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.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Phosphatidylcholines chemistry   metabolism   MeSH
• Ion Channel Gating genetics   MeSH
• Genetic Vectors chemistry   metabolism   MeSH
• HEK293 Cells MeSH
• Protein Interaction Domains and Motifs MeSH
• Protein Conformation, alpha-Helical MeSH
• Protein Conformation, beta-Strand MeSH
• Humans MeSH
• Liposomes chemistry   metabolism   MeSH
• Luminescent Proteins genetics   metabolism   MeSH
• Patch-Clamp Techniques MeSH
• Mutation MeSH
• Neoplasm Proteins chemistry   genetics   metabolism   MeSH
• ORAI1 Protein chemistry   genetics   metabolism   MeSH
• Stromal Interaction Molecule 1 chemistry   genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Genes, Reporter MeSH
• Molecular Dynamics Simulation MeSH
• Amino Acid Substitution MeSH
• Calcium metabolism   MeSH
• Calcium Signaling * MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The tetrahydroisoquinoline trabectedin is a marine compound with approved activity against human soft-tissue sarcoma. It exerts antiproliferative activity mainly by specific binding to the DNA and inducing DNA double-strand breaks (DSB). As homologous recombination repair (HRR)-deficient tumors are more susceptible to trabectedin, hyperthermia-mediated on-demand induction of HRR deficiency represents a novel and promising strategy to boost trabectedin treatment. For the first time, we demonstrate enhancement of trabectedin effectiveness in human sarcoma cell lines by heat and characterize cellular events and molecular mechanisms related to heat-induced effects. Hyperthermic temperatures (41.8 or 43°C) enhanced significantly trabectedin-related clonogenic cell death and G2/M cell cycle arrest followed by cell type-dependent induction of apoptosis or senescence. Heat combination increased accumulation of γH2AX foci as key marker of DSBs. Expression of BRCA2 protein, an integral protein of the HRR machinery, was significantly decreased by heat. Consequently, recruitment of downstream RAD51 to γH2AX-positive repair foci was almost abolished indicating relevant impairment of HRR by heat. Accordingly, enhancement of trabectedin effectiveness was significantly augmented in BRCA2-proficient cells by hyperthermia and alleviated in BRCA2 knockout or siRNA-transfected BRCA2 knockdown cells. In peripheral blood mononuclear cells isolated from sarcoma patients, increased numbers of nuclear γH2AX foci were detected after systemic treatment with trabectedin and hyperthermia of the tumor region. The findings establish BRCA2 degradation by heat as a key factor for a novel treatment strategy that allows targeted chemosensitization to trabectedin and other DNA damaging antitumor drugs by on-demand induction of HRR deficiency.

• MeSH
• Antineoplastic Agents, Alkylating pharmacology   MeSH
• Apoptosis drug effects   radiation effects   MeSH
• Models, Biological MeSH
• Drug Resistance, Neoplasm radiation effects   MeSH
• Dioxoles pharmacology   MeSH
• Histones metabolism   MeSH
• Hyperthermia, Induced * MeSH
• Caspases metabolism   MeSH
• Cell Cycle Checkpoints drug effects   radiation effects   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• BRCA2 Protein metabolism   MeSH
• Proteolysis drug effects   radiation effects   MeSH
• Recombinational DNA Repair drug effects   radiation effects   MeSH
• Rad51 Recombinase metabolism   MeSH
• Sarcoma metabolism   pathology   therapy   MeSH
• Tetrahydroisoquinolines pharmacology   MeSH
• Protein Transport MeSH
• Protein Binding MeSH
• Cell Survival drug effects   radiation effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

