Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induces permeation of Orai Ca2+ channels upon endoplasmic reticulum Ca2+-store depletion. A drop in luminal Ca2+ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca2+ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca2+-release-activated Ca2+ currents (ICRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors microphthalmia-associated transcription factor (MITF) and transcription factor EB (TFEB) into the nucleus. These STIM1 mutations additionally stimulated an enhanced production of autophagosomes. In summary, mutations in STIM1 that cause structural unfolding promoted Ca2+ down-stream activation of autophagic processes.

• Klíčová slova
• Ca2+, EF-hand, MITF, Orai, SOCE, STIM, TFEB, hydrophobic pocket, tubular aggregate myopathy,
• MeSH
• autofagie * MeSH
• kationty dvojmocné metabolismus   MeSH
• konformace proteinů, alfa-helix MeSH
• lidé MeSH
• motivy EF-ruky MeSH
• mutace MeSH
• myopatie strukturální vrozené genetika   metabolismus   MeSH
• nádorové proteiny chemie   genetika   metabolismus   MeSH
• protein STIM1 chemie   genetika   metabolismus   MeSH
• rozbalení proteinů MeSH
• simulace molekulární dynamiky MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kationty dvojmocné MeSH
• nádorové proteiny MeSH
• protein STIM1 MeSH
• STIM1 protein, human MeSH Prohlížeč
• vápník MeSH

The stromal interaction molecule 1 (STIM1) has two important functions, Ca2+ sensing within the endoplasmic reticulum and activation of the store-operated Ca2+ channel Orai1, enabling plasma-membrane Ca2+ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca2+-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca2+ ions. In MD simulations, a single Ca2+ ion was sufficient to stabilize the luminal STIM1 complex. Ca2+ store depletion destabilized the two EF hands, triggering disassembly of the hydrophobic cleft that they form together with the stable SAM domain. Point mutations associated with tubular aggregate myopathy or cancer that targeted the canonical EF hand, and the hydrophobic cleft yielded constitutively clustered STIM1, which was associated with activation of Ca2+ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca2+ binding and refine the currently known initial steps of STIM1 activation on a molecular level.

• MeSH
• algoritmy MeSH
• buněčná membrána metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• HEK293 buňky MeSH
• hydrofobní a hydrofilní interakce MeSH
• konfokální mikroskopie MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• motivy EF-ruky MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny chemie   genetika   metabolismus   MeSH
• protein ORAI1 chemie   metabolismus   MeSH
• protein STIM1 chemie   genetika   metabolismus   MeSH
• proteinové domény * MeSH
• rozbalení proteinů * MeSH
• simulace molekulární dynamiky * MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorové proteiny MeSH
• protein ORAI1 MeSH
• protein STIM1 MeSH
• STIM1 protein, human MeSH Prohlížeč
• vápník MeSH

The detailed functional mechanism of recoverin, which acts as a myristoyl switch at the rod outer-segment disk membrane, is elucidated by direct and replica-exchange molecular dynamics. In accord with NMR structural evidence and calcium binding assays, simulations point to the key role of enhanced calcium binding to the EF3 loop of the semiopen state of recoverin as compared to the closed state. This 2-4-order decrease in calcium dissociation constant stabilizes the semiopen state in response to the increase of cytosolic calcium concentration in the vicinity of recoverin. A second calcium ion then binds to the EF2 loop and, consequently, the structure of the protein changes from the semiopen to the open state. The latter has the myristoyl chain extruded to the cytosol, ready to act as a membrane anchor of recoverin.

