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BACKGROUND: Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a member of the Ca2+/calmodulin-dependent kinase (CaMK) family, functions as an upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase. Thus, CaMKK2 is involved in the regulation of several key physiological and pathophysiological processes. Previous studies have suggested that Ca2+/CaM binding may cause unique conformational changes in the CaMKKs compared with other CaMKs. However, the underlying mechanistic details remain unclear. METHODS: In this study, hydrogen-deuterium exchange coupled to mass spectrometry, time-resolved fluorescence spectroscopy, small-angle x-ray scattering and chemical cross-linking were used to characterize Ca2+/CaM binding-induced structural changes in CaMKK2. RESULTS: Our data suggest that: (i) the CaMKK2 kinase domain interacts with the autoinhibitory region (AID) through the N-terminal lobe of the kinase domain including the RP insert, a segment important for targeting downstream substrate kinases; (ii) Ca2+/CaM binding affects the structure of several regions surrounding the ATP-binding pocket, including the activation segment; (iii) although the CaMKK2:Ca2+/CaM complex shows high conformational flexibility, most of its molecules are rather compact; and (iv) AID-bound Ca2+/CaM transiently interacts with the CaMKK2 kinase domain. CONCLUSIONS: Interactions between the CaMKK2 kinase domain and the AID differ from those of other CaMKs. In the absence of Ca2+/CaM binding the autoinhibitory region inhibits CaMKK2 by both blocking access to the RP insert and by affecting the structure of the ATP-binding pocket. GENERAL SIGNIFICANCE: Our results corroborate the hypothesis that Ca2+/CaM binding causes unique conformational changes in the CaMKKs relative to other CaMKs.
- MeSH
- fosforylace MeSH
- kalmodulin metabolismus MeSH
- kinasa proteinkinasy závislé na vápníku a kalmodulinu antagonisté a inhibitory chemie metabolismus MeSH
- konformace proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- proteinové domény MeSH
- vápník metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- antigeny CD36 genetika MeSH
- chromozomy genetika MeSH
- finanční podpora výzkumu jako téma MeSH
- hybridizace in situ fluorescenční metody využití MeSH
- krysa rodu rattus MeSH
- mapování chromozomů metody využití MeSH
- potkani inbrední SHR genetika MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
Recombination of antibody genes in B cells can involve distant genomic loci and contribute a foreign antigen-binding element to form hybrid antibodies with broad reactivity for Plasmodium falciparum. So far, antibodies containing the extracellular domain of the LAIR1 and LILRB1 receptors represent unique examples of cross-chromosomal antibody diversification. Here, we devise a technique to profile non-VDJ elements from distant genes in antibody transcripts. Independent of the preexposure of donors to malaria parasites, non-VDJ inserts were detected in 80% of individuals at frequencies of 1 in 104 to 105 B cells. We detected insertions in heavy, but not in light chain or T cell receptor transcripts. We classify the insertions into four types depending on the insert origin and destination: 1) mitochondrial and 2) nuclear DNA inserts integrated at VDJ junctions; 3) inserts originating from telomere proximal genes; and 4) fragile sites incorporated between J-to-constant junctions. The latter class of inserts was exclusively found in memory and in in vitro activated B cells, while all other classes were already detected in naïve B cells. More than 10% of inserts preserved the reading frame, including transcripts with signs of antigen-driven affinity maturation. Collectively, our study unravels a mechanism of antibody diversification that is layered on the classical V(D)J and switch recombination.
- MeSH
- B-lymfocyty * imunologie MeSH
- CD antigeny imunologie MeSH
- genomika MeSH
- geny pro imunoglobuliny * MeSH
- imunoglobulinový receptor leukocytů B1 imunologie MeSH
- inzerční mutageneze MeSH
- lehké řetězce imunoglobulinů genetika MeSH
- lidé MeSH
- Plasmodium falciparum MeSH
- protilátky protozoální genetika MeSH
- receptory antigenů T-buněk genetika MeSH
- receptory imunologické imunologie MeSH
- rozmanitost protilátek * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
While the mechanisms governing DNA damage response and repair are fundamentally conserved, cross-kingdom comparisons indicate that they differ in many aspects due to differences in life-styles and developmental strategies. In photosynthetic organisms these differences have not been fully explored because gene-discovery approaches are mainly based on homology searches with known DDR/DNA repair proteins. Here we performed a forward genetic screen in the green algae Chlamydomonas reinhardtii to identify genes deficient in DDR/DNA repair. We isolated five insertional mutants that were sensitive to various genotoxic insults and two of them exhibited altered efficiency of transgene integration. To identify genomic regions disrupted in these mutants, we established a novel adaptor-ligation strategy for the efficient recovery of the insertion flanking sites. Four mutants harbored deletions that involved known DNA repair factors, DNA Pol zeta, DNA Pol theta, SAE2/COM1, and two neighbouring genes encoding ERCC1 and RAD17. Deletion in the last mutant spanned two Chlamydomonas-specific genes with unknown function, demonstrating the utility of this approach for discovering novel factors involved in genome maintenance.
- MeSH
- bakteriální transformace účinky léků MeSH
- Chlamydomonas reinhardtii účinky léků genetika MeSH
- genetické vektory genetika MeSH
- hydroxymočovina farmakologie toxicita MeSH
- inzerční mutageneze * MeSH
- mutace MeSH
- oprava DNA * MeSH
- pořadí genů genetika MeSH
- poškození DNA * účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Analysis of plants bearing a T-DNA insertion is a potent tool of modern molecular biology, providing valuable information about the function and involvement of genes in metabolic pathways. A collection of 12 Arabidopsis thaliana lines with T-DNA insertions in the gene coding for the catalytic subunit of telomerase (AtTERT) and in adjacent regions was screened for telomerase activity [telomere repeat amplification protocol (TRAP) assay], telomere length (terminal restriction fragments), and AtTERT transcription (quantitative reverse transcription-PCR). Lines with the insertion located upstream of the start codon displayed unchanged telomere stability and telomerase activity, defining a putative minimal AtTERT promoter and the presence of a regulatory element linked to increased transcription in the line SALK_048471. Lines bearing a T-DNA insertion inside the protein-coding region showed telomere shortening and lack of telomerase activity. Transcription in most of these lines was unchanged upstream of the T-DNA insertion, while it was notably decreased downstream. The expression profile varied markedly in mutant lines harbouring insertions at the 5' end of AtTERT which showed increased transcription and abolished tissue specificity. Moreover, the line FLAG_385G01 (T-DNA insertion inside intron 1) revealed the presence of a highly abundant downstream transcript with normal splicing but without active telomerase. The role of regulatory elements found along the AtTERT gene is discussed in respect to natural telomerase expression and putative intron-mediated enhancement.
- MeSH
- Arabidopsis genetika MeSH
- DNA bakterií genetika MeSH
- genotyp MeSH
- inzerční mutageneze MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proteiny huseníčku genetika MeSH
- regulace genové exprese u rostlin MeSH
- regulační oblasti nukleových kyselin genetika MeSH
- telomerasa genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