The aim of the present study was to investigate the subcellular localization of proteins participating in the double-strand break response pathway - p53, Mdm2, p21 and Chk2. MOLT-4 cells were pre-treated with mitoxantrone in concentrations 1 nmol/l and 5 nmol/l. The trypan blue technique was used to determine cell viability and proliferation. Western blotting was used to evaluate changes in p53, Mdm2 and Chk2 protein expression and sandwich ELISA was used to evaluate changes in the p21 protein amount. After 1 nmol/l mitoxantrone cells did not die, but their ability to proliferate was decreased. The p53 protein was activated and phosphorylated at serines 15 and 392 and accumulated in the nucleus after 24 and 48 h. The Mdm2 protein was present in the cytoplasm with its maximal level after 8 and 16 h. The p21 protein was detected in the nucleus after 24 and 48 h. Increased levels of phosphorylated Chk2 at threonine 68 were observed in the cytoplasmic fraction after 24 and 48 h of mitoxantrone treatment. We used mitoxantrone as an inducer of double-strand breaks to bring new data about the subcellular distribution of proteins responding to DNA damage. In MOLT-4 cells, the p53 protein was activated. p53 was phosphorylated at serines 15 and 392 and accumulated in the nucleus. The Mdm2 protein was activated in advance to p53 and occurred in the cytoplasm. The p21 protein was present in the nucleus. Chk2 kinase was activated by the phosphorylation at threonine 68 and we observed increased levels of this protein in the cytoplasmic fraction.

• MeSH
• Precursor Cell Lymphoblastic Leukemia-Lymphoma pathology   MeSH
• Cell Division drug effects   MeSH
• Checkpoint Kinase 2 MeSH
• Cytoplasm chemistry   MeSH
• DNA Breaks, Double-Stranded * MeSH
• Phosphorylation MeSH
• Cyclin-Dependent Kinase Inhibitor p21 analysis   MeSH
• Humans MeSH
• Mitoxantrone toxicity   MeSH
• Cell Line, Tumor MeSH
• Neoplasm Proteins analysis   MeSH
• Tumor Suppressor Protein p53 analysis   MeSH
• DNA Repair * MeSH
• Protein Processing, Post-Translational MeSH
• Antineoplastic Agents toxicity   MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• Subcellular Fractions drug effects   MeSH
• Protein Transport MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

53BP1 is a key component of the genome surveillance network activated by DNA double strand breaks (DSBs). Despite its known accumulation at the DSB sites, the spatiotemporal aspects of 53BP1 interaction with DSBs and the role of other DSB regulators in this process remain unclear. Here, we used real-time microscopy to study the DSB-induced redistribution of 53BP1 in living cells. We show that within minutes after DNA damage, 53BP1 becomes progressively, yet transiently, immobilized around the DSB-flanking chromatin. Quantitative imaging of single cells revealed that the assembly of 53BP1 at DSBs significantly lagged behind Mdc1/NFBD1, another DSB-interacting checkpoint mediator. Furthermore, short interfering RNA-mediated ablation of Mdc1/NFBD1 drastically impaired 53BP1 redistribution to DSBs and triggered premature dissociation of 53BP1 from these regions. Collectively, these in vivo measurements identify Mdc1/NFBD1 as a key upstream determinant of 53BP1's interaction with DSBs from its dynamic assembly at the DSB sites through sustained retention within the DSB-flanking chromatin up to the recovery from the checkpoint.

• MeSH
• Chromatin physiology   MeSH
• DNA-Binding Proteins physiology   genetics   MeSH
• Phosphoproteins physiology   genetics   MeSH
• Intracellular Signaling Peptides and Proteins physiology   genetics   MeSH
• Nuclear Proteins physiology   genetics   MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• RNA, Small Interfering physiology   MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• DNA Damage * MeSH
• Recombinant Fusion Proteins genetics   metabolism   MeSH
• Trans-Activators physiology   genetics   MeSH
• Protein Transport MeSH
• Protein Binding MeSH
• Green Fluorescent Proteins genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH

N-methyl-D-aspartate (NMDA) receptors are glutamate ion channels that are critically involved in excitatory synaptic transmission and plasticity. The functional NMDA receptor is a heterotetramer composed mainly of GluN1 and GluN2 subunits. It is generally thought that only correctly assembled NMDA receptors can pass the quality control checkpoint in the endoplasmic reticulum (ER) and are transported to the cell surface membranes. The molecular mechanisms underlying these processes remain poorly understood. Using chimeric and mutated GluN1 subunits expressed in heterologous cells, we identified a single amino acid residue within the fourth membrane domain (M4) of GluN1 subunit, L830, that regulates the surface number of NMDA receptors. Our experiments show that this residue is not critical for the interaction between GluN1 and GluN2 subunits or for the formation of functional receptors, but rather that it regulates the forward trafficking of the NMDA receptors. The surface expression of both GluN2A- and GluN2B-containing receptors is regulated by the L830 residue in a similar manner. We also found that the L830 residue is not involved in the trafficking of individually expressed GluN1 subunits. Our data reveal a critical role of the single amino acid residue within the GluN1 M4 domain in the surface delivery of functional NMDA receptors.

• MeSH
• Amino Acids genetics   MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Endoplasmic Reticulum metabolism   MeSH
• HEK293 Cells MeSH
• Protein Structure, Quaternary physiology   MeSH
• Humans MeSH
• Mutagenesis physiology   MeSH
• Receptors, Cell Surface chemistry   genetics   metabolism   MeSH
• Receptors, N-Methyl-D-Aspartate chemistry   genetics   metabolism   MeSH
• Protein Structure, Tertiary physiology   MeSH
• Protein Transport physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The protein kinases ataxia-telangiectasia mutated (ATM) and ATM-Rad3 related (ATR) are activated in response to DNA damage, genotoxic stress and virus infections. Here we show that during infection with wild-type adenovirus, ATR and its cofactors RPA32, ATRIP and TopBP1 accumulate at viral replication centres, but there is minimal ATR activation. We show that the Mre11/Rad50/Nbs1 (MRN) complex is recruited to viral centres only during infection with adenoviruses lacking the early region E4 and ATR signaling is activated. This suggests a novel requirement for the MRN complex in ATR activation during virus infection, which is independent of Mre11 nuclease activity and recruitment of RPA/ATR/ATRIP/TopBP1. Unlike other damage scenarios, we found that ATM and ATR signaling are not dependent on each other during infection. We identify a region of the viral E4orf3 protein responsible for immobilization of the MRN complex and show that this prevents ATR signaling during adenovirus infection. We propose that immobilization of the MRN damage sensor by E4orf3 protein prevents recognition of viral genomes and blocks detrimental aspects of checkpoint signaling during virus infection.

• MeSH
• Adenoviridae MeSH
• Adenoviridae Infections * metabolism   MeSH
• Adenovirus E4 Proteins chemistry   metabolism   MeSH
• Ataxia Telangiectasia Mutated Proteins MeSH
• Cell Line MeSH
• DNA-Binding Proteins * metabolism   MeSH
• DNA Repair Enzymes * metabolism   MeSH
• Phosphorylation MeSH
• Nuclear Proteins * metabolism   MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Multiprotein Complexes * metabolism   MeSH
• Tumor Suppressor Proteins metabolism   MeSH
• Protein Serine-Threonine Kinases * metabolism   MeSH
• Cell Cycle Proteins * metabolism   MeSH
• Virus Replication MeSH
• Amino Acid Sequence MeSH
• Signal Transduction * MeSH
• Protein Transport MeSH
• Check Tag
• Humans MeSH

