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Yeast models of mutations in the mitochondrial ATP6 gene found in human cancer cells
Get PDFSince the discovery of somatic mtDNA mutations in tumor cells, multiple studies have focused on establishing a
causal relationship between those changes and alterations in energy metabolism, a hallmark of cancer cells. Yet
the consequences of these mutations on mitochondrial function remain largely unknown. In this study, Saccharomyces cerevisiae has been used as a model to investigate the functional consequences of four cancer-associated missense mutations (8914CNA, 8932CNT, 8953ANG, 9131TNC) found in the mitochondrial MT-ATP6 gene. This
gene encodes the a-subunit of F1FO-ATP synthase, which catalyzes the last steps of ATP production in mitochondria. Although the four studied mutations affected well-conserved residues of the a-subunit, only one of them (8932CNT) had a significant impact on mitochondrial function, due to a less efficient incorporation of the asubunit into ATP synthase. Our findings indicate that these ATP6 genetic variants found in human tumors are neutral mitochondrial genome substitutions with a limited, if any, impact on the energetic function of mitochondria
Type III CRISPR complexes from Thermus thermophilus
Get PDFPathogen-specific acquired immunity in bacteria is mediated
by the CRISPR (clustered regularly interspaced short
palindromic repeats)-Cas systems. Thermus thermophilus
strain HB8 contains CRISPR systems of several major
subtypes (type I, IIIA and IIIB), and has become a widely
studied model for CRISPR biology. We have selected two
highly expressed CRISPR spacers, crRNA 2.1 and crRNA
2.2, and have enriched endogenous T. thermophilus proteins
that co-purify with these crRNAs. Mass spectroscopy
indicates that the chromatography protocol enriches
predominantly Csm complex subunits, but also Cmr subunits.
After several chromatographic steps, size exclusion
chromatography indicated a molecular mass of the
crRNA associated complex of 265±69 kDa. In agreement
with earlier work, crRNAs of different lengths (containing
the selected spacers) were observed. Most of these were
completely lost when several T. thermophilus csm genes
were ablated
Pentatricopeptide Motifs in the N-Terminal Extension Domain of Yeast Mitochondrial RNA Polymerase Rpo41p Are Not Essential for Its Function
Get PDFThe core mitochondrial RNA polymerase is a single-subunit enzyme that in yeast Saccharomyces cerevisiae is encoded by the nuclear RPO41 gene. It is an evolutionary descendant of the bacteriophage RNA polymerases, but it includes an additional unconserved N-terminal extension (NTE) domain that is unique to the organellar enzymes. This domain mediates interactions between the polymerase and accessory regulatory factors, such as yeast Sls1p and Nam1p. Previous studies demonstrated that deletion of the entire NTE domain results only in a temperature-dependent respiratory deficiency. Several sequences related to the pentatricopeptide (PPR) motifs were identified in silico in Rpo41p, three of which are located in the NTE domain. PPR repeat proteins are a large family of organellar RNA-binding factors, mostly involved in posttranscriptional gene expression mechanisms. To study their function, we analyzed the phenotype of strains bearing Rpo41p variants where each of these motifs was deleted. We found that deletion of any of the three PPR motifs in the NTE domain does not affect respiratory growth at normal temperature, and it results in a moderate decrease in mtDNA stability. Steady-state levels of COX1 and COX2 mRNAs are also moderately affected. Only the deletion of the second motif results in a partial respiratory deficiency, manifested only at elevated temperature. Our results thus indicate that the PPR motifs do not play an essential role in the function of the NTE domain of the mitochondrial RNA polymerase
Antiangiogenic activity and plasma stability study of peptidomimetics containing unnatural proline analogs
Get PDFBiologically active peptides are often considered as leading compounds in the drugs development process. However, the peptide bond is susceptible to enzymatic hydrolysis, thus the peptides may not be entirely stable in body fluids. One of the way to reduce this effect is to design new analogs with modified structure and properties. In general, such compounds are similar to the parent peptide sequence (to preserve biological activity), but structural changes ensure higher
degradation resistance due to the enzyme failure of recognizing
cleavage site
