N-methyl-d-aspartate receptors (NMDARs) play an essential role in regulating glutamatergic neurotransmission. Recently, pathogenic missense mutations were identified in genes encoding NMDAR subunits; however, their effect on NMDAR activity is often poorly understood. Here, we examined whether three previously identified pathogenic mutations (M641I, A645S, and Y647S) in the M3 domain of the GluN1 subunit affect the receptor's surface delivery, agonist sensitivity, Mg2+ block, and/or inhibition by the FDA-approved NMDAR blocker memantine. When expressed in HEK293 cells, we found reduced surface expression of GluN1-M641I/GluN2A, GluN1-Y647S/GluN2A, and GluN1-Y647S/GluN2B receptors; other mutation-bearing NMDAR combinations, including GluN1/GluN3A receptors, were expressed at normal surface levels. When expressed in rat hippocampal neurons, we consistently found reduced surface expression of the GluN1-M641I and GluN1-Y647S subunits when compared with wild-type GluN1 subunit. At the functional level, we found that GluN1-M641I/GluN2 and GluN1-A645S/GluN2 receptors expressed in HEK293 cells have wild-type EC50 values for both glutamate and glycine; in contrast, GluN1-Y647S/GluN2 receptors do not produce glutamate-induced currents. In the presence of a physiological concentration of Mg2+, we found that GluN1-M641I/GluN2 receptors have a lower memantine IC50 and slower offset kinetics, whereas GluN1-A645S/GluN2 receptors have a higher memantine IC50 and faster offset kinetics when compared to wild-type receptors. Finally, we found that memantine was the most neuroprotective in hippocampal neurons expressing GluN1-M641I subunits, followed by neurons expressing wild-type GluN1 and then GluN1-A645S subunits in an NMDA-induced excitotoxicity assay. These results indicate that specific pathogenic mutations in the M3 domain of the GluN1 subunit differentially affect the trafficking and functional properties of NMDARs.
- MeSH
- agonisté excitačních aminokyselin aplikace a dávkování MeSH
- antagonisté excitačních aminokyselin aplikace a dávkování MeSH
- HEK293 buňky MeSH
- hipokampus účinky léků fyziologie MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lidé MeSH
- mutace účinky léků genetika MeSH
- podjednotky proteinů agonisté antagonisté a inhibitory genetika MeSH
- potkani Wistar MeSH
- povrchové vlastnosti účinky léků MeSH
- proteiny nervové tkáně agonisté antagonisté a inhibitory genetika MeSH
- receptory N-methyl-D-aspartátu agonisté antagonisté a inhibitory genetika MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Paragangliomas and pheochromocytomas (PPGLs) are chromaffin tumors associated with severe catecholamine-induced morbidities. Surgical removal is often curative. However, complete resection may not be an option for patients with succinate dehydrogenase subunit A-D (SDHx) mutations. SDHx mutations are associated with a high risk for multiple recurrent, and metastatic PPGLs. Treatment options in these cases are limited and prognosis is dismal once metastases are present. Identification of new therapeutic targets and candidate drugs is thus urgently needed. Previously, we showed elevated expression of succinate receptor 1 (SUCNR1) in SDHB PPGLs and SDHD head and neck paragangliomas. Its ligand succinate has been reported to accumulate due to SDHx mutations. We thus hypothesize that autocrine stimulation of SUCNR1 plays a role in the pathogenesis of SDHx mutation-derived PPGLs. We confirmed elevated SUCNR1 expression in SDHx PPGLs and after SDHB knockout in progenitor cells derived from a human pheochromocytoma (hPheo1). Succinate significantly increased viability of SUCNR1-transfected PC12 and ERK pathway signaling compared to control cells. Candidate SUCNR1 inhibitors successfully reversed proliferative effects of succinate. Our data reveal an unrecognized oncometabolic function of succinate in SDHx PPGLs, providing a growth advantage via SUCNR1.
- MeSH
- feochromocytom farmakoterapie enzymologie genetika metabolismus MeSH
- krysa rodu rattus MeSH
- kyselina jantarová metabolismus MeSH
- lidé MeSH
- mutace MeSH
- myši MeSH
- paragangliom farmakoterapie enzymologie genetika metabolismus MeSH
- podjednotky proteinů genetika metabolismus MeSH
- receptory spřažené s G-proteiny antagonisté a inhibitory genetika metabolismus MeSH
- sukcinátdehydrogenasa nedostatek genetika MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
While >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.
- MeSH
- dítě MeSH
- DNA řízené RNA-polymerasy chemie genetika MeSH
- dospělí MeSH
- fibroblasty metabolismus patologie MeSH
- genetická transkripce * MeSH
- kojenec MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- mitochondriální DNA genetika MeSH
- mitochondrie genetika MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mutace genetika MeSH
- nemoci nervového systému genetika patologie MeSH
- oxidativní fosforylace MeSH
- podjednotky proteinů metabolismus MeSH
- proteinové domény MeSH
- rodokmen MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.
