- MeSH
- interferon gama genetika imunologie MeSH
- Leishmania braziliensis imunologie MeSH
- Leishmania donovani imunologie MeSH
- Leishmania major imunologie MeSH
- leishmanióza kožní imunologie patologie MeSH
- lidé MeSH
- makrofágy imunologie MeSH
- monocyty imunologie MeSH
- myši inbrední BALB C MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- přirozená imunita genetika imunologie MeSH
- proteiny vázající GTP genetika metabolismus MeSH
- transkriptom genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.
BACKGROUND: Leishmania virulence factors responsible for the complicated epidemiology of the various leishmaniases remain mainly unidentified. This study is a characterization of a gene previously identified as upregulated in two of three overlapping datasets containing putative factors important for Leishmania's ability to establish mammalian intracellular infection and to colonize the gut of an insect vector. METHODOLOGY/PRINCIPAL FINDINGS: The investigated gene encodes ATP/GTP binding motif-containing protein related to Leishmania development 1 (ALD1), a cytosolic protein that contains a cryptic ATP/GTP binding P-loop. We compared differentiation, growth rates, and infective abilities of wild-type and ALD1 null mutant cell lines of L. mexicana. Loss of ALD1 results in retarded growth kinetics but not defects in differentiation in axenic culture. Similarly, when mice and the sand fly vector were infected with the ALD1 null mutant, the primary difference in infection and colonization phenotype relative to wild type was an inability to achieve maximal host pathogenicity. While ability of the ALD1 null mutant cells to infect macrophages in vitro was not affected, replication within macrophages was clearly curtailed. CONCLUSIONS/SIGNIFICANCE: L. mexicana ALD1, encoding a protein with no assigned functional domains or motifs, was identified utilizing multiple comparative analyses with the related and often experimentally overlooked monoxenous flagellates. We found that it plays a role in Leishmania infection and colonization in vitro and in vivo. Results suggest that ALD1 functions in L. mexicana's general metabolic network, rather than function in specific aspect of virulence as anticipated from the compared datasets. This result validates our comparative genomics approach for finding relevant factors, yet highlights the importance of quality laboratory-based analysis of genes tagged by these methods.
- MeSH
- hmyz - vektory parazitologie MeSH
- Leishmania mexicana genetika patogenita MeSH
- leishmanióza kožní parazitologie MeSH
- makrofágy parazitologie MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- proteiny vázající GTP genetika metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- Psychodidae parazitologie MeSH
- virulence MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Variants in the ATL1 gene have been repeatedly described as the second most frequent cause of hereditary spastic paraplegia (HSP), a motor neuron disease manifested by progressive lower limb spasticity and weakness. Variants in ATL1 have been described mainly in patients with early onset HSP. We performed Sanger sequencing of all coding exons and adjacent intron regions of the ALT1 gene in 111 Czech patients with pure form of HSP and additional Multiplex-Ligation Probe Analysis (MLPA) testing targeting the ATL1 gene in 56 of them. All patients except seven were previously tested by Sanger sequencing of the SPAST gene with negative results. ATL1 diagnostic testing revealed only five missense variants in the ATL1 gene. Four of them are novel, but we suppose only two of them to be pathogenic and causal. The remaining variants are assumed to be benign. MLPA testing in 56 of sequence variant negative patients revealed no gross deletion in the ATL1 gene. Variants in the ATL1 gene are more frequent in patients with early onset HSP, but in general the occurrence of pathogenic variants in the ATL1 gene is low in our cohort, less than 4.5% and less than 11.1% in patients with onset before the age of ten. Variants in the ATL1 gene are a less frequent cause of HSP among Czech patients than has been previously reported among other populations.
