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The angiotensin metabolite His-Leu is a strong copper chelator forming highly redox active species
Get PDFHis-Leu is a hydrolytic byproduct of angiotensin metabolism, whose concentration in the bloodstream could be at least micromolar. This encouraged us to investigate its Cu(II) binding properties and the concomitant redox reactivity. The Cu(II) binding constants were derived from isothermal titration calorimetry and potentiometry, while identities and structures of complexes were obtained from ultraviolet–visible, circular dichroism, and room-temperature electronic paramagnetic resonance spectroscopies. Four types of Cu(II)/His-Leu complexes were detected. The histamine-like complexes prevail at low pH. At neutral and mildly alkaline pH and low Cu(II):His-Leu ratios, they are superseded by diglycine-like complexes involving the deprotonated peptide nitrogen. At His-Leu:Cu(II) ratios of ≥2, bis-complexes are formed instead. Above pH 10.5, a diglycine-like complex containing the equatorially coordinated hydroxyl group predominates at all ratios tested. Cu(II)/His-Leu complexes are also strongly redox active, as demonstrated by voltammetric studies and the ascorbate oxidation assay. Finally, numeric competition simulations with human serum albumin, glycyl-histydyl-lysine, and histidine revealed that His-Leu might be a part of the low-molecular weight Cu(II) pool in blood if its abundance is >10 μM. These results yield further questions, such as the biological relevance of ternary complexes containing His-Leu
Determining the Identity Nucleotides and the Energy of Binding of tRNAs to Their Aminoacyl-tRNA Synthetases Using a Simple Logistic Model.
Get PDFThis study showed that the predictor in logistic regression can be applied to estimating the Gibbs free energy of tRNAs’ recognition of and binding to their aminoacyl-tRNA synthetases. Then, 24 linear logistic regression models predicting different classes of tRNAs loaded with a corresponding amino acid were trained in a machine learning classification method, reducing the misclassification error to zero. The models were based on minimal subsets of Boolean explanatory variables describing the favorite presence of nucleotides or nucleosides localized in the different parts of the tRNA. In 90% of cases, they agree with the components of the consensus strand in a class of tRNAs loaded by a given amino acid. According to the proposed theoretical model, the values of the free energy for the entry of the recognition state in the process of tRNA charging were obtained, and the inputs from identity nucleotides and the tRNA strand backbone were distinguished. Almost all the resulting models indicated leading anticodon tandems defining the first and second positions of the anticodon (positions 35 and 36 of the tRNA strand) and the small sets (up to six positions) of the other nucleotides as the natural identity nucleotides most influential in the free energy balance. The magnitude of their input to this energy depends on the position in the strand, favoring positions −1, 35, and 36. The role of position 34 is relatively smaller. These identity attributes may not always be fully arranged in a real single adaptor molecule but were comprehensively present in a given tRNA class. A detailed analysis of the resulting models showed that the absolute value of the energy of binding the tandem 35–36 decreases with the number of identity positions, as well as with the decreasing number of possible hydrogen bonds. On the other hand, in these conditions, the absolute value of the energy of binding of other identity nucleotides increases. All the models indicate that the nucleotide-independent energy of the repulsion tRNA backbone decreases with the number of identity nucleotides. It was also shown that the total free energy change in entering the recognition state increases with the amino acid mass, making this process less spontaneous, which may have an evolutionary reference
Badania nad mechanizmami działania i rozwoju oporności na bakteriocyny klasy II u bakterii Gram-dodatnich
Get PDFBakteriocyny to grupa peptydów lub białek wytwarzanych przez bakterie w celu zabijania lub hamowania wzrostu innych bakterii zasiedlających tę samą niszę ekologiczną. Zainteresowanie bakteriocynami wynika z ich potencjalnego zastosowania m.in. w konserwacji żywności i terapii zakażeń wywołanych przez antybiotykooporne szczepy bakterii chorobotwórczych. Liczba publikacji identyfikujących nowe szczepy bakterii produkujących bakteriocyny nieustannie wzrasta. Jednocześnie, zauważalny jest brak badań opisujących mechanizmy działania większości nowo zidentyfikowanych bakteriocyn, a także mechanizmy nabywania oporności na te bakteriocyny i oporności krzyżowej na antybiotyki. Dokładne poznanie tych zagadnień pozwoli na opracowanie wytycznych zapewniających najbardziej efektywne, bezpieczne i długotrwałe stosowanie bakteriocyn bez ryzyka rozwoju oporności. W niniejszej pracy opisano główne założenia rozprawy doktorskiej dr inż. Aleksandry Tymoszewskiej, której celem była identyfikacja mechanizmów działania i rozwoju oporności na bakteriocyny klasy II u bakterii Gram-dodatnich. Wykorzystując jako model badawczy komórki bakterii Lactococcus lactis, zbadano dwie grupy bakteriocyn: (i) garwicyny Q, A, B i C, oraz BacSJ; oraz (ii) bakteriocyny aureocyno A53 (AurA53)- i enterocyno L50 (EntL50)- podobne. Pokazano, że bakteriocyny grupy (i) rozpoznają komórki wrażliwe i tworzą pory wewnątrz błony komórkowej bakterii wykorzystując specyficzny receptor, system mannozo-specyficznej fosfotransferazy (Man-PTS), a także, że bakterie wrażliwe nabywają oporność na badane bakteriocyny poprzez modyfikacje struktury Man-PTS. Z kolei nabywanie oporności na bakteriocyny grupy (ii), tworzące pory bezpośrednio w błonie komórkowej bakterii, zachodzi poprzez zmiany w strukturze ściany i błony komórkowej wywołane zmianami w ekspresji białek zaangażowanych w metabolizm lipidów lub stanowiących elementy systemu regulacyjnego YsaCB-KinG-LlrG. Otrzymane wyniki rzucają nowe światło na dotychczasowe poglądy dotyczące mechanizmów działania bakteriocyn i szeroko otwierają możliwości ich dalszych badań
Decoding Arabidopsis thaliana CPK/SnRK Superfamily Kinase Client Signaling Networks Using Peptide Library and Mass Spectrometry
Get PDFMembers of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein
kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of
client specificity of the members of this superfamily is fragmentary. As this family is represented by
over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client
relationships is crucial to our understanding the nuances of this large family of kinases as directed
towards signal transduction pathways. Herein, we used the kinase client (KiC) assay—a relative,
quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay—to identify and
characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four
SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that
contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation
sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase,
CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence
analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS,
that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK-
and SnRK-specific and overlapping signaling network architectures and recapitulate many known
in vivo relationships validating this large-scale approach towards discovering kinase targets
Whole-genome sequencing and characterization of human fecal isolate Lacticaseibacillus casei LC130 from NORDBIOTIC collection
Get PDFWe report the complete genome sequence of Lacticaseibacillus casei LC130, isolated from a healthy human fecal sample and part of the NORDBIOTIC collection. The 2.969 Mb genome of LC130 includes genes potentially involved in lactose metabolism and the production of bacteriocins, peptidases, and polyamines, suggesting potential health benefits.
KEYWORDS probiotics, prohealth effects, bacteriocins, gluten-degrading peptidases, polyamines, lactose metabolism, lactic acid bacteria, fecal human isolat
Polar-Region Soils as Novel Reservoir of Lactic Acid Bacteria from the Genus Carnobacterium
Get PDFPolar habitats offer excellent sites to isolate unique bacterial strains due to their diverse physical, geochemical, and biological factors. We hypothesize that the unique environmental conditions of polar regions select for distinct strains of lactic acid bacteria (LAB) with novel biochemical properties. In this study, we characterized ten strains of psychrotrophic LAB isolated from hitherto poorly described sources—High Arctic and maritime Antarctic soils and soil-like materials, including ornithogenic soils, cryoconites, elephant seal colonies, and postglacial moraines. We evaluated the physiological and biochemical properties of the isolates. Based on 16S rRNA and housekeeping genes, the four LAB strains were assigned to three Carnobacterium species: C. alterfunditum, C. maltaromaticum, and C. jeotgali. The remaining strains may represent three new species of the Carnobacterium genus. All isolates were neutrophilic and halophilic psychrotrophs capable of fermenting various carbohydrates, organic acids, and alcohols. The identified metabolic properties of the isolated Carnobacterium strains suggest possible syntrophic interactions with other microorganisms in polar habitats. Some showed antimicrobial activity against food pathogens such as Listeria monocytogenes and human pathogens like Staphylococcus spp. Several isolates exhibited unique metabolic traits with potential biotechnological applications that could be more effectively exploited under less stringent technological conditions compared to thermophilic LAB strains, such as lower temperatures and reduced nutrient concentrations. Analysis of extrachromosomal genetic elements revealed 13 plasmids ranging from 4.5 to 79.5 kb in five isolates, featuring unique genetic structures and high levels of previously uncharacterized genes. This work is the first comprehensive study of the biochemical properties of both known and new Carnobacterium species and enhances our understanding of bacterial communities in harsh and highly selective polar soil ecosystems
ICESp1109, a novel hybrid Integrative-Conjugative Element of macrolide-resistant Streptococcus pyogenes serotype M77 collected between 2003 and 2017 in Poland
Get PDFBackground. The antibiotic resistance determinants and associated mobile genetic elements (MGEs) were detected among
Streptococcus pyogenes (group A streptococci [GAS]) clinical isolates of an M77 serotype collected in Poland between 2003 and 2017.