• MeSH
• komplexní sloučeniny chemie   metabolismus   MeSH
• konformace proteinů MeSH
• magnetická rezonanční spektroskopie MeSH
• motivy EF-ruky MeSH
• mutace MeSH
• rekoverin chemie   genetika   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• skot MeSH
• termodynamika MeSH
• vápník chemie   metabolismus   MeSH
• vazba proteinů MeSH
• změna skupenství MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• komplexní sloučeniny MeSH
• rekoverin MeSH
• vápník MeSH

Trehalases hydrolyze the non-reducing disaccharide trehalose amassed by cells as a universal protectant and storage carbohydrate. Recently, it has been shown that the activity of neutral trehalase Nth1 from Saccharomyces cerevisiae is mediated by the 14-3-3 protein binding that modulates the structure of both the catalytic domain and the region containing the EF-hand-like motif, whose role in the activation of Nth1 is unclear. In this work, the structure of the Nth1·14-3-3 complex and the importance of the EF-hand-like motif were investigated using site-directed mutagenesis, hydrogen/deuterium exchange coupled to mass spectrometry, chemical cross-linking, and small angle x-ray scattering. The low resolution structural views of Nth1 alone and the Nth1·14-3-3 complex show that the 14-3-3 protein binding induces a significant structural rearrangement of the whole Nth1 molecule. The EF-hand-like motif-containing region forms a separate domain that interacts with both the 14-3-3 protein and the catalytic trehalase domain. The structural integrity of the EF-hand like motif is essential for the 14-3-3 protein-mediated activation of Nth1, and calcium binding, although not required for the activation, facilitates this process by affecting its structure. Our data suggest that the EF-hand like motif-containing domain functions as the intermediary through which the 14-3-3 protein modulates the function of the catalytic domain of Nth1.

• Klíčová slova
• 14–3-3, Bmh, Calcium, Enzyme Mechanisms, H/D Exchange, Mass Spectrometry (MS), Neutral Trehalase, Protein Cross-linking, Protein Structure, SAXS,
• MeSH
• aktivace enzymů MeSH
• katalytická doména MeSH
• molekulární modely MeSH
• motivy EF-ruky * MeSH
• proteiny 14-3-3 chemie   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae enzymologie   MeSH
• sekvence aminokyselin MeSH
• trehalasa chemie   metabolismus   MeSH
• vápník metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BMH1 protein, S cerevisiae MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• trehalasa MeSH
• vápník MeSH

The transient receptor potential channel A1 (TRPA1) is unique among ion channels of higher vertebrates in that it harbors a large ankyrin repeat domain. The TRPA1 channel is expressed in the inner ear and in nociceptive neurons. It is involved in hearing as well as in the perception of pungent and irritant chemicals. The ankyrin repeat domain has special mechanical properties, which allows it to function as a soft spring that can be extended over a large range while maintaining structural integrity. A calcium-binding site has been experimentally identified within the ankyrin repeats. We built a model of the N-terminal 17 ankyrin repeat structure, including the calcium-binding EF-hand. In our simulations we find the calcium-bound state to be rigid as compared to the calcium-free state. While the end-to-end distance can change by almost 50% in the apo form, these fluctuations are strongly reduced by calcium binding. This increase in stiffness that constraints the end-to-end distance in the holo form is predicted to affect the force acting on the gate of the TRPA1 channel, thereby changing its open probability. Simulations of the transmembrane domain of TRPA1 show that residue N855, which has been associated with familial episodic pain syndrome, forms a strong link between the S4-S5 connecting helix and S1, thereby creating a direct force link between the N-terminus and the gate. The N855S mutation weakens this interaction, thereby reducing the communication between the N-terminus and the transmembrane part of TRPA1.

• MeSH
• ankyrinová repetice fyziologie   MeSH
• kationtové kanály TRP chemie   fyziologie   MeSH
• kationtový kanál TRPA1 MeSH
• lidé MeSH
• molekulární modely MeSH
• motivy EF-ruky fyziologie   MeSH
• proteiny nervové tkáně chemie   fyziologie   MeSH
• simulace molekulární dynamiky MeSH
• vápník metabolismus   MeSH
• vápníkové kanály chemie   fyziologie   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kationtové kanály TRP MeSH
• kationtový kanál TRPA1 MeSH
• proteiny nervové tkáně MeSH
• TRPA1 protein, human MeSH Prohlížeč
• vápník MeSH
• vápníkové kanály MeSH

