Selenoproteome Expression Studied by Non-Radioactive Isotopic Selenium-Labeling in Human Cell Lines.
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ABSTRACT:
SUBMITTER: Sonet J
PROVIDER: S-EPMC8306042 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Selenium (Se), as an essential trace element, plays crucial roles in many organisms including humans. The biological functions of selenium are mainly mediated by selenoproteins, a unique class of selenium-containing proteins in which selenium is inserted in the form of selenocysteine. Due to their low abundance and uneven tissue distribution, detection of selenoproteins within proteomes is very challenging, and therefore functional studies of these proteins are limited. In this study, we developed a computational method, named as selenium-encoded isotopic signature targeted profiling (SESTAR), which utilizes the distinct natural isotopic distribution of selenium to assist detection of trace selenium-containing signals from shotgun-proteomic data. SESTAR can detect femtomole quantities of synthetic selenopeptides in a benchmark test and dramatically improved detection of native selenoproteins from tissue proteomes in a targeted profiling mode. By applying SESTAR to screen publicly available datasets from Human Proteome Map, we provide a comprehensive picture of selenoprotein distributions in human primary hematopoietic cells and tissues. We further demonstrated that SESTAR can aid chemical-proteomic strategies to identify additional selenoprotein targets of RSL3, a canonical inducer of cell ferroptosis. We believe SESTAR not only serves as a powerful tool for global profiling of native selenoproteomes, but can also work seamlessly with chemical-proteomic profiling strategies to enhance identification of target proteins, post-translational modifications, or protein-protein interactions.
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Project description:Selenoprotein P (Sel P) is a selenium-rich glycoprotein believed to play a key role in selenium (Se) transport throughout the body. Development of a Sel P knockout mouse model has supported this notion and initial studies have indicated that selenium supply to various tissues is differentially affected by genetic deletion of Sel P. Se in the form of the amino acid, selenocysteine, is incorporated into selenoproteins at UGA codons. Thus, Se availability affects not only selenoprotein levels, but also the turnover of selenoprotein mRNAs via the nonsense-mediated decay pathway. We investigated how genetic deletion of Sel P in mice affected levels of the mRNAs encoding all known members of the murine selenoprotein family, as well as three non-selenoprotein factors involved in their synthesis, selenophosphate synthetase 1 (SPS1), SECIS-binding protein 2 (SBP2) and SECp43. Our findings present a comprehensive description of selenoprotein mRNA expression in the following murine tissues: brain, heart, intestine, kidney, liver, lung, spleen and testes. We also describe how abundance of selenoproteins and selenoprotein-synthesis factors are affected by genetic deletion of Sel P in some of these tissues, providing insight into how the presence of this selenoprotein influences selenoprotein mRNA levels, and thus, the selenoproteome.
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Project description:NMR studies of human integral membrane proteins provide unique opportunities to probe structure and dynamics at specific locations and on multiple timescales, often with significant implications for disease mechanism and drug development. Since membrane proteins such as G protein-coupled receptors (GPCRs) are highly dynamic and regulated by ligands or other perturbations, NMR methods are potentially well suited to answer basic functional questions (such as addressing the biophysical basis of ligand efficacy) as well as guiding applications (such as novel ligand design). However, such studies on eukaryotic membrane proteins have often been limited by the inability to incorporate optimal isotopic labels for NMR methods developed for large protein/lipid complexes, including methyl TROSY. We review the different expression systems for production of isotopically labeled membrane proteins and highlight the use of the yeast Pichia pastoris to achieve perdeuteration and 13C methyl probe incorporation within isoleucine sidechains. We further illustrate the use of this method for labeling of several biomedically significant GPCRs.
