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A comprehensive method for determining cellular uptake of purine nucleoside phosphorylase and adenylosuccinate synthetase inhibitors by
Get PDFDue to the growing number of Helicobacter pylori strains resistant to currently available antibiotics, there is an urgent need to design new drugs utilizing different molecular mechanisms than those that have been used up to now. Enzymes of the purine salvage pathway are possible targets of such new antibiotics because H. pylori is not able to synthetize purine nucleotides de novo. The bacterium’s recovery of purines and purine nucleotides from the environment is the only source of these essential DNA and RNA building blocks. We have identified formycins and hadacidin as potent inhibitors of purine nucleoside phosphorylase (PNP) and adenylosuccinate synthetase (AdSS) from H. pylori — two key enzymes of the purine salvage pathway. However, we have found that these compounds are not effective in H. pylori cell cultures. To address this issue, we have developed a universal comprehensive method for assessing H. pylori cell penetration by drug candidates, with three alternative detection assays. These include liquid chromatography tandem mass spectrometry, UV absorption, and inhibition of the target enzyme by the tested compound. Using this approach, we have shown that cellular uptake by H. pylori of formycins and hadacidin is very poor, which reveals why their in vitro inhibition of PNP and AdSS and their effect on H. pylori cell cultures are so different. The cell penetration assessment method developed here will be extremely useful for validating the cellular uptake of other drug candidates, facilitating the design of new potent therapeutic agents against H. pylori
Intermediate Cu(II)-Thiolate Species in the Reduction of Cu(II)GHK by Glutathione: A Handy Chelate for Biological Cu(II) Reduction
Get PDFGly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV–vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter
Expanding Diversity of Firmicutes Single-Strand Annealing Proteins: a Putative Role of Bacteriophage-Host Arms Race
Get PDFBacteriophage-encoded single strand annealing proteins (SSAPs) are recombinases
which can substitute the classical, bacterial RecA and manage the DNA metabolism
at different steps of phage propagation. SSAPs have been shown to efficiently promote
recombination between short and rather divergent DNA sequences and were exploited
for in vivo genetic engineering mainly in Gram-negative bacteria. In opposition to the
conserved and almost universal bacterial RecA protein, SSAPs display great sequence
diversity. The importance for SSAPs in phage biology and phage-bacteria evolution is
underlined by their role as key players in events of horizontal gene transfer (HGT). All
of the above provoke a constant interest for the identification and study of new phage
recombinase proteins in vivo, in vitro as well as in silico. Despite this, a huge body
of putative ssap genes escapes conventional classification, as they are not properly
annotated. In this work, we performed a wide-scale identification, classification and
analysis of SSAPs encoded by the Firmicutes bacteria and their phages. By using
sequence similarity network and gene context analyses, we created a new high quality
dataset of phage-related SSAPs, substantially increasing the number of annotated
SSAPs. We classified the identified SSAPs into seven distinct families, namely RecA,
Gp2.5, RecT/Redb, Erf, Rad52/22, Sak3, and Sak4, organized into three superfamilies.
