shown as the most antigenic SARS-CoV-2 protein, has been widely selected as the target of choice for DNA/RNA vaccines. Vaccination campaigns have reported high vaccination rates and protection, but numerous unintended effects, ranging from muscle pain to death, have led to concerns about the safety of RNA/DNA vaccines. In parallel to these studies, several open reading frames (ORFs) have been found to be overlapping SARS-CoV-2 accessory genes, two of which, ORF2b and ORF-Sh, overlap the spike protein sequence. Thus, the presence of these, and potentially other ORFs on SARS-CoV-2 DNA/RNA vaccines, could lead to the translation of undesired proteins during vaccination. Herein, we discuss the translation of overlapping genes in connection with DNA/RNA vaccines. Two mRNA vaccine spike protein sequences, which have been made publicly-available, were compared to the wild-type sequence in order to uncover possible differences in putative overlapping ORFs. Notably, the Moderna mRNA-1273 vaccine sequence is predicted to contain no frameshifted ORFs on the positive sense strand, which highlights the utility of codon optimization in DNA/RNA vaccine design to remove undesired overlapping ORFs. Since little information is available on ORF2b or ORF-Sh, we use structural bioinformatics techniques to investigate the structure-function relationship of these proteins. The presence of putative ORFs on DNA/RNA vaccine candidates implies that overlapping genes may contribute to the translation of smaller peptides, potentially leading to unintended clinical outcomes, and that the protein-coding potential of DNA/RNA vaccines should be rigorously examined prior to administration.
- MeSH
- DNA vakcíny škodlivé účinky genetika MeSH
- glykoprotein S, koronavirus genetika MeSH
- kodon MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- messenger RNA MeSH
- mRNA vakcíny škodlivé účinky genetika MeSH
- otevřené čtecí rámce MeSH
- překrývající se geny * MeSH
- proteinové domény MeSH
- proteosyntéza MeSH
- vakcíny proti COVID-19 škodlivé účinky genetika MeSH
- virové geny * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
[Figure: see text].
- MeSH
- adaptorové proteiny signální transdukční chemie metabolismus MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- elongace genetické transkripce * MeSH
- exprese genu MeSH
- interakční proteinové domény a motivy genetika MeSH
- lidé MeSH
- mapy interakcí proteinů MeSH
- molekulární modely MeSH
- mutace MeSH
- nádorové buněčné linie MeSH
- proteinové domény MeSH
- proteiny vázající RNA chemie genetika metabolismus MeSH
- RNA-polymerasa II chemie metabolismus MeSH
- transkripční elongační faktory chemie metabolismus MeSH
- transkripční faktory chemie genetika metabolismus MeSH
- vazba proteinů MeSH
- vnitřně neuspořádané proteiny chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.
- MeSH
- buněčné linie MeSH
- fosforylace MeSH
- genetická transkripce MeSH
- genový knockdown MeSH
- lidé MeSH
- myši knockoutované MeSH
- neurony chemie metabolismus MeSH
- posttranskripční úpravy RNA MeSH
- proteinové domény MeSH
- regulace genové exprese MeSH
- RNA-polymerasa II chemie genetika metabolismus MeSH
- RNA * chemie genetika metabolismus MeSH
- stabilita RNA MeSH
- transkripční faktory genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Surgery is an efficient way to treat localized prostate cancer (PCa), however, it is challenging to demarcate rapidly and accurately the tumor boundary intraoperatively, as existing tumor detection methods are seldom performed in real-time. To overcome those limitations, we develop a fluorescent molecular rotor that specifically targets the prostate-specific membrane antigen (PSMA), an established marker for PCa. The probes have picomolar affinity (IC50 = 63-118 pM) for PSMA and generate virtually instantaneous onset of robust fluorescent signal proportional to the concentration of the PSMA-probe complex. In vitro and ex vivo experiments using PCa cell lines and clinical samples, respectively, indicate the utility of the probe for biomedical applications, including real-time monitoring of endocytosis and tumor staging. Experiments performed in a PCa xenograft model reveal suitability of the probe for imaging applications in vivo.
- MeSH
- antigeny povrchové chemie metabolismus MeSH
- buňky PC-3 MeSH
- endocytóza MeSH
- fluorescenční spektrometrie metody MeSH
- glutamátkarboxypeptidasa II chemie metabolismus MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sondy chemie metabolismus MeSH
- myši inbrední BALB C MeSH
- myši nahé MeSH
- nádorové buněčné linie MeSH
- nádory prostaty diagnóza metabolismus MeSH
- optické zobrazování metody MeSH
- proteinové domény MeSH
- transplantace heterologní MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