New insights into interactions between the nucleotide-binding domain of CFTR and keratin 8
Get PDFThe intermediate filament protein keratin 8 (K8) interacts with the nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane regulator (CFTR) with phenylalanine 508 deletion (ΔF508), and this interaction hampers the biogenesis of functional ΔF508-CFTR and its insertion into the plasma membrane. Interruption of this interaction may constitute a new therapeutic target for cystic fibrosis patients bearing the ΔF508 mutation. Here we aimed to determine the binding surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify the NBD1 fragments perturbed by the ΔF508 mutation, we used hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS) on recombinant wild-type (wt) NBD1 and ΔF508-NBD1 of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head domain, and extended this investigation using bioinformatics procedures and surface plasmon resonance, which revealed regions affected by the peptide binding in both wt-NBD1 and ΔF508-NBD1. Finally, we performed HDX-MS analysis of the NBD1 molecules and full-length K8, revealing hydrogen-bonding network changes accompanying complex formation. In conclusion, we have localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also confirm the stronger binding of K8 to ΔF508-NBD1, which is supported by an additional binding site located in the vicinity of the ΔF508 mutation in NBD1. This article is protected by copyright. All rights reserved
Adhesion of the genome-sequenced Lactococcus lactis subsp. cremoris IBB477 strain is mediated by specific molecular determinants
Get PDFUnderstanding the nature of mucus-microbe interactions will provide important information that can help to elucidate the mechanisms underlying probiotic adhesion. This study focused on the adhesive properties of the Lactococcus lactis subsp. cremoris IBB477 strain, previously shown to persist in the gastrointestinal tract of germ-free rats. The shear flow-induced detachment of L. lactis cells was investigated under laminar flow conditions. Such a dynamic approach demonstrated increased adhesion to bare and mucin-coated polystyrene for IBB477, compared to that observed for the MG1820 control strain. To identify potential genetic determinants giving adhesive properties to IBB477, the improved high-quality draft genome sequence comprising chromosome and five plasmids was obtained and analysed. The number of putative adhesion proteins was determined on the basis of surface/extracellular localisation and/or the presence of adhesion domains. To identify proteins essential for the IBB477 specific adhesion property, nine deletion mutants in chromosomal genes have been constructed and analysed using adhesion tests on bare polystyrene as well as mucin-, fibronectin- or collagen IV-coated polystyrene plates in comparison to the wild-type strain. These experiments demonstrated that gene AJ89_07570 encoding a protein containing DUF285, MucBP and four Big_3 domains is involved in adhesion to bare and mucin-coated polystyrene. To summarise, in the present work, we characterised the adhesion of IBB477 under laminar flow conditions; identified the putative adherence factors present in IBB477, which is the first L. lactis strain exhibiting adhesive and mucoadhesive properties to be sequenced and demonstrated that one of the proteins containing adhesion domains contributes to adhesion
Revisiting Mitochondrial pH with an Improved Algorithm for Calibration of the Ratiometric 5(6)-carboxy-SNARF-1 Probe Reveals Anticooperative Reaction with H+ Ions and Warrants Further Studies of Organellar pH
Get PDFFluorescence measurements of pH and other analytes in the cell rely on accurate calibrations,
but these have routinely used algorithms that inadequately describe the properties of
indicators. Here, we have established a more accurate method for calibrating and analyzing
data obtained using the ratiometric probe 5(6)-carboxy-SNARF-1. We tested the implications
of novel approach to measurements of pH in yeast mitochondria, a compartment containing
a small number of free H+ ions. Our findings demonstrate that 5(6)-carboxy-SNARF-
1 interacts with H+ ions inside the mitochondria in an anticooperative manner (Hill coefficient
n of 0.5) and the apparent pH inside the mitochondria is ~0.5 unit lower than had been generally
assumed. This result, at odds with the current consensus on the mechanism of
energy generation in the mitochondria, is in better agreement with theoretical considerations
and warrants further studies of organellar pH
Arteriovenous oscillations of the redox potential: Is the redox state influencing blood flow?