Methods. The genomes of 136 M77 GAS isolates were sequenced using short- and selected with long-read approach; whole
genome sequences were analyzed to determine the genetic context of macrolide resistance determinants.
Results. The analysed strains were collected from in- and outpatients. Sequencing data analysis revealed that all strains carried
the tet(O) gene. They were classified as a single sequence type, ST63. The unique erythromycin-resistance determinant, the
erm(TR), was detected in 76.5% (n = 104) of isolates. It was found predominantly (n = 74) within a novel hybrid integrative
conjugative element composed of the ICESp1108-like sequence and ICESp2906 variant, which was then named ICESp1109.
However, in strains isolated before 2008, erm(TR) was located within ICESp2905 (n = 27) and in 3 strains - within stand-alone
ICESp1108-like sequences.
Conclusions. Based on phylogenetic analysis results, the clonal dissemination of the macrolide-resistant S pyogenes M77/ST63
strain with hybrid ICESp1109 was observed between 2008 and 2017. ICESp1109 is the novel hybrid ICE in gram-positive bacteria
Decipher syntrophies within C2-C4 organic acids-degrading anaerobic microbiomes: A multi-omic exploration
Get PDFIn methanogenic ecosystems, carboxylic acid degradation is a crucial process facilitated by specialized bacteria working in syntrophy with methanogens. This study explores the transcriptomic responses in 178 metagenome-assembled genomes upon changes in feeding composition, specifically lactate, butyrate, propionate, and acetate, in four lab-scale bioreactors. Methanothrix soehngenii and Methanoculleus sp. emerged as key biomethanation contributors. Through metatranscriptomics, machine learning-based functional annotation, and flux balance analysis the underlying microbial interactions and dynamics were deciphered. Syntrophomonadaceae and Mesotoga species maintained mutualistic metabolite exchanges with hydrogenotrophic methanogens, degrading primarily butyrate and lactate, respectively. Acetate was mainly consumed by Smithellaceae sp., in competition with M. soehngenii in all reactors. Furthermore, the acetoclastic archaeon exhibited a previously undocumented capability to metabolize lactate, thereby confirming the prevalence of acetotrophic pathway. Transcriptomic profiles revealed an additional layer of complexity, where propionate dismutation and beta-oxidation pathways are interconnected by the exchanges of butyrate between putative syntrophs, including Syntrophomonadaceae and Smithellaceae species. Integrating multi-omics data with genome-scale metabolic modeling enabled the accurate reconstruction of dynamics within the controlled ecosystem. This composite novel approach was applied to the AD system to unveil the intricate relationships operating within the microbiome to promote thriving. Results elucidated the still poorly explored organization of the so-called microbial dark matter
E3 ubiquitin ligase RNF2 protects polymerase ι from destabilization
Get PDFHuman DNA polymerase ι (Polι) belongs to the Y-family of specialized DNA polymerases engaged in the DNA damage tolerance pathway of translesion DNA synthesis that is crucial to the maintenance of genome integrity. The extreme infidelity of Polι and the fact that both its up- and down-regulation correlate with various cancers indicate that Polι expression and access to the replication fork should be strictly controlled. Here, we identify RNF2, an E3 ubiquitin ligase, as a new interacting partner of Polι that is responsible for Polι stabilization in vivo. Interestingly, while we report that RNF2 does not directly ubiquitinate Polι, inhibition of the E3 ubiquitin ligase activity of RNF2 affects the cellular level of Polι thereby protecting it from destabilization. Additionally, we indicate that this mechanism is more general, as DNA polymerase η, another Y-family polymerase and the closest paralogue of Polι, share similar features