ADAR RNA editing enzymes are high-affinity dsRNA-binding proteins that deaminate adenosines to inosines in pre-mRNA hairpins and also exert editing-independent effects. We generated a Drosophila AdarE374A mutant strain encoding a catalytically inactive Adar with CRISPR/Cas9. We demonstrate that Adar adenosine deamination activity is necessary for normal locomotion and prevents age-dependent neurodegeneration. The catalytically inactive protein, when expressed at a higher than physiological level, can rescue neurodegeneration in Adar mutants, suggesting also editing-independent effects. Furthermore, loss of Adar RNA editing activity leads to innate immune induction, indicating that Drosophila Adar, despite being the homolog of mammalian ADAR2, also has functions similar to mammalian ADAR1. The innate immune induction in fly Adar mutants is suppressed by silencing of Dicer-2, which has a RNA helicase domain similar to MDA5 that senses unedited dsRNAs in mammalian Adar1 mutants. Our work demonstrates that the single Adar enzyme in Drosophila unexpectedly has dual functions.
- MeSH
- adenosindeaminasa chemie genetika MeSH
- adenosinmonofosfát metabolismus MeSH
- bodová mutace genetika MeSH
- degenerace nervu patologie MeSH
- Drosophila melanogaster genetika imunologie MeSH
- editace RNA genetika MeSH
- katalýza MeSH
- lokomoce MeSH
- messenger RNA genetika metabolismus MeSH
- mozek metabolismus MeSH
- přirozená imunita genetika MeSH
- proteinové domény MeSH
- proteiny Drosophily chemie genetika metabolismus MeSH
- regulace genové exprese MeSH
- ribonukleasa III metabolismus MeSH
- RNA-helikasy metabolismus MeSH
- stárnutí patologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Glucan particles derived from yeast have been recently proposed as potential drug delivery carriers. Here, we demonstrate the potential of glucan particles for protein delivery in vivo, using the insect Drosophila melanogaster as a model organism. By employing genetic tools, we demonstrate the capacity of yeast glucan particles to spread efficiently through the Drosophila body, to enter macrophages and to deliver an active transcription factor protein successfully. Moreover, the glucan particles were nontoxic and induced only minimal immune response. The injection of glucan particles did not impair the ability of Drosophila to fight and survive infection by pathogenic bacteria. From this study, Drosophila emerges as an excellent model to test and develop drug delivery systems based on glucan particles, specifically aimed to regulate macrophages.
- MeSH
- Drosophila melanogaster imunologie metabolismus MeSH
- glukany chemie metabolismus MeSH
- kvasinky chemie MeSH
- makrofágy cytologie imunologie metabolismus MeSH
- nosiče léků chemie metabolismus MeSH
- proteiny Drosophily chemie metabolismus MeSH
- systémy cílené aplikace léků * MeSH
- transkripční faktory chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: De novo mutations in PURA have recently been described to cause PURA syndrome, a neurodevelopmental disorder characterised by severe intellectual disability (ID), epilepsy, feeding difficulties and neonatal hypotonia. OBJECTIVES: To delineate the clinical spectrum of PURA syndrome and study genotype-phenotype correlations. METHODS: Diagnostic or research-based exome or Sanger sequencing was performed in individuals with ID. We systematically collected clinical and mutation data on newly ascertained PURA syndrome individuals, evaluated data of previously reported individuals and performed a computational analysis of photographs. We classified mutations based on predicted effect using 3D in silico models of crystal structures of Drosophila-derived Pur-alpha homologues. Finally, we explored genotype-phenotype correlations by analysis of both recurrent mutations as well as mutation classes. RESULTS: We report mutations in PURA (purine-rich element binding protein A) in 32 individuals, the largest cohort described so far. Evaluation of clinical data, including 22 previously published cases, revealed that all have moderate to severe ID and neonatal-onset symptoms, including hypotonia (96%), respiratory problems (57%), feeding difficulties (77%), exaggerated startle response (44%), hypersomnolence (66%) and hypothermia (35%). Epilepsy (54%) and gastrointestinal (69%), ophthalmological (51%) and endocrine problems (42%) were observed frequently. Computational analysis of facial photographs showed subtle facial dysmorphism. No strong genotype-phenotype correlation was identified by subgrouping mutations into functional classes. CONCLUSION: We delineate the clinical spectrum of PURA syndrome with the identification of 32 additional individuals. The identification of one individual through targeted Sanger sequencing points towards the clinical recognisability of the syndrome. Genotype-phenotype analysis showed no significant correlation between mutation classes and disease severity.
- MeSH
- abnormality očí genetika MeSH
- DNA vazebné proteiny chemie genetika MeSH
- genetické asociační studie MeSH
- lidé MeSH
- mentální retardace genetika MeSH
- mutace * MeSH
- novorozenec MeSH
- obličej abnormality MeSH
- proteiny Drosophily chemie genetika MeSH
- strukturní homologie proteinů MeSH
- svalová hypotonie etiologie genetika MeSH
- syndrom MeSH
- těhotenství MeSH
- transkripční faktory chemie genetika MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy. The preference for low occupancy complexes at saturating LC8 concentrations with both human and Drosophila ASCIZ indicates that negative cooperativity is an important feature of ASCIZ-LC8 interactions. The prevalence of intrinsic disorder and multivalency among transcription factors suggests that formation of heterogeneous, dynamic complexes is a widespread mechanism for tuning transcriptional regulation.