The expression, cellular distribution, and subcellular sorting of the microtubule (MT)-nucleating γ-tubulin small complex (γTuSC) proteins, GCP2 and GCP3, were studied in human glioblastoma cell lines and in clinical tissue samples representing all histologic grades of adult diffuse astrocytic gliomas (n = 54). Quantitative real-time polymerase chain reaction revealed a significant increase in the expression of GCP2 and GCP3 transcripts in glioblastoma cells versus normal human astrocytes; these were associated with higher amounts of both γTuSC proteins. GCP2 and GCP3 were concentrated in the centrosomes in interphase glioblastoma cells, but punctate and diffuse localizations were also detected in the cytosol and nuclei/nucleoli. Nucleolar localization was fixation dependent. GCP2 and GCP3 formed complexes with γ-tubulin in the nucleoli as confirmed by reciprocal immunoprecipitation experiments and immunoelectron microscopy. GCP2 and GCP3 depletion caused accumulation of cells in G2/M and mitotic delay but did not affect nucleolar integrity. Overexpression of GCP2 antagonized the inhibitory effect of the CDK5 regulatory subunit-associated tumor suppressor protein 3 (C53) on DNA damage G2/M checkpoint activity. Tumor cell GCP2 and GCP3 immunoreactivity was significantly increased over that in normal brains in glioblastoma samples; it was also associated with microvascular proliferation. These findings suggest that γTuSC protein dysregulation in glioblastomas may be linked to altered transcriptional checkpoint activity or interaction with signaling pathways associated with a malignant phenotype.

• MeSH
• Cell Nucleolus metabolism   MeSH
• Cell Cycle physiology   MeSH
• Zebrafish MeSH
• Glioblastoma metabolism   pathology   ultrastructure   MeSH
• Chickens MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 metabolism   MeSH
• Brain Neoplasms metabolism   pathology   ultrastructure   MeSH
• DNA Damage genetics   MeSH
• Microtubule-Associated Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Neoplastic physiology   MeSH
• Protein Transport MeSH
• Tubulin metabolism   MeSH
• Anura MeSH
• Animals MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

γ-Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules from microtubule organizing centers. Using immunolocalization and cell fractionation techniques in combination with siRNAi and expression of FLAG-tagged constructs, we have obtained evidence that γ-tubulin is also present in nucleoli of mammalian interphase cells of diverse cellular origins. Immunoelectron microscopy has revealed γ-tubulin localization outside fibrillar centers where transcription of ribosomal DNA takes place. γ-Tubulin was associated with nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear γ-tubulin, making it unlikely that rapid active transport via nuclear pores participates in the transport of γ-tubulin into the nucleus. This finding was confirmed by heterokaryon assay and time-lapse imaging of photoconvertible protein Dendra2 tagged to γ-tubulin. Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an association of γ-tubulin with tumor suppressor protein C53 located at multiple subcellular compartments including nucleoli. The notion of an interaction between γ-tubulin and C53 was corroborated by pull-down and co-immunoprecipitation experiments. Overexpression of γ-tubulin antagonized the inhibitory effect of C53 on DNA damage G(2) /M checkpoint activation. The combined results indicate that aside from its known role in microtubule nucleation, γ-tubulin may also have nuclear-specific function(s).

• MeSH
• Astrocytes metabolism   MeSH
• Cell Nucleolus metabolism   MeSH
• Cell Nucleus metabolism   MeSH
• Time-Lapse Imaging MeSH
• Fluorescent Antibody Technique MeSH
• Glioblastoma metabolism   MeSH
• Mass Spectrometry MeSH
• Microscopy, Immunoelectron MeSH
• Immunoprecipitation MeSH
• Intracellular Signaling Peptides and Proteins metabolism   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Humans MeSH
• Microtubules metabolism   MeSH
• Mitosis physiology   MeSH
• Cell Line, Tumor MeSH
• Brain Neoplasms metabolism   MeSH
• Nerve Tissue Proteins metabolism   MeSH
• Protein Transport physiology   MeSH
• Tubulin metabolism   MeSH
• Genes, Tumor Suppressor MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Cell Biology MeSH
• Genetic Predisposition to Disease MeSH
• Molecular Biology MeSH
• Neoplastic Processes MeSH
• Neoplasms MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Publication type
• monografie

• MeSH
• Cells * MeSH
• Molecular Biology MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• molekulární biologie, molekulární medicína
• NML Publication type
• učebnice vysokých škol