Get PDFOBJECTIVE
Studies on the regulation of human blood flow revealed several modes of oscillations with frequencies ranging from 0.005 to 1 Hz. Several mechanisms were proposed that might influence these oscillations, such as the activity of vascular endothelium, the neurogenic activity of vessel wall, the intrinsic activity of vascular smooth muscle, respiration, and heartbeat. These studies relied typically on non-invasive techniques, for example, laser Doppler flowmetry. Oscillations of biochemical markers were rarely coupled to blood flow.
METHODS
The redox potential difference between the artery and the vein was measured by platinum electrodes placed in the parallel homonymous femoral artery and the femoral vein of ventilated anesthetized pigs.
RESULTS
Continuous measurement at 5 Hz sampling rate using a digital nanovoltmeter revealed fluctuating signals with three basic modes of oscillations: ∼ 1, ∼ 0.1 and ∼ 0.01 Hz. These signals clearly overlap with reported modes of oscillations in blood flow, suggesting coupling of the redox potential and blood flow.
DISCUSSION
The amplitude of the oscillations associated with heart action was significantly smaller than for the other two modes, despite the fact that heart action has the greatest influence on blood flow. This finding suggests that redox potential in blood might be not a derivative but either a mediator or an effector of the blood flow control system
Synthesis of derivatives of methoxydibenzo[b, f]oxepine in the presence of sodium azide
Get PDFDibenzo[b,f]oxepine is an important scaffold in medicinal chemistry and its derivatives occur in several medicinally important plants. A new approach to methoxydibenzo[b,f]oxepines (15–21) proceeding under mild reaction conditions, has been developed. Notably, the use of sodium azide in the reaction allows access to new substituted dibenzo[b,f]oxepines. In order to study their shape and conformation, the optimum structures of these compounds were calculated using the DFT B3LYP/6-311++G(2d,p) method. A docking simulation was performed to insert compound 20 into the crystal structure of tubulin at the colchicine binding site to determine the probable binding model. The information from this work can be helpful for the investigation of new tubulin polymerization inhibitors exhibiting stronger activity
Genomic and Functional Characterization of the Unusual pLOCK 0919 Plasmid Harboring the spaCBA Pili Cluster in Lactobacillus casei LOCK 0919.
Get PDFHere, we report the extensive bioinformatic and functional analyses of the unusual pLOCK 0919, a plasmid originating from the probiotic Lactobacillus (L.) casei LOCK 0919 strain. This plasmid is atypical because it harbors the spaCBA-srtC gene cluster encoding SpaCBA pili. We show that all other spaCBA-srtC sequences of the Lactobacillus genus that have been previously described and deposited in GenBank are present in the chromosomal DNA. Another important observation for pLOCK 0919 is that the spaCBA-srtC gene cluster and its surrounding genes are highly similar to the respective DNA region that is present in the most well-known and active SpaCBA pili producer, the probiotic L. rhamnosus GG strain. Our results demonstrate that the spaCBA-srtC clusters of pLOCK 0919 and L. rhamnosus GG are genealogically similar, located in DNA regions that are rich in transposase genes and are poorly conserved among the publicly available sequences of Lactobacillus sp. In contrast to chromosomally-localized pilus gene clusters from L. casei and L. paracasei, the plasmidic spaC of L. casei LOCK 0919 is expressed and undergoes a slight glucose-induced repression. Moreover, results of series of in vitro tests demonstrate that L. casei LOCK 0919 has an adhesion potential, which is largely determined by the presence of the pLOCK 0919 plasmid. In particular, the plasmid occurrence positively influenced the hydrophobicity and aggregation abilities of L. casei LOCK 0919. Moreover, in vivo studies indicate that among the three Lactobacillus strains used to colonize the gastrointestinal tract of germ-free (GF) mice, already after two days of colonization, L. casei LOCK 0919 became the dominant strain and persisted there for at least 48 days