- MeSH
- cytoplazmatické dyneiny chemie genetika metabolismus MeSH
- Drosophila melanogaster růst a vývoj metabolismus fyziologie MeSH
- dyneiny chemie genetika metabolismus MeSH
- lidé MeSH
- proteiny Drosophily chemie genetika metabolismus MeSH
- regulace genové exprese * MeSH
- transkripční faktory chemie genetika metabolismus MeSH
- vnitřně neuspořádané proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Biogenic amines are common biologically active substances extended within the whole animal kingdom where they play vital roles as signal transducer as well as regulator of cell functions. One of these biogenic amines called octopamine (OA) is synthesized from tyramine (TA) by the catalysis of tyramine-β-hydroxylase (TβH) originated in the insect nervous system. Both TA and OA act as neurotransmitters, neurohormones and neuromodulators in the arthropod nervous system. Herein, the inhibitory activity of 1-arylimidazole-2(3H)-thiones (AITs) was tested on cloned Drosophila tyramine-β-hydroxylase (DmTβH) expressed in Bombyx mori strain. Radiolabelled (3)H-TA was used to analyze the activity of AITs exhibited inhibitory effects on DmTβH, whose ID50 values range from 0.02 to 2511nM where DmTβH was inhibited in a dose-dependent manner at pH 7.6 and 25°C during a 30min of incubation. To understand the catalytic role of the TβH, a three dimensional structure of the TβH from Drosophila melanogaster was constructed by homology modeling using the Phyre2 web server with 100% confidence. The modeled three-dimensional structure of TβH was used to perform the docking study with AITs. This may give more insights to precise design of inhibitors for TβH to control insect's population.
- MeSH
- Drosophila melanogaster enzymologie MeSH
- inhibitory enzymů chemie MeSH
- katalytická doména MeSH
- konformace proteinů, beta-řetězec MeSH
- oxygenasy se smíšenou funkcí antagonisté a inhibitory chemie MeSH
- proteiny Drosophily antagonisté a inhibitory chemie MeSH
- sekvence aminokyselin MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články 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
- 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
Intramembrane proteases cleave membrane proteins in their transmembrane helices to regulate a wide range of biological processes. They catalyse hydrolytic reactions within the hydrophobic environment of lipid membranes where water is normally excluded. How? Do the different classes of intramembrane proteases share any mechanistic principles? In this review these questions will be discussed in view of the crystal structures of prokaryotic members of the three known catalytic types of intramembrane proteases published over the past 7 years. Rhomboids, the intramembrane serine proteases that are the best understood family, will be the initial area of focus, and the principles that have arisen from a number of structural and biochemical studies will be considered. The site-2 metalloprotease and GXGD-type aspartyl protease structures will then be discussed, with parallels drawn and differences highlighted between these enzymes and the rhomboids. Despite the significant advances achieved so far, to obtain a detailed understanding of the mechanism of any intramembrane protease, high-resolution structural information on the substrate-enzyme complex is required. This remains a major challenge for the field.
- MeSH
- aminokyselinové motivy MeSH
- aspartátové proteasy chemie metabolismus MeSH
- bakteriální proteiny chemie metabolismus MeSH
- DNA vazebné proteiny chemie MeSH
- endopeptidasy chemie metabolismus MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- membránové proteiny chemie metabolismus MeSH
- metaloendopeptidasy chemie metabolismus MeSH
- molekulární evoluce MeSH
- molekulární sekvence - údaje MeSH
- preseniliny chemie metabolismus MeSH
- proteiny Drosophily chemie metabolismus MeSH
- proteiny z Escherichia coli chemie MeSH
- proteolýza MeSH
- sekvence aminokyselin MeSH
- serinové proteasy chemie metabolismus MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
We are using a candidate gene approach to identify genes contributing to cancer through somatic mutation. Somatic mutations were found in breast cancer samples in the human casein kinase I epsilon (CKIepsilon) gene, a homolog of the Drosophila gene dco in which certain point mutations lead to imaginal disc overgrowth. We therefore created fly genotypes in which the dco gene carried point mutations homologous to those discovered in CKIepsilon, and tested them in vivo. The results show that the most frequent mutation discovered in breast cancer, L39Q, causes a striking overgrowth phenotype in flies. Further experiments show that this mutation affects the newly recognized Fat/Warts signaling pathway, which controls organ size and shape in both flies and mammals. Another mutation, S101R, modifies the mutant phenotype so that the affected tissue disintegrates, mimicking more aggressive forms of breast cancer. Our results thus strongly support the conclusion that CKIepsilon mutations play important roles in breast carcinogenesis.
- MeSH
- alely MeSH
- Drosophila embryologie genetika růst a vývoj MeSH
- fenotyp MeSH
- kasein kinasa 1 epsilon chemie genetika fyziologie MeSH
- larva genetika MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- mutace MeSH
- nádory prsu genetika MeSH
- proliferace buněk MeSH
- proteiny Drosophily chemie genetika fyziologie MeSH
- sekvence aminokyselin MeSH
- signální transdukce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- Drosophila melanogaster enzymologie genetika MeSH
- glykolipidy metabolismus MeSH
- krevní skupiny - systém P genetika MeSH
- lidé MeSH
- N-acetylglukosaminyltransferasy genetika chemie MeSH
- proteiny Drosophily genetika chemie MeSH
- sekvence aminokyselin účinky léků MeSH
- sekvenční homologie aminokyselin MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