Analysis of the relationships between the revealed protein clusters led us to recognize
Sak3-like proteins as a new distinct SSAP family. Our analysis showed an irregular
phylogenetic distribution of ssap genes among different bacterial phyla and specific
phages, which can be explained by the high rates of ssap HGT. We propose that
the evolution of phage recombinases could be tightly linked to the dissemination
of bacterial phage-resistance mechanisms (e.g., abortive infection and CRISPR/Cas
systems) targeting ssap genes and be a part of the constant phage-bacteria arms race
Reprogramming mRNA Expression in Response to Defect in RNA Polymerase III Assembly in the Yeast Saccharomyces cerevisiae
Get PDFThe coordinated transcription of the genome is the fundamental mechanism in molecular biology. Transcription in eukaryotes is carried out by three main RNA polymerases: Pol I, II, and III. One basic problem is how a decrease in tRNA levels, by downregulating Pol III efficiency, influences the expression pattern of protein-coding genes. The purpose of this study was to determine the mRNA levels in the yeast mutant rpc128-1007 and its overdose suppressors, RBS1 and PRT1. The rpc128-1007 mutant prevents assembly of the Pol III complex and functionally mimics similar mutations in human Pol III, which cause hypomyelinating leukodystrophies. We applied RNAseq followed by the hierarchical clustering of our complete RNA-seq transcriptome and functional analysis of genes from the clusters. mRNA upregulation in rpc128-1007 cells was generally stronger than downregulation. The observed induction of mRNA expression was mostly indirect and resulted from the derepression of general transcription factor Gcn4, differently modulated by suppressor genes. rpc128-1007 mutation, regardless of the presence of suppressors, also resulted in a weak increase in the expression of ribosome biogenesis genes. mRNA genes that were downregulated by the reduction of Pol III assembly comprise the proteasome complex. In summary, our results provide the regulatory links affected by Pol III assembly that contribute differently to cellular fitness
Global view on the metabolism of RNA poly(A) tails in yeast Saccharomyces cerevisiae
No full textThe polyadenosine tail (poly[A]-tail) is a universal modification of eukaryotic messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). In budding yeast, Pap1-synthesized mRNA poly(A) tails enhance export and translation, whereas Trf4/5-mediated polyadenylation of ncRNAs facilitates degradation by the exosome. Using direct RNA sequencing, we decipher the extent of poly(A) tail dynamics in yeast defective in all relevant exonucleases, deadenylases, and poly(A) polymerases. Predominantly ncRNA poly(A) tails are 20-60 adenosines long. Poly(A) tails of newly transcribed mRNAs are 50 adenosine long on average, with an upper limit of 200. Exonucleolysis by Trf5-assisted nuclear exosome and cytoplasmic deadenylases trim the tails to 40 adenosines on average. Surprisingly, PAN2/3 and CCR4-NOT deadenylase complexes have a large pool of non-overlapping substrates mainly defined by expression level. Finally, we demonstrate that mRNA poly(A) tail length strongly responds to growth conditions, such as heat and nutrient deprivation
The Lactococcal dgkB (yecE) and dxsA Genes for Lipid Metabolism Are Involved in the Resistance to Cell Envelope-Acting Antimicrobials
Get PDFAbstract: The emergence of antibiotic-resistant bacteria led to an urgent need for next-generation antimicrobial agents with novel mechanisms of action. The use of positively charged antimicro-bial peptides that target cytoplasmic membrane is an especially promising strategy since essen-tial functions and the conserved structure of the membrane hinder the development of bacterial resistance. Aureocin A53- and enterocin L50-like bacteriocins are highly cationic, mem-brane-targeting antimicrobial peptides that have potential as next-generation antibiotics. How-ever, the mechanisms of resistance to these bacteriocins and cross-resistance against antibiotics must be examined before application to ensure their safe use. Here, in the model bacterium Lac-tococcus lactis, we studied the development of resistance to selected aureocin A53- and enterocin L50-like bacteriocins and its correlation with antibiotics. First, to generate spontaneous resistant mutants, L. lactis was exposed to bacteriocin BHT-B. Sequencing of their genomes revealed single nucleotide polymorphisms (SNPs) in the dgkB (yecE) and dxsA genes encoding diacylglycerol kinase and 1-deoxy-D-xylulose 5-phosphate synthase, respectively. Then, selected mutants un-derwent susceptibility tests with a wide array of bacteriocins and antibiotics. The highest altera-tions in the sensitivity of studied mutants were seen in the presence of cytoplasmic membrane targeting bacteriocins (K411, Ent7, EntL50, WelM, SalC, nisin) and antibiotics (daptomycin and gramicidin) as well as lipid II cycle-blocking bacteriocins (nisin and Lcn972) and antibiotics (bac-itracin). Interestingly, decreased via the SNPs accumulation sensitivity to membrane-active bac-teriocins and antibiotics resulted in the concurrently increased vulnerability to bacitracin, car-benicillin or chlortetracycline. It is suspected that SNPs may result in alterations to the efficiency of the nascent enzymes rather than a total loss of their function as neither deletion nor overex-pression of dxsA restored the phenotype observed in spontaneous mutants