- MeSH
- krystalografie rentgenová MeSH
- kvantová teorie MeSH
- molekulární modely MeSH
- mutace genetika MeSH
- mutantní proteiny metabolismus MeSH
- podjednotky proteinů antagonisté a inhibitory chemie metabolismus MeSH
- proteinové domény MeSH
- pyridiny chemie farmakologie MeSH
- pyrimidiny chemie farmakologie MeSH
- pyrroly chemie farmakologie MeSH
- RNA-dependentní RNA-polymerasa antagonisté a inhibitory chemie metabolismus MeSH
- termodynamika MeSH
- virová léková rezistence účinky léků MeSH
- virové proteiny antagonisté a inhibitory chemie metabolismus MeSH
- virus chřipky A účinky léků enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
The oxidative phosphorylation (OXPHOS) system localized in the inner mitochondrial membrane secures production of the majority of ATP in mammalian organisms. Individual OXPHOS complexes form supramolecular assemblies termed supercomplexes. The complexes are linked not only by their function but also by interdependency of individual complex biogenesis or maintenance. For instance, cytochrome c oxidase (cIV) or cytochrome bc1 complex (cIII) deficiencies affect the level of fully assembled NADH dehydrogenase (cI) in monomeric as well as supercomplex forms. It was hypothesized that cI is affected at the level of enzyme assembly as well as at the level of cI stability and maintenance. However, the true nature of interdependency between cI and cIV is not fully understood yet. We used a HEK293 cellular model where the COX4 subunit was completely knocked out, serving as an ideal system to study interdependency of cI and cIV, as early phases of cIV assembly process were disrupted. Total absence of cIV was accompanied by profound deficiency of cI, documented by decrease in the levels of cI subunits and significantly reduced amount of assembled cI. Supercomplexes assembled from cI, cIII, and cIV were missing in COX4I1 knock-out (KO) due to loss of cIV and decrease in cI amount. Pulse-chase metabolic labeling of mitochondrial DNA (mtDNA)-encoded proteins uncovered a decrease in the translation of cIV and cI subunits. Moreover, partial impairment of mitochondrial protein synthesis correlated with decreased content of mitochondrial ribosomal proteins. In addition, complexome profiling revealed accumulation of cI assembly intermediates, indicating that cI biogenesis, rather than stability, was affected. We propose that attenuation of mitochondrial protein synthesis caused by cIV deficiency represents one of the mechanisms, which may impair biogenesis of cI.
- MeSH
- glykolýza MeSH
- HEK293 buňky MeSH
- lidé MeSH
- mitochondriální nemoci metabolismus MeSH
- mitochondriální proteiny biosyntéza MeSH
- oxidativní fosforylace MeSH
- podjednotky proteinů metabolismus MeSH
- proteosyntéza * MeSH
- respirační komplex IV metabolismus MeSH
- spotřeba kyslíku MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytochrome c oxidase (COX), the terminal enzyme of mitochondrial electron transport chain, couples electron transport to oxygen with generation of proton gradient indispensable for the production of vast majority of ATP molecules in mammalian cells. The review summarizes current knowledge of COX structure and function of nuclear-encoded COX subunits, which may modulate enzyme activity according to various conditions. Moreover, some nuclear-encoded subunits posess tissue-specific and development-specific isoforms, possibly enabling fine-tuning of COX function in individual tissues. The importance of nuclear-encoded subunits is emphasized by recently discovered pathogenic mutations in patients with severe mitopathies. In addition, proteins substoichiometrically associated with COX were found to contribute to COX activity regulation and stabilization of the respiratory supercomplexes. Based on the summarized data, a model of three levels of quaternary COX structure is postulated. Individual structural levels correspond to subunits of the i) catalytic center, ii) nuclear-encoded stoichiometric subunits and iii) associated proteins, which may constitute several forms of COX with varying composition and differentially regulated function.
- MeSH
- buněčné jádro enzymologie genetika MeSH
- genom MeSH
- lidé MeSH
- mitochondriální nemoci enzymologie patologie MeSH
- mitochondrie enzymologie genetika MeSH
- orgánová specificita MeSH
- podjednotky proteinů MeSH
- respirační komplex IV genetika metabolismus MeSH
- signální transdukce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Kinetoplastid flagellates are known for several unusual features, one of which is their complex mitochondrial genome, known as kinetoplast (k) DNA, composed of mutually catenated maxi- and minicircles. Trypanosoma lewisi is a member of the Stercorarian group of trypanosomes which is, based on human infections and experimental data, now considered a zoonotic pathogen. By assembling a total of 58 minicircle classes, which fall into two distinct categories, we describe a novel type of kDNA organization in T. lewisi. RNA-seq approaches allowed us to map the details of uridine insertion and deletion editing events upon the kDNA transcriptome. Moreover, sequencing of small RNA molecules enabled the identification of 169 unique guide (g) RNA genes, with two differently organized minicircle categories both encoding essential gRNAs. The unprecedented organization of minicircles and gRNAs in T. lewisi broadens our knowledge of the structure and expression of the mitochondrial genomes of these human and animal pathogens. Finally, a scenario describing the evolution of minicircles is presented.