- MeSH
- dítě MeSH
- exony MeSH
- introny MeSH
- kojenec MeSH
- lidé středního věku MeSH
- lidé MeSH
- membránové proteiny genetika MeSH
- missense mutace MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mutační analýza DNA MeSH
- proteiny vázající GTP genetika MeSH
- rodokmen MeSH
- spastická paraplegie dědičná genetika MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
BACKGROUND: Primary ciliary dyskinesia (PCD) is a multigenic autosomal recessive condition affecting respiratory tract and other organs where ciliary motility is required. The extent of its genetic heterogeneity is remarkable. The aim of the study was to develop a cost-effective pipeline for genetic diagnostics using a combination of Sanger and next generation sequencing (NGS). MATERIALS AND METHODS: Data and samples of 33 families with 38 affected subjects with PCD diagnosed in childhood were collected over the territory of the Czech Republic. A panel of 18 PCD causative or candidate genes was implemented into an Illumina TruSeq Custom Amplicon NGS assay, and three ancestral mutations in SPAG1 were screened by conventional Sanger sequencing, which was also used for the confirmation of the NGS results and for the analysis of familial segregation. RESULTS: The causative gene was DNAH5 in 11/33 (33%) probands, SPAG1 in 8/33 (24%), and DNAI1, CCDC40, LRRC6 in one family each. If the high proportion of subjects with bi-allelic ancestral mutations in SPAG1 is corroborated in other Caucasian populations, a simple Sanger sequencing test for these three mutations may serve as an effective pre-screening step, being followed by an NGS panel for other, much larger, PCD genes. CONCLUSIONS: We present a combination of Sanger sequencing with an NGS panel for known and candidate PCD genes, implemented in a moderate-size national collection of patients. This strategy has proven to be cost-effective, rapid and reliable, and was able to detect the causative gene in two thirds of our PCD patients.
- MeSH
- alely MeSH
- antigeny povrchové genetika MeSH
- dítě MeSH
- Kartagenerův syndrom diagnóza genetika MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mutace * MeSH
- předškolní dítě MeSH
- proteiny vázající GTP genetika MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Drug resistance is the major obstacle to successful cancer therapy. Our study focuses on resistance to Aurora kinase inhibitors tested as anti-cancer drugs in clinical trials. We have used 2D electrophoresis in the pH ranges of 4-7 and 6-11 followed by protein identification using MALDI-TOF/TOF to compare the protein composition of HCT116 colon cancer cells either sensitive to CYC116 and ZM447439 inhibitors or resistant toward these drugs. The analysis also included p53(+/+) and p53(-/-) phenotypes of HCT116 cells. Our findings demonstrate that platelet-activating factor acetylhydrolase and GTP-binding nuclear protein Ran contribute to the development of resistance to ZM447439 only where resistance is related to p53. On the other hand, serine hydroxymethyltransferase was found to promote the tumor growth in cells resistant to CYC116 without the influence of p53. Computer modeling of interaction networks highlighted a direct link of the p53-independent mechanism of resistance to CYC116 with autophagy. Importantly, serine hydroxymethyltransferase, serpin B5, and calretinin represent the target proteins that may help overcome resistance in combination therapies. In addition, serpin B5 and calretinin appear to be candidate biomarkers that may be accessible in patients for monitoring of cancer therapy with ease.
- MeSH
- 1-alkyl-2-acetylglycerofosfocholinesterasa genetika metabolismus MeSH
- apoptóza účinky léků MeSH
- benzamidy farmakologie MeSH
- chemorezistence * účinky léků genetika MeSH
- chinazoliny farmakologie MeSH
- cílená molekulární terapie MeSH
- HCT116 buňky MeSH
- inhibitory proteinkinas farmakologie MeSH
- lidé MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- nádory * farmakoterapie genetika MeSH
- protein-serin-threoninkinasy antagonisté a inhibitory metabolismus MeSH
- proteiny vázající GTP genetika metabolismus MeSH
- protinádorové látky farmakologie MeSH
- pyrimidiny farmakologie MeSH
- thiazoly farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Several subunits of the multifunctional eukaryotic translation initiation factor 3 (eIF3) contain well-defined domains. Among them is the conserved bipartite PCI domain, typically serving as the principal scaffold for multisubunit 26S proteasome lid, CSN and eIF3 complexes, which constitutes most of the C-terminal region of the c/NIP1 subunit. Interestingly, the c/NIP1-PCI domain is exceptional in that its deletion, despite being lethal, does not affect eIF3 integrity. Here, we show that a short C-terminal truncation and two clustered mutations directly disturbing the PCI domain produce lethal or slow growth phenotypes and significantly reduce amounts of 40S-bound eIF3 and eIF5 in vivo. The extreme C-terminus directly interacts with blades 1-3 of the small ribosomal protein RACK1/ASC1, which is a part of the 40S head, and, consistently, deletion of the ASC1 coding region likewise affects eIF3 association with ribosomes. The PCI domain per se shows strong but unspecific binding to RNA, for the first time implicating this typical protein-protein binding domain in mediating protein-RNA interactions also. Importantly, as our clustered mutations severely reduce RNA binding, we conclude that the c/NIP1 C-terminal region forms an important intermolecular bridge between eIF3 and the 40S head region by contacting RACK1/ASC1 and most probably 18S rRNA.
- MeSH
- adaptorové proteiny signální transdukční chemie genetika metabolismus MeSH
- delece genu MeSH
- eukaryotický iniciační faktor 3 chemie genetika metabolismus MeSH
- iniciace translace peptidového řetězce MeSH
- interakční proteinové domény a motivy MeSH
- malé podjednotky ribozomu eukaryotické chemie metabolismus MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- podjednotky proteinů chemie metabolismus MeSH
- proteiny vázající GTP chemie genetika metabolismus MeSH
- RNA ribozomální 18S metabolismus MeSH
- Saccharomyces cerevisiae - proteiny biosyntéza chemie genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- transkripční faktory bZIP biosyntéza genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins.
- MeSH
- cirkulární dichroismus MeSH
- fosforylace MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- maloúhlový rozptyl MeSH
- proteiny 14-3-3 chemie genetika metabolismus MeSH
- proteiny aktivující GTPasu chemie genetika metabolismus MeSH
- proteiny vázající GTP chemie genetika metabolismus MeSH
- rezonanční přenos fluorescenční energie MeSH
- sekundární struktura proteinů MeSH
- signální transdukce MeSH
- terciární struktura proteinů MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) for the alpha-subunit of heterotrimeric G proteins. Several RGS proteins have been found to interact with 14-3-3 proteins. The 14-3-3 protein binding inhibits the GAP function of RGS proteins presumably by blocking their interaction with G(alpha) subunit. Since RGS proteins interact with G(alpha) subunits through their RGS domains, it is reasonable to assume that the 14-3-3 protein can either sterically occlude the G(alpha) interaction surface of RGS domain and/or change its structure. In this work, we investigated whether the 14-3-3 protein binding affects the structure of RGS3 using the time-resolved tryptophan fluorescence spectroscopy. Two single-tryptophan mutants of RGS3 were used to study conformational changes of RGS3 molecule. Our measurements revealed that the 14-3-3 protein binding induces structural changes in both the N-terminal part and the C-terminal RGS domain of phosphorylated RGS3 molecule. Experiments with the isolated RGS domain of RGS3 suggest that this domain alone can, to some extent, interact with the 14-3-3 protein in a phosphorylation-independent manner. In addition, a crystal structure of the RGS domain of RGS3 was solved at 2.3A resolution. The data obtained from the resolution of the structure of the RGS domain suggest that the 14-3-3 protein-induced conformational change affects the region within the G(alpha)-interacting portion of the RGS domain. This can explain the inhibitory effect of the 14-3-3 protein on GAP activity of RGS3.
- MeSH
- fluorescenční spektrometrie MeSH
- fosforylace MeSH
- interakční proteinové domény a motivy MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- molekulární modely MeSH
- multiproteinové komplexy MeSH
- mutageneze cílená MeSH
- podjednotky proteinů MeSH
- proteiny 14-3-3 chemie metabolismus MeSH
- proteiny aktivující GTPasu chemie genetika metabolismus MeSH
- proteiny vázající GTP chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- rezonanční přenos fluorescenční energie MeSH
- sekvence aminokyselin MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- stabilita proteinů MeSH
- substituce aminokyselin MeSH
- techniky in vitro MeSH
- terciární struktura proteinů MeSH
- tryptofan chemie MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