An arsenic (ars) four-gene operon, containing genes encoding a putative membrane permease (ArsP), a transcriptional repressor (ArsR), an arsenate reductase (ArsC) and an arsenical-resistance membrane transporter (Acr3) was first identified in urease-positive thermophilic Campylobacter (UPTC) isolate, CF89-12. UPTC CF89-12 and some other Campylobacter lari isolates contained their ars four-genes, similarly, differing from that in the reference C. lari RM2100 strain. Two putative promoters and a putative terminator were identified for the operon in UPTC CF89-12. In vivo transcription of the operon was confirmed in the UPTC cells. PCR experiments using two primer pairs designed in silico to amplify two arsR and arsC-acr3 segments, respectively, generated two amplicons, approximately 200 and 350 base pairs, with all 31 of 31 and 19 of 31 C. lari isolates (n = 17 for UPTC; n = 14 for UN C. lari), respectively. An inverted repeat forming a dyad structure, a potential binding site for a transcriptional repressor, was identified in the promoter region. Within the deduced 61 amino acids sequence of the putative arsR open reading frame from the UPTC CF89-12, a metal binding box and a DNA-binding helix-turn-helix motif were identified. The UPTC CF89-12 and some other UPTC isolates isolated from natural environment were resistant to arsenate.

• MeSH
• arsen * MeSH
• arsenátreduktasy genetika   MeSH
• bakteriální geny * MeSH
• bakteriální RNA genetika   MeSH
• Campylobacter lari genetika   izolace a purifikace   MeSH
• DNA bakterií genetika   MeSH
• DNA primery MeSH
• genetické lokusy MeSH
• konformace nukleové kyseliny MeSH
• molekulární sekvence - údaje MeSH
• motiv helix-turn-helix genetika   MeSH
• operon genetika   MeSH
• otevřené čtecí rámce MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• sekvenční seřazení MeSH
• ureasa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• arsen * MeSH
• arsenátreduktasy MeSH
• bakteriální RNA MeSH
• DNA bakterií MeSH
• DNA primery MeSH
• ureasa MeSH

Association of the NEUROD Ala45Thr polymorphism with Type 1 diabetes mellitus (DM) has been found in some but not all populations. We performed a study on the association of two NEUROD exon 2 polymorphisms, the Ala45Thr and the Pro197His, with childhood-onset Type 1 DM in the Czech population. We compared 285 children with Type 1 DM diagnosed under the age of 15 years with 289 non-diabetic control children. The genotypes were determined using novel real-time allele-specific PCR assays in the TaqMan format, and data were analysed using logistic regression. The numbers of subjects with codon 45 genotypes Ala/Ala, Ala/Thr, Thr/Thr were 95, 145, 45 among cases and 117, 130, 42 among controls. Thr45 phenotypic positivity was associated with a significant risk of Type 1 DM (OR=2.01, CI 95% 1.25-3.24) in a multivariate logistic regression model involving also the insulin gene -23HphI genotype and the presence of Type 1 DM-associated HLA-DQB1*0302-DQA1*03 (DQ8) and DQB1*0201-DQA1*05 (DQ2) molecules. No association was observed for the Pro197His mutation which was carried by 5.3% cases and 5.9% controls. Our results confirm that the NEUROD Ala45Thr polymorphism is associated with childhood-onset Type 1 DM.

• MeSH
• alanin MeSH
• diabetes mellitus 1. typu genetika   MeSH
• dítě MeSH
• DNA primery MeSH
• frekvence genu MeSH
• HLA-DQ antigeny genetika   MeSH
• inzulin genetika   MeSH
• lidé MeSH
• missense mutace * MeSH
• mladiství MeSH
• motiv helix-loop-helix MeSH
• polymerázová řetězová reakce metody   MeSH
• proteiny nervové tkáně genetika   MeSH
• regresní analýza MeSH
• sekvence nukleotidů MeSH
• threonin MeSH
• transkripční faktory bHLH MeSH
• věk při počátku nemoci MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• alanin MeSH
• DNA primery MeSH
• HLA-DQ antigeny MeSH
• inzulin MeSH
• Neurogenic differentiation factor 1 MeSH Prohlížeč
• proteiny nervové tkáně MeSH
• threonin MeSH
• transkripční faktory bHLH MeSH