Ionizing radiation is a harsh environmental factor that could induce plant senescence. We hypothesized that radiation-related senescence remodels proteome, particularly by triggering the accumulation ...of prion-like proteins in plant tissues. The object of this study, pea (
L.), is an agriculturally important legume. Research on the functional importance of amyloidogenic proteins was never performed on this species.
Pea seeds were irradiated in the dose range 5-50 Gy of X-rays. Afterward, Fourier-transform infrared spectroscopy (FTIR) was used to investigate changes in the secondary structure of proteins in germinated 3-day-old seedlings. Specifically, we evaluated the ratio between the amide I and II peaks. Next, we performed protein staining with Congo red to compare the presence of amyloids in the samples. In parallel, we profiled the detergent-resistant proteome fraction by ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS). Differentially accumulated proteins were functionally analyzed in MapMan software, and the PLAAC tool was used to predict putative prion-like proteins.
We showed a reduced germination rate but higher plant height and faster appearance of reproductive organs in the irradiated at dose of 50 Gy group compared with the control; furthermore, we demonstrated more β-sheets and amyloid aggregates in the roots of stressed plants. We detected 531 proteins in detergent-resistant fraction extracted from roots, and 45 were annotated as putative prion-like proteins. Notably, 29 proteins were significantly differentially abundant between the irradiated and the control groups. These proteins belong to several functional categories: amino acid metabolism, carbohydrate metabolism, cytoskeleton organization, regulatory processes, protein biosynthesis, and RNA processing. Thus, the discovery proteomics provided deep data on novel aspects of plant stress biology.
Our data hinted that protein accumulation stimulated seedlings' growth as well as accelerated ontogenesis and, eventually, senescence, primarily through translation and RNA processing. The increased abundance of primary metabolism-related proteins indicates more intensive metabolic processes triggered in germinating pea seeds upon X-ray exposure. The functional role of detected putative amyloidogenic proteins should be validated in overexpression or knockout follow-up studies.
Processes that regulate gene transcription are directly under the influence of the genome organization. The epigenome contains additional information that is not brought by DNA sequence, and ...generates spatial and functional constraints that complement genetic instructions. DNA methylation on CpGs constitutes an epigenetic mark generally correlated with transcriptionally silent condensed chromatin. Replication of methylation patterns by DNA methyltransferases maintains genome stability through cell division. Here we present evidence of an unanticipated dynamic role for DNA methylation in gene regulation in human cells. Periodic, strand-specific methylation/demethylation occurs during transcriptional cycling of the pS2/TFF1 gene promoter on activation by oestrogens. DNA methyltransferases exhibit dual actions during these cycles, being involved in CpG methylation and active demethylation of 5mCpGs through deamination. Inhibition of this process precludes demethylation of the pS2 gene promoter and its subsequent transcriptional activation. Cyclical changes in the methylation status of promoter CpGs may thus represent a critical event in transcriptional achievement.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The phosphorylation of proteins modulates various functions of proteins and plays an important role in the regulation of cell signaling. In recent years, label-free quantitative (LFQ) ...phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. Despite the great progress, the studies of protein phosphorylation are still limited in throughput, robustness, and reproducibility, hampering analyses that involve multiple perturbations, such as those needed to follow the dynamics of phosphoproteomes. To address these challenges, we introduce here the LFQ phosphoproteomics workflow that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies. We applied this workflow to analyze the whole-cell phosphoproteome of the fission yeast
. Using this strategy, we identified 8353 phosphosites from which 1274 were newly identified. This provides a significant addition to the
phosphoproteome. The results of our study highlight that combining of PGC and SAX fractionation strategies substantially increases the robustness and specificity of LFQ phosphoproteomics. Overall, the presented LFQ phosphoproteomics workflow opens the door for studies that would get better insight into the complexity of the protein kinase functions of the fission yeast
.