Phototropin interactions with SUMO proteins
Get PDFThe disruption of the sumoylation pathway affects processes controlled by the two phototropins (phots) of Arabidopsis thaliana, phot1 and phot2. Phots, plant UVA/blue light photoreceptors, regulate growth responses and fast movements aimed at optimizing photosynthesis, such as phototropism, chloroplast relocations and stomatal opening. Sumoylation is a posttranslational modification, consisting of the addition of a SUMO (SMALL UBIQUITIN-RELATED MODIFIER) protein to a lysine residue in the target protein. In addition to affecting the stability of proteins, it regulates their activity, interactions and subcellular localization. We examined physiological responses controlled by phots, phototropism and chloroplast movements, in sumoylation pathway mutants. Chloroplast accumulation in response to both continuous and pulse light was enhanced in the E3 ligase siz1 mutant, in a manner dependent on phot2. A significant decrease in phot2 protein abundance was observed in this mutant after blue light treatment both in seedlings and mature leaves. Using plant transient expression and yeast two-hybrid assays, we found that phots interacted with SUMO proteins mainly through their N-terminal parts, which contain the photosensory LOV domains. The covalent modification in phots by SUMO was verified using an Arabidopsis sumoylation system reconstituted in bacteria followed by the mass spectrometry analysis. Lys 297 was identified as the main target of SUMO3 in the phot2 molecule. Finally, sumoylation of phot2 was detected in Arabidopsis mature leaves upon light or heat stress treatment
Unique properties of alpha-helical DNA-binding KfrA protein of RA3 plasmid from IncU incompatibility group and its host-dependent role in plasmid maintenance.
Get PDFKfrA, encoded on the broad-host-range RA3 plasmid, is an alphahelical DNA-binding protein that acts as a transcriptional autoregulator. The KfrARA3 operator site overlaps the kfrA promoter and is composed of five 9-bp direct repeats (DRs). Here, the biological properties of KfrA were studied using both in vivo and in vitro approaches. Localization of the DNA-binding helix-turn-helix
motif (HTH) was mapped to the N29-R52 region by protein structure modeling and confirmed by alanine scanning. KfrA repressor ability depended on the number and orientation of DRs in the operator, as well as the ability of the protein
to oligomerize. The long alpha-helical tail from residues 54 to 355 was shown to be involved in self-interactions, whereas the region from residue 54 to 177 was involved in heterodimerization with KfrC, another RA3-encoded alphahelical protein. KfrA also interacted with the segrosome proteins IncC (ParA) and KorB (ParB), representatives of the class Ia active partition systems. Deletion of the kfr genes from the RA3 stability module decreased the plasmid retention in diverse hosts in a species-dependent manner. The specific interactions of KfrA with DNA are essential not only for the transcriptional regulatory function but also for the accessory role of KfrA in stable plasmid maintenance
Proteomic Profile of Saliva in Parkinson’s Disease Patients: A Proof of Concept Study
No full textParkinson’s disease (PD) is a progressive neurodegenerative disorder. It affects many organs. Lewy bodies—a histopathological “hallmark” of PD—are detected in about 75% of PD submandibular gland samples. We hypothesize that saliva can be a source of biomarkers of PD. The aim of the study was to evaluate and compare the salivary proteome of PD patients and healthy controls (HC). Salivary samples from 39 subjects (24 PD patients, mean age 61.6 ± 8.2; 15 HC, mean age 60.9 ± 6.7) were collected. Saliva was collected using RNA-Pro-Sal kits. Label-free LC-MS/MS mass spectrometry was performed to characterize the proteome of the saliva. IPA analysis of upstream inhibitors was performed. A total of 530 proteins and peptides were identified. We observed lower concentrations of S100-A16, ARP2/3, and VPS4B in PD group when compared to HC. We conclude that the salivary proteome composition of PD patients is different than that of healthy controls. We observed a lower concentration of proteins involved in inflammatory processes, exosome formation, and adipose tissue formation. The variability of expression of proteins between the two groups needs to be considered
