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| 2. |
- Böiers, Charlotta, et al.
(författare)
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Lymphomyeloid Contribution of an Immune-Restricted Progenitor Emerging Prior to Definitive Hematopoietic Stem Cells.
- 2013
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Ingår i: Cell Stem Cell. - : Elsevier BV. - 1934-5909 .- 1875-9777. ; 13:5, s. 535-548
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Tidskriftsartikel (refereegranskat)abstract
- In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the innate immune system are formed early in development, whereas lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we demonstrate that the embryonic lymphoid commitment process initiates earlier than previously appreciated, prior to emergence of definitive HSCs, through establishment of a previously unrecognized entirely immune-restricted and lymphoid-primed progenitor. Notably, this immune-restricted progenitor appears to first emerge in the yolk sac and contributes physiologically to the establishment of lymphoid and some myeloid components of the immune-system, establishing the lymphomyeloid lineage restriction process as an early and physiologically important lineage-commitment step in mammalian hematopoiesis.
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| 3. |
- Aleksandrova, Elena V, et al.
(författare)
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Structural basis of Cfr-mediated antimicrobial resistance and mechanisms for its evasion
- 2023
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The ribosome is an essential drug target as many classes of clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent mechanisms of resistance to PTC-acting drugs is C8-methylation of the universally conserved adenine residue 2503 (A2503) of the 23S rRNA by the methyltransferase Cfr. Despite its clinical significance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. In this work, we developed a method to express a functionally-active Cfr-methyltransferase in the thermophilic bacterium Thermus thermophilus and report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-tRNAs. Our structures reveal that an allosteric rearrangement of nucleotide A2062 upon Cfr-methylation of A2503 is likely responsible for the inability of some PTC inhibitors to bind to the ribosome, providing additional insights into the Cfr resistance mechanism. Lastly, by determining the structures of the Cfr-methylated ribosome in complex with the antibiotics iboxamycin and tylosin, we provide the structural bases behind two distinct mechanisms of evading Cfr-mediated resistance.
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| 4. |
- Aleksandrova, Elena V., et al.
(författare)
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Structural basis of Cfr-mediated antimicrobial resistance and mechanisms to evade it
- 2024
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Ingår i: Nature Chemical Biology. - : Springer Nature. - 1552-4450 .- 1552-4469.
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Tidskriftsartikel (refereegranskat)abstract
- The bacterial ribosome is an essential drug target as many clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent resistance mechanisms to PTC-acting drugs in Gram-positive bacteria is C8-methylation of the universally conserved A2503 nucleobase by Cfr methylase in 23S ribosomal RNA. Despite its clinical importance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. Here, we report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-transfer RNAs. These structures reveal an allosteric rearrangement of nucleotide A2062 upon Cfr-mediated methylation of A2503 that likely contributes to the reduced potency of some PTC inhibitors. Additionally, we provide the structural bases behind two distinct mechanisms of engaging the Cfr-methylated ribosome by the antibiotics iboxamycin and tylosin.
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| 5. |
- Obana, Nozomu, et al.
(författare)
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Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria : VmlR2, Ard1 and CplR
- 2023
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 51:9, s. 4536-4554
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Tidskriftsartikel (refereegranskat)abstract
- Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.
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| 6. |
- Ainelo, Andres, et al.
(författare)
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The structure of DarB in complex with RelNTD reveals nonribosomal activation of Rel stringent factors
- 2023
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 9:3, s. 1-14
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Tidskriftsartikel (refereegranskat)abstract
- Rel stringent factors are bifunctional ribosome-associated enzymes that catalyze both synthesis and hydrolysis of the alarmones (p)ppGpp. Besides the allosteric control by starved ribosomes and (p)ppGpp, Rel is regulated by various protein factors depending on specific stress conditions, including the c-di-AMP-binding protein DarB. However, how these effector proteins control Rel remains unknown. We have determined the crystal structure of the DarB2:RelNTD2 complex, uncovering that DarB directly engages the SYNTH domain of Rel to stimulate (p)ppGpp synthesis. This association with DarB promotes a SYNTH-primed conformation of the N-terminal domain region, markedly increasing the affinity of Rel for ATP while switching off the hydrolase activity of the enzyme. Binding to c-di-AMP rigidifies DarB, imposing an entropic penalty that precludes DarB-mediated control of Rel during normal growth. Our experiments provide the basis for understanding a previously unknown mechanism of allosteric regulation of Rel stringent factors independent of amino acid starvation.
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| 7. |
- Ajawatanawong, Pravech, et al.
(författare)
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SeqFIRE : a web application for automated extraction of indel regions and conserved blocks from protein multiple sequence alignments
- 2012
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:W1, s. W340-W347
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Tidskriftsartikel (refereegranskat)abstract
- Analyses of multiple sequence alignments generally focus on well-defined conserved sequence blocks, while the rest of the alignment is largely ignored or discarded. This is especially true in phylogenomics, where large multigene datasets are produced through automated pipelines. However, some of the most powerful phylogenetic markers have been found in the variable length regions of multiple alignments, particularly insertions/deletions (indels) in protein sequences. We have developed Sequence Feature and Indel Region Extractor (SeqFIRE) to enable the automated identification and extraction of indels from protein sequence alignments. The program can also extract conserved blocks and identify fast evolving sites using a combination of conservation and entropy. All major variables can be adjusted by the user, allowing them to identify the sets of variables most suited to a particular analysis or dataset. Thus, all major tasks in preparing an alignment for further analysis are combined in a single flexible and user-friendly program. The output includes a numbered list of indels, alignments in NEXUS format with indels annotated or removed and indel-only matrices. SeqFIRE is a user-friendly web application, freely available online at www.seqfire.org/.
