The 6-oxopurine phosphoribosyltransferases (PRTs) are drug targets for the treatment of parasitic diseases. This is due to the fact that parasites are auxotrophic for the 6-oxopurine bases relying on salvage enzymes for the synthesis of their 6-oxopurine nucleoside monophosphates. In Trypanosoma brucei, the parasite that is the aetiological agent for sleeping sickness, there are three 6-oxopurine PRT isoforms. Two are specific for hypoxanthine and guanine, whilst the third, characterized here, uses all three naturally occurring bases with similar efficiency. Here, we have determined crystal structures for TbrHGXPRT in complex with GMP, XMP and IMP to investigate the structural basis for substrate specificity. The results show that Y201 and E208, not commonly observed within the purine binding pocket of 6-oxopurine PRTs, contribute to the versatility of this enzyme. The structures further show that a nearby water can act as an adaptor to facilitate the binding of XMP and GMP. When GMP binds, a water can accept a proton from the 2-amino group but when XMP binds, the equivalent water can donate its proton to the 2-oxo group. However, when IMP is bound, no water molecule is observed at that location. DATABASE: Coordinates and structure factors were submitted to the Protein Data Bank and have accession codes of 6MXB, 6MXC, 6MXD and 6MXG for the TbrHGXPRT.XMP complex, TbrHGXPRT.GMP complex, TbrHGXPRT.IMP complex, and TbrHGPRT.XMP complex, respectively.
- MeSH
- inosinmonofosfát chemie metabolismus MeSH
- konformace proteinů MeSH
- kyselina 5'-guanylová chemie metabolismus MeSH
- pentosyltransferasy chemie metabolismus MeSH
- ribonukleotidy chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- substrátová specifita MeSH
- Trypanosoma brucei brucei enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Structural details of changes accompanying interaction between insulin-related hormones and their binding partners are often enigmatic. Here, cross-linking/mass spectrometry could complement structural techniques and reveal details of these protein-protein interfaces. We used such approach to clarify missing structural description of the interface in human insulin-like growth factor (IGF-1): Drosophila melanogaster imaginal morphogenesis protein-late 2 protein (Imp-L2) complex which we studied previously by X-ray crystallography. We crosslinked these proteins by heterobifunctional cross-linker sulfosuccinimidyl 4,4'-azidopentanoate (Sulfo-SDA) for the subsequent mass spectrometry (MS) analysis. The MS analysis revealed IGF-1:Imp-L2 interactions which were not resolved in the crystal structure of this assembly, and they converged with X-ray results, indicating the importance of the IGF-1 N-terminus interaction with the C-terminal (185-242) part of the Imp-L2 for stability of this complex. Here, we also showed the advantage and reliability of MS approach in solving details of protein-protein interactions that are too flexible for solid state structural methods.
- Publikační typ
- časopisecké články MeSH
The complete nucleotide sequences of six IMP-4-encoding plasmids recovered from Enterobacteriaceae isolates of wildlife origin were characterized. Sequencing data showed that plasmids of different incompatibility groups (IncM, IncI1, IncF, and nontypeable [including an IncX5_2 and two pPrY2001-like]) carried the blaIMP-4-carrying integrons In809 or In1460. Most of the plasmids carried an mph(A) region, and chrA-like, aac(3)-IId, and blaTEM-1b genes. Finally, plasmid analysis revealed the involvement of two different IS26- and Tn1696-associated mechanisms in the mobilization of IMP-4-encoding integrons.
Inosine-5'-monophosphate dehydrogenase catalyzes the critical step in the de novo synthesis of guanosine nucleotides: the oxidation of inosine monophosphate to xanthosine monophosphate. This reaction can be inhibited by specific inhibitors, such as ribavirin or mycophenolic acid, which are widely used in clinical treatment when required to inhibit the proliferation of viruses or cells. However, it was recently found that such an inhibition affects the cells, leading to a redistribution of IMPDH2 and the appearance of IMPDH2 inclusions in the cytoplasm. According to their shape, these inclusions have been termed "Rods and Rings" (R&R). In this work, we focused on the subcellular localization of IMPDH2 protein and the ultrastructure of R&R inclusions. Using microscopy and western blot analysis, we show the presence of nuclear IMPDH2 in human cells. We also show that the nuclear pool has an ability to form Rod structures after inhibition by ribavirin. Concerning the ultrastructure, we observed that R&R inclusions in cellulo correspond to the accumulation of fibrous material that is not surrounded by a biological membrane. The individual fibers are composed of regularly repeating subunits with a length of approximately 11 nm. Together, our findings describe the localization of IMPDH2 inside the nucleus of human cells as well as the ultrastructure of R&R inclusions.