- MeSH
- adenosintrifosfatasy genetika MeSH
- editace RNA MeSH
- fylogeneze MeSH
- genom mitochondriální MeSH
- guide RNA, Kinetoplastida genetika MeSH
- mitochondrie genetika MeSH
- podjednotky proteinů genetika MeSH
- protozoální DNA genetika MeSH
- RNA protozoální genetika MeSH
- Trypanosoma lewisi genetika MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.
- MeSH
- Bardetův-Biedlův syndrom genetika metabolismus patologie MeSH
- buněčné linie MeSH
- cilie metabolismus MeSH
- CRISPR-Cas systémy genetika MeSH
- cytoplazma metabolismus MeSH
- editace genu MeSH
- fluorescenční mikroskopie MeSH
- FRAP MeSH
- lidé MeSH
- mutace MeSH
- podjednotky proteinů genetika metabolismus MeSH
- proteiny asociované s mikrotubuly nedostatek genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Precise guided pollen tube growth by the female gametophyte is a prerequisite for successful sexual reproduction in flowering plants. Cysteine-rich proteins (CRPs) secreted from the embryo sac are known pollen tube attractants perceived by pollen tube receptor-like kinases. How pre-mRNA splicing facilitates this cell-to-cell communication is not understood. Here, we report a novel function of Pre-mRNA PROCESSING factor 8 paralogs, PRP8A and PRP8B, as regulators of pollen tube attraction. Double mutant prp8a prp8b ovules cannot attract pollen tubes, and prp8a prp8b pollen tubes fail to sense the ovule's attraction signals. Only 3% of ovule-expressed genes were misregulated in prp8a prp8b Combination of RNA sequencing and the MYB98/LURE1.2-YFP reporter revealed that the expression of MYB98, LUREs and 49 other CRPs were downregulated, suggesting loss of synergid cell fate. Differential exon usage and intron retention analysis revealed autoregulation of PPR8A/PRP8B splicing. In vivo, PRP8A co-immunoprecipitates with splicing enhancer AtSF3A1, suggesting involvement of PRP8A in 3'-splice site selection. Our data hint that the PRP8A/PRP8B module exhibits spliceosome autoregulation to facilitate pollen tube attraction via transcriptional regulation of MYB98, CRPs and LURE pollen tube attractants.
- MeSH
- Arabidopsis metabolismus MeSH
- fluorescenční mikroskopie MeSH
- geneticky modifikované rostliny metabolismus MeSH
- místa sestřihu RNA MeSH
- mutageneze MeSH
- podjednotky proteinů genetika metabolismus MeSH
- proteiny huseníčku chemie genetika metabolismus MeSH
- proteiny vázající RNA chemie genetika metabolismus MeSH
- pylová láčka růst a vývoj metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- sestřihové faktory genetika metabolismus MeSH
- spliceozomy metabolismus MeSH
- transkripční faktory genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Transcription factors exert their regulatory potential on RNA polymerase II machinery through a multiprotein complex called Mediator complex or Mediator. The Mediator complex integrates regulatory signals from cell regulatory cascades with the regulation by transcription factors. The Mediator complex consists of 25 subunits in Saccharomyces cerevisiae and 30 or more subunits in multicellular eukaryotes. Mediator subunit 28 (MED28), along with MED30, MED23, MED25 and MED26, belong to presumably evolutionarily new subunits that seem to be absent in unicellular eukaryotes and are likely to have evolved together with multicellularity and cell differentiation. Previously, we have shown that an originally uncharacterized predicted gene, F28F8.5, is the true MED28 orthologue in Caenorhabditis elegans (mdt-28) and showed that it is involved in a spectrum of developmental processes. Here, we studied the proteomic interactome of MDT-28 edited as GFP::MDT-28 using Crispr/Cas9 technology or MDT-28::GFP expressed from extrachromosomal arrays in transgenic C. elegans exploiting the GFPTRAP system and mass spectrometry. The results show that MDT-28 associates with the Head module subunits MDT-6, MDT-8, MDT-11, MDT-17, MDT- 20, MDT-22, and MDT-30 and the Middle module subunit MDT-14. The analyses also identified additional proteins as preferential MDT-28 interactants, including chromatin-organizing proteins, structural proteins and enzymes. The results provide evidence for MDT-28 engagement in the Mediator Head module and support the possibility of physical (direct or indirect) interaction of MDT-28 with additional proteins, reflecting the transcription-regulating potential of primarily structural and enzymatic proteins at the level of the Mediator complex.
- MeSH
- alely MeSH
- Caenorhabditis elegans metabolismus MeSH
- jaderné proteiny metabolismus MeSH
- mediátorový komplex metabolismus MeSH
- podjednotky proteinů metabolismus MeSH
- proteiny Caenorhabditis elegans metabolismus MeSH
- proteomika * MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH