Amelogenesis imperfecta (AI) is a heterogeneous group of genetic rare diseases disrupting enamel development (Smith et al., Front Physiol, 2017a, 8, 333). The clinical enamel phenotypes can be described as hypoplastic, hypomineralized or hypomature and serve as a basis, together with the mode of inheritance, to Witkop's classification (Witkop, J Oral Pathol, 1988, 17, 547-553). AI can be described in isolation or associated with others symptoms in syndromes. Its occurrence was estimated to range from 1/700 to 1/14,000. More than 70 genes have currently been identified as causative. Objectives: We analyzed using next-generation sequencing (NGS) a heterogeneous cohort of AI patients in order to determine the molecular etiology of AI and to improve diagnosis and disease management. Methods: Individuals presenting with so called "isolated" or syndromic AI were enrolled and examined at the Reference Centre for Rare Oral and Dental Diseases (O-Rares) using D4/phenodent protocol (www.phenodent.org). Families gave written informed consents for both phenotyping and molecular analysis and diagnosis using a dedicated NGS panel named GenoDENT. This panel explores currently simultaneously 567 genes. The study is registered under NCT01746121 and NCT02397824 (https://clinicaltrials.gov/). Results: GenoDENT obtained a 60% diagnostic rate. We reported genetics results for 221 persons divided between 115 AI index cases and their 106 associated relatives from a total of 111 families. From this index cohort, 73% were diagnosed with non-syndromic amelogenesis imperfecta and 27% with syndromic amelogenesis imperfecta. Each individual was classified according to the AI phenotype. Type I hypoplastic AI represented 61 individuals (53%), Type II hypomature AI affected 31 individuals (27%), Type III hypomineralized AI was diagnosed in 18 individuals (16%) and Type IV hypoplastic-hypomature AI with taurodontism concerned 5 individuals (4%). We validated the genetic diagnosis, with class 4 (likely pathogenic) or class 5 (pathogenic) variants, for 81% of the cohort, and identified candidate variants (variant of uncertain significance or VUS) for 19% of index cases. Among the 151 sequenced variants, 47 are newly reported and classified as class 4 or 5. The most frequently discovered genotypes were associated with MMP20 and FAM83H for isolated AI. FAM20A and LTBP3 genes were the most frequent genes identified for syndromic AI. Patients negative to the panel were resolved with exome sequencing elucidating for example the gene involved ie ACP4 or digenic inheritance. Conclusion: NGS GenoDENT panel is a validated and cost-efficient technique offering new perspectives to understand underlying molecular mechanisms of AI. Discovering variants in genes involved in syndromic AI (CNNM4, WDR72, FAM20A … ) transformed patient overall care. Unravelling the genetic basis of AI sheds light on Witkop's AI classification.

• Klíčová slova
• NGS, amelogenesis imperfecta, enamel, genetics, next-generation sequencing, rare diseases,
• Publikační typ
• časopisecké články MeSH

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.

• Klíčová slova
• AND-gate, CRAC channel, Electrophysiology, Gating, Gating checkpoints, Opening-permissive conformation, Orai1, STIM1, Signal propagation,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• fosfatidylcholiny chemie   metabolismus   MeSH
• gating iontového kanálu genetika   MeSH
• genetické vektory chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• lidé MeSH
• liposomy chemie   metabolismus   MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• metoda terčíkového zámku MeSH
• mutace MeSH
• nádorové proteiny chemie   genetika   metabolismus   MeSH
• protein ORAI1 chemie   genetika   metabolismus   MeSH
• protein STIM1 chemie   genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• reportérové geny MeSH
• simulace molekulární dynamiky MeSH
• substituce aminokyselin MeSH
• vápník metabolismus   MeSH
• vápníková signalizace * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1-palmitoyl-2-oleoylphosphatidylcholine MeSH Prohlížeč
• bakteriální proteiny MeSH
• enhanced cyan fluorescent protein MeSH Prohlížeč
• fosfatidylcholiny MeSH
• liposomy MeSH
• luminescentní proteiny MeSH
• nádorové proteiny MeSH
• ORAI1 protein, human MeSH Prohlížeč
• protein ORAI1 MeSH
• protein STIM1 MeSH
• rekombinantní proteiny MeSH
• STIM1 protein, human MeSH Prohlížeč
• vápník MeSH
• yellow fluorescent protein, Bacteria MeSH Prohlížeč
• zelené fluorescenční proteiny MeSH

Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73-91; Orai3 amino acids 48-65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.

• Klíčová slova
• atomic force microscopy (AFM), calcium release-activated calcium channel protein 1 (ORAI1), electrophysiology, signal transduction, stromal interaction molecule 1 (STIM1),
• MeSH
• HEK293 buňky MeSH
• lidé MeSH
• nádorové proteiny chemie   genetika   metabolismus   MeSH
• protein ORAI1 chemie   genetika   metabolismus   MeSH
• protein STIM1 chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• sekundární struktura proteinů MeSH
• vápníkové kanály chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorové proteiny MeSH
• ORAI1 protein, human MeSH Prohlížeč
• Orai3 protein, human MeSH Prohlížeč
• protein ORAI1 MeSH
• protein STIM1 MeSH
• STIM1 protein, human MeSH Prohlížeč
• vápníkové kanály MeSH

The channel Orai1 requires Ca2+ store depletion in the endoplasmic reticulum and an interaction with the Ca2+ sensor STIM1 to mediate Ca2+ signaling. Alterations in Orai1-mediated Ca2+ influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation. Our analysis showed that certain Orai1 mutations were clustered in the transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca2+ influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region. Together, these results enabled us to identify a cluster of Orai1 mutations that trigger Ca2+ permeation associated with gene transcription and provide a gating mechanism for Orai1.

• MeSH
• aktivace transkripce genetika   MeSH
• arginin metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• Drosophila melanogaster MeSH
• gating iontového kanálu genetika   MeSH
• genomika MeSH
• HCT116 buňky MeSH
• HEK293 buňky MeSH
• lidé MeSH
• metoda terčíkového zámku MeSH
• mutace MeSH
• nádorové proteiny genetika   metabolismus   MeSH
• nádory metabolismus   MeSH
• nemoci svalů metabolismus   MeSH
• protein ORAI1 genetika   metabolismus   MeSH
• protein STIM1 genetika   metabolismus   MeSH
• sekundární struktura proteinů genetika   MeSH
• simulace molekulární dynamiky MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• arginin MeSH
• nádorové proteiny MeSH
• ORAI1 protein, human MeSH Prohlížeč
• protein ORAI1 MeSH
• protein STIM1 MeSH
• STIM1 protein, human MeSH Prohlížeč
• vápník MeSH

The Ca(2+) release-activated Ca(2+) channel mediates Ca(2+) influx in a plethora of cell types, thereby controlling diverse cellular functions. The channel complex is composed of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum Ca(2+)-sensing protein, and Orai1, a plasma membrane Ca(2+) channel. Channels composed of STIM1 and Orai1 mediate Ca(2+) influx even at low extracellular Ca(2+) concentrations. We investigated whether the activity of Orai1 adapted to different environmental Ca(2+) concentrations. We used homology modeling and molecular dynamics simulations to predict the presence of an extracellular Ca(2+)-accumulating region (CAR) at the pore entrance of Orai1. Furthermore, simulations of Orai1 proteins with mutations in CAR, along with live-cell experiments, or simulations and electrophysiological recordings of the channel with transient, electrostatic loop3 interacting with loop1 (the site of CAR) determined that CAR enhanced Ca(2+) permeation most efficiently at low external Ca(2+) concentrations. Consistent with these results, cells expressing Orai1 CAR mutants exhibited impaired gene expression stimulated by the Ca(2+)-activated transcription factor nuclear factor of activated T cells (NFAT). We propose that the Orai1 channel architecture with a close proximity of CAR to the selectivity filter, which enables Ca(2+)-selective ion permeation, enhances the local extracellular Ca(2+) concentration to maintain Ca(2+)-dependent gene regulation even in environments with relatively low Ca(2+)concentrations.

• MeSH
• Drosophila melanogaster MeSH
• genetická transkripce fyziologie   MeSH
• HEK293 buňky MeSH
• iontový transport fyziologie   MeSH
• lidé MeSH
• membránové proteiny * chemie   genetika   metabolismus   MeSH
• permeabilita buněčné membrány fyziologie   MeSH
• protein ORAI1 MeSH
• protein STIM1 MeSH
• proteiny Drosophily * chemie   genetika   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny * MeSH
• olf186-F protein, Drosophila MeSH Prohlížeč
• protein ORAI1 MeSH
• protein STIM1 MeSH
• proteiny Drosophily * MeSH
• Stim protein, Drosophila MeSH Prohlížeč
• vápník MeSH