- MeSH
- aktivní transport - buněčné jádro účinky léků MeSH
- buněčné jádro enzymologie metabolismus ultrastruktura MeSH
- cytoplazma enzymologie ultrastruktura MeSH
- HeLa buňky MeSH
- IMP-dehydrogenasa antagonisté a inhibitory chemie metabolismus ultrastruktura MeSH
- inhibitory enzymů farmakologie MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The objective of this study was to perform molecular surveillance for assessing the spread of carbapenemase-producing Pseudomonas aeruginosa in Czech hospitals. One hundred thirty-six carbapenemase-producing isolates were recovered from 22 hospitals located throughout the country. Sequence type 357 (ST357) dominated (n = 120) among carbapenemase producers. One hundred seventeen isolates produced IMP-type (IMP-7 [n = 116] and IMP-1 [n = 1]) metallo-β-lactamases (MβLs), 15 produced the VIM-2 MβL, and the remaining isolates expressed the GES-5 enzyme. The blaIMP-like genes were located in three main integron types, with In-p110-like being the most prevalent (n = 115). The two other IMP-encoding integrons (In1392 and In1393) have not been described previously. blaVIM-2-carrying integrons included In59-like, In56, and a novel element (In1391). blaGES-5 was carried by In717. Sequencing data showed that In-p110-like was associated with a Tn4380-like transposon inserted in genomic island LESGI-3 in the P. aeruginosa chromosome. The other integrons were also integrated into the P. aeruginosa chromosome. These findings indicated the clonal spread of ST357 P. aeruginosa, carrying the IMP-7-encoding integron In-p110, in Czech hospitals. Additionally, the sporadic emergence of P. aeruginosa producing different carbapenemase types, associated with divergent or novel integrons, punctuated the ongoing evolution of these bacteria.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální chromozomy chemie MeSH
- beta-laktamasy genetika metabolismus MeSH
- epidemiologické monitorování MeSH
- exprese genu MeSH
- genomové ostrovy MeSH
- genotyp MeSH
- incidence MeSH
- integrony MeSH
- izoenzymy genetika metabolismus MeSH
- karbapenemy farmakologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- mnohočetná bakteriální léková rezistence genetika MeSH
- multilokusová sekvenční typizace MeSH
- nemocnice MeSH
- pseudomonádové infekce epidemiologie mikrobiologie MeSH
- Pseudomonas aeruginosa účinky léků enzymologie genetika izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- Geografické názvy
- Česká republika epidemiologie MeSH
OBJECTIVES: The objective of this study was to investigate the silver gull as an indicator of environmental contamination by salmonellae and carbapenemase-producing Enterobacteriaceae (CPE) in south-east Australia. METHODS: A total of 504 cloacal samples were collected from gull chicks at three nesting colonies in New South Wales, Australia [White Bay (n = 144), Five Islands (n = 200) and Montague Island (n = 160)] and were examined for salmonellae and CPE. Isolates were tested for carbapenemase genes and susceptibility to 14 antibiotics. Clonality was determined by PFGE and MLST. Genetic context and conjugative transfer of the carbapenemase gene were determined. RESULTS: A total of 120 CPE of 10 species, mainly Escherichia coli (n = 85), carrying the gene blaIMP-4, blaIMP-38 or blaIMP-26 were obtained from 80 (40%) gulls from Five Islands. Thirty percent of birds from this colony were colonized by salmonellae. Most isolates contained the gene within a class 1 integron showing a blaIMP-4-qacG-aacA4-catB3 array. The blaIMP gene was carried by conjugative plasmids of variable sizes (80-400 kb) and diverse replicons, including HI2-N (n = 30), HI2 (11), A/C (17), A/C-Y (2), L/M (5), I1 (1) and non-typeable (6). Despite the overall high genetic variability, common clones and plasmid types were shared by different birds and bacterial isolates, respectively. CONCLUSIONS: Our data demonstrate a large-scale transmission of carbapenemase-producing bacteria into wildlife, likely as a result of the feeding habits of the birds at a local waste depot. The isolates from gulls showed significant similarities with clinical isolates from Australia, suggesting the human origin of the isolates. The sources of CPE for gulls on Five Islands should be explored and proper measures applied to stop the transmission into the environment.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální proteiny analýza genetika MeSH
- beta-laktamasy analýza genetika MeSH
- Charadriiformes mikrobiologie MeSH
- Enterobacteriaceae enzymologie izolace a purifikace MeSH
- enterobakteriální infekce epidemiologie mikrobiologie veterinární MeSH
- genotyp MeSH
- kloaka mikrobiologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- molekulární epidemiologie MeSH
- molekulární typizace MeSH
- nemoci ptáků epidemiologie mikrobiologie MeSH
- ostrovy epidemiologie MeSH
- prevalence MeSH
- ptáci MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Nový Jižní Wales epidemiologie MeSH
- ostrovy epidemiologie MeSH
IMP-8 metallo-β-lactamase was identified in Klebsiella pneumoniae sequence type 252 (ST252), isolated in a Portuguese hospital in 2009. blaIMP-8 was the first gene cassette of a novel class 3 integron, In1144, also carrying the blaGES-5, blaBEL-1, and aacA4 cassettes. In1144 was located on a ColE1-like plasmid, pKP-M1144 (12,029 bp), with a replication region of limited nucleotide similarity to those of other RNA-priming plasmids, such as pJHCMW1. In1144 and pKP-M1144 represent an interesting case of evolution of resistance determinants in Gram-negative bacteria.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální proteiny genetika MeSH
- beta-laktamasy genetika MeSH
- DNA bakterií genetika MeSH
- infekce bakteriemi rodu Klebsiella mikrobiologie MeSH
- karbapenemy farmakologie MeSH
- Klebsiella pneumoniae účinky léků genetika izolace a purifikace MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- mnohočetná bakteriální léková rezistence genetika MeSH
- molekulární sekvence - údaje MeSH
- plazmidy účinky léků genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Portugalsko MeSH