Complex I (NADH dehydrogenase) is the first enzyme in the respiratory chain. It catalyses the electron transfer from NADH to ubiquinone that is associated with proton pumping out of the matrix. In ...this study, we characterized NADH dehydrogenase activity in seven monoxenous trypanosomatid species: Blechomonas ayalai, Herpetomonas tarakana, Kentomonas sorsogonicus, Leptomonas seymouri, Novymonas esmeraldas, Sergeia podlipaevi and Wallacemonas raviniae. We also investigated the subunit composition of the complex I in dixenous Phytomonas serpens, in which its presence and activity have been previously documented. In addition to P. serpens, the complex I is functionally active in N. esmeraldas and S. podlipaevi. We also identified 24–32 subunits of the complex I in individual species by using mass spectrometry. Among them, for the first time, we recognized several proteins of the mitochondrial DNA origin.
•Molecular mechanism of deterioration in walnut kernels was studied by proteomics.•Accumulation of folding, translation, proteolysis, detoxification and glycolysis related proteins occurred during ...aging.•Gluconeogenesis and amino acid metabolism related proteins were less abundant in aged kernels.•Lipase activation in aged kernels was due to lipase post-translational regulatory mechanisms.•Failure in lipid gluconeogenesis and amino acid metabolism leads to oxidative stress in aging nuts.
Little is known on the molecular mechanisms of deterioration in aging edible nuts. Proteomic differences between aging and germinating nuts may unravel these mechanisms. Walnut (Juglans regia) kernels have physiological dormancy which can be alleviated by cyanide accompanying with enhanced gluconeogenesis. Warm/moist conditions however, promote kernel deterioration marked by reduced germination associated with increased lipase activity. The hypotheses that aging i) brings about increased lipase activity and enhanced lipid mobilization through increased abundance of lipase protein, ii) compromises gluconeogenesis of lipid reserves, iii) enhances proteolysis and amino acid catabolism, and finally iv) leads to oxidative stress were tested in a proteomic study of kernels aged by controlled deterioration, and those dormant non-aged and cyanide-treated ones all with 15% moisture content; having low, medium and high germination potentials, respectively. This revealed 155 differentially abundant proteins out of 930 identified ones compared to dry (6% moisture content) kernels. In deteriorated nuts, proteins with increased abundance were mainly involved in protein folding, translation and degradation, stress response/detoxification and glycolysis, but those with decreased abundance were related to gluconeogenesis and amino acid metabolism. Contrastingly, in non-aged cyanide-treated kernels, the proteins belonged to gluconeogenesis and oxidative pentose phosphate pathway showed increased abundance. Unlike aging seeds of many other plants, the deteriorated kernels accumulated various chaperons and detoxification proteins. Thus, deteriorating kernels experienced perturbations in lipid gluconeogenesis and amino acid metabolism associated with enhanced respiration and oxidative stress. As no lipase protein increased during kernel aging, the aging-induced lipase activation is possibly regulated by post-translational mechanisms.
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Endometrial cancer belongs to the most common gynecologic cancer types globally, with increasing incidence. There are numerous ways of classifying different cases. The most recent decade has brought ...advances in molecular classification, which show more accurate prognostic factors and the possibility of personalised adjuvant treatment. In addition, diagnostic approaches lag behind these advances, with methods causing patients discomfort while lacking the reproducibility of tissue sampling for biopsy. Minimally invasive liquid biopsies could therefore represent an alternative screening and diagnostic approach in patients with endometrial cancer. The method could potentially detect molecular changes in this cancer type and identify patients at early stages. In this pilot study, we tested such a detection method based on circulating tumour DNA isolated from the peripheral blood plasma of 21 Slovak endometrial cancer patients. We successfully detected oncomutations in the circulating DNA of every single patient, although the prognostic value of the detected mutations failed to offer certainty. Furthermore, we detected changes associated with clonal hematopoiesis, including
mutations, which were present in the majority of circulating tumour DNA samples.