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| 8. |
- Atkinson, Gemma C., et al.
(författare)
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An evolutionary ratchet leading to loss of elongation factors in eukaryotes
- 2014
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Ingår i: BMC Evolutionary Biology. - : Springer Science and Business Media LLC. - 1471-2148. ; 14, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- Background: The GTPase eEF1A is the eukaryotic factor responsible for the essential, universal function of aminoacyl-tRNA delivery to the ribosome. Surprisingly, eEF1A is not universally present in eukaryotes, being replaced by the paralog EFL independently in multiple lineages. The driving force behind this unusually frequent replacement is poorly understood. Results: Through sequence searching of genomic and EST databases, we find a striking association of eEF1A replacement by EFL and loss of eEF1A's guanine exchange factor, eEF1Bα, suggesting that EFL is able to spontaneously recharge with GTP. Sequence conservation and homology modeling analyses indicate several sequence regions that may be responsible for EFL's lack of requirement for eEF1Bα. Conclusions: We propose that the unusual pattern of eEF1A, eEF1Bα and EFL presence and absence can be explained by a ratchet-like process: if either eEF1A or eEF1Bα diverges beyond functionality in the presence of EFL, the system is unable to return to the ancestral, eEF1A:eEFBα-driven state.
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| 9. |
- Atkinson, Gemma C., et al.
(författare)
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Evolution of nonstop, no-go and nonsense-mediated mRNA decay and their termination factor-derived components
- 2008
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Ingår i: BMC Evolutionary Biology. - : Springer Science and Business Media LLC. - 1471-2148. ; 8, s. 290-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Members of the eukaryote/archaea specific eRF1 and eRF3 protein families have central roles in translation termination. They are also central to various mRNA surveillance mechanisms, together with the eRF1 paralogue Dom34p and the eRF3 paralogues Hbs1p and Ski7p. We have examined the evolution of eRF1 and eRF3 families using sequence similarity searching, multiple sequence alignment and phylogenetic analysis. Results: Extensive BLAST searches confirm that Hbs1p and eRF3 are limited to eukaryotes, while Dom34p and eRF1 (a/eRF1) are universal in eukaryotes and archaea. Ski7p appears to be restricted to a subset of Saccharomyces species. Alignments show that Dom34p does not possess the characteristic class-1 RF minidomains GGQ, NIKS and YXCXXXF, in line with recent crystallographic analysis of Dom34p. Phylogenetic trees of the protein families allow us to reconstruct the evolution of mRNA surveillance mechanisms mediated by these proteins in eukaryotes and archaea. Conclusion: We propose that the last common ancestor of eukaryotes and archaea possessed Dom34p-mediated no-go decay (NGD). This ancestral Dom34p may or may not have required a trGTPase, mostly like a/eEF1A, for its delivery to the ribosome. At an early stage in eukaryotic evolution, eEF1A was duplicated, giving rise to eRF3, which was recruited for translation termination, interacting with eRF1. eRF3 evolved nonsense-mediated decay (NMD) activity either before or after it was again duplicated, giving rise to Hbs1p, which we propose was recruited to assist eDom34p in eukaryotic NGD. Finally, a third duplication within ascomycete yeast gave rise to Ski7p, which may have become specialised for a subset of existing Hbs1p functions in non-stop decay (NSD). We suggest Ski7p-mediated NSD may be a specialised mechanism for counteracting the effects of increased stop codon read-through caused by prion-domain [ PSI+] mediated eRF3 precipitation.
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| 10. |
- Beljantseva, Jelena, et al.
(författare)
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Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA
- 2017
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:14, s. 3726-3731
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Tidskriftsartikel (refereegranskat)abstract
- The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, pathogenicity, and antibiotic tolerance. We show that the tetrameric small alarmone synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis is a sequence-specific RNA-binding protein. RelQ's enzymatic and RNA binding activities are subject to intricate allosteric regulation. (p)ppGpp synthesis is potently inhibited by the binding of single-stranded RNA. Conversely, RelQ's enzymatic activity destabilizes the RelQ: RNA complex. pppGpp, an allosteric activator of the enzyme, counteracts the effect of RNA. Tetramerization of RelQ is essential for this regulatory mechanism, because both RNA binding and enzymatic activity are abolished by deletion of the SAS-specific C-terminal helix 5 alpha. The interplay of pppGpp binding, (p)ppGpp synthesis, and RNA binding unites two archetypal regulatory paradigms within a single protein. The mechanism is likely a prevalent but previously unappreciated regulatory switch used by the widely distributed bacterial SAS enzymes.
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