The last decade has witnessed a renewed interest in selenium (Se) as an element able to prevent a range of illnesses in humans, mainly through supplementation. However, such supplementation relies on ...species such as sodium selenite or selenomethionine, which proved to have limited solubility and bioavailability, thus leading to limited activity. To overcome this limitation, other selenium species need to be explored, such as phthalic selenoanhydride (R-Se), which is soluble in physiological media. R-Se releases various reactive selenium species (RSeS), including hydrogen selenide (H
2
Se), that can interact with cellular components, such as glutathione (GSH) and hydrogen sulfide (H
2
S). This interplay between R-Se and the cellular components provides a sophisticated biochemical release mechanism that could be behind the noteworthy biological activities observed for this compound. In order to investigate the interactions of phthalic chalcogen anhydrides with H
2
S or GSH, we have employed UV-vis spectrophotometry, electron paramagnetic resonance spectroscopy (EPR) and plasmid DNA (pDNA) cleavage assay. We found that apart from R-Se, the other analogues do not have the ability to scavenge the &z.rad;cPTIO radical or to cleave pDNA on their own. In contrast, the scavenging potency for the &z.rad;cPTIO radical and for the O
2
&z.rad;
−
radical exerted by R-Se and its sulfur analogue (R-S) significantly increased when they were evaluated in the presence of H
2
S. However, GSH only changed the radical scavenging activity of R-Se. These new discoveries may explain some of the biological activities associated with this class of compounds and open a new approach to ascertain the possible mechanisms underlying their biological actions.
The last decade has witnessed a renewed interest in selenium (Se) as an element able to prevent a range of illnesses in humans, mainly through supplementation.
Protein kinases are important enzymes involved in the regulation of various cellular processes. To function properly, each protein kinase phosphorylates only a limited number of proteins among the ...thousands present in the cell. This provides a rapid and dynamic regulatory mechanism that controls biological functions of the proteins. Despite the importance of protein kinases, most of their substrates remain unknown. Recently, the advances in the fields of protein engineering, chemical genetics, and mass spectrometry have boosted studies on identification of bona fide substrates of protein kinases. Among the various methods in protein kinase specific substrate identification, genetically engineered protein kinases and quantitative phosphoproteomics have become promising tools. Herein, we review the current advances in the field of chemical genetics in analog-sensitive protein kinase mutants and highlight selected strategies for identifying protein kinase substrates and studying the dynamic nature of protein phosphorylation.
Pre-mRNA splicing plays a fundamental role in securing protein diversity by generating multiple transcript isoforms from a single gene. Recently, it has been shown that specific G-patch ...domain-containing proteins are critical cofactors involved in the regulation of splicing processes. In this study, using the knock-out strategy, affinity purification and the yeast-two-hybrid assay, we demonstrated that the spliceosome-associated G-patch protein Gpl1 of the fission yeast S. pombe mediates interactions between putative RNA helicase Gih35 (SPAC20H4.09) and WD repeat protein Wdr83, and ensures their binding to the spliceosome. Furthermore, RT-qPCR analysis of the splicing efficiency of deletion mutants indicated that the absence of any of the components of the Gpl1-Gih35-Wdr83 complex leads to defective splicing of fet5 and pwi1, the reference genes whose unspliced isoforms harboring premature stop codons are targeted for degradation by the nonsense-mediated decay (NMD) pathway. Together, our results shed more light on the functional interactome of G-patch protein Gpl1 and revealed that the Gpl1-Gih35-Wdr83 complex plays an important role in the regulation of pre-mRNA splicing in S. pombe.
Lysosomal storage disorders (LSD) are a group of over 70 rare inherited metabolic disorders that are caused mostly by the deficiency of specific lysosomal enzymes. Lack of these enzymes leads to the ...interference with cellular functions due to excessive accumulation of undegraded substrate in cells and tissues. Effective treatment of these diseases, if it is available, relies on early and accurate diagnostics. Several traditional methods for diagnostics of LSD are used worldwide; however, new techniques, methods and instruments need to be applied to the diagnostic process to increase its sensitivity, repeatability and reliability. In this review, diagnostic methods based on mass spectrometry and their respective sample preparation steps and eventual separation by liquid chromatography are discussed, emphasizing specific biomarkers of each lysosomal storage disorder subclass. Up-to-date evaluation of research conducted in the areas of diagnostics of lysosomal storage disorders by mass spectrometry is comprehensively summarized in this study.
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