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Project description:Gpx (glutathione peroxidase)-1 enzyme activity and mRNA levels decrease dramatically in Se (selenium) deficiency, whereas other selenoproteins are less affected by Se deficiency. This hierarchy of Se regulation is not understood, but the position of the UGA selenocysteine codon is thought to play a major role in making selenoprotein mRNAs susceptible to nonsense-mediated decay. Thus in the present paper we studied the complete selenoproteome in the mouse to uncover additional selenoprotein mRNAs that are highly regulated by Se status. Mice were fed on Se-deficient, Se-marginal and Se-adequate diets (0, 0.05 and 0.2 microg of Se/g respectively) for 35 days, and selenoprotein mRNA levels in liver and kidney were determined using microarray analysis and quantitative real-time PCR analysis. Se-deficient mice had liver Se concentrations and liver Gpx1 and thioredoxin reductase activities that were 4, 3 and 3% respectively of the levels in Se-adequate mice, indicating that the mice were Se deficient. mRNAs for Selh (selenoprotein H) and Sepw1 (selenoprotein W) as well as Gpx1 were decreased by Se deficiency to <40% of Se-adequate levels. Five and two additional mRNAs were moderately down-regulated in Sedeficient liver and kidney respectively. Importantly, nine selenoprotein mRNAs in liver and fifteen selenoprotein mRNAs in the kidney were not significantly regulated by Se deficiency, clearly demonstrating that Se regulation of selenoprotein mRNAs is not a general phenomenon. The similarity of the response to Se deficiency suggests that there is one underlying mechanism responsible. Importantly, the position of the UGA codon did not predict susceptibility to Se regulation, clearly indicating that additional features are involved in causing selenoprotein mRNAs to be sensitive to Se status.
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Project description:Nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the principle techniques of structural biology. It is not only a powerful method for elucidating the three-dimensional structures under near physiological conditions but also a convenient method for studying protein-ligand interactions and protein dynamics. A major drawback of macromolecular NMR is its size limitation, caused by slower tumbling rates and greater complexity of the spectra as size increases. Segmental isotopic labeling allows for specific segment(s) within a protein to be selectively examined by NMR, thus significantly reducing the spectral complexity for large proteins and allowing for the application of a variety of solution-based NMR strategies. Two related approaches are generally used in the segmental isotopic labeling of proteins: expressed protein ligation and protein trans-splicing. Here, we describe the methodology and recent application of expressed protein ligation and protein trans-splicing for NMR structural studies of proteins and protein complexes. We also describe the protocol used in our lab for the segmental isotopic labeling of a 50-kDa protein Csk (C-terminal Src kinase) using expressed protein ligation methods.
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Project description:Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mm glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2 and CID-MS3 by neutral loss scan monitoring of two selective neutral losses (162.05 and 84.04 Da for the glucose cleavage and an intermediate rearrangement of the glucose moiety). On the other hand, quantitative analysis was based on labeling of proteins with [(13)C(6)]glucose incubation to evaluate the native glycated proteins labeled with [(12)C(6)]glucose. As glycation is chemoselective, it is exclusively occurring in potential targets for in vivo modifications. This approach, named glycation isotopic labeling, enabled differentiation of glycated peptides labeled with both isotopic forms resulting from enzymatic digestion by mass spectrometry (6-Da mass shift/glycation site). The strategy was then applied to a reference plasma sample, revealing the detection of 50 glycated proteins and 161 sugar attachment positions with identification of preferential glycation sites for each protein. A predictive approach was also tested to detect potential glycation sites under high glucose concentration.
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Project description:BackgroundSelenium (Se) is an essential oligonutrient, as a component of several Se-containing proteins (selenoproteins), which exert important biological functions within an organism. In livestock, Se-enriched products have been proposed as dietary supplements to be included into functional feeds for animal preventive health care. To this end, it is important to understand the optimal range of concentrations for supplementation and how long it takes to be assimilated into the organism.MethodsIn this study, rainbow trout (Oncorhynchus mykiss) were fed a control diet containing 0.9 g Kg-1 Se or the same diet supplemented with a Se-Yeast product (Sel-Plex) to achieve Se concentrations ranging from 1.5-8.9 g Kg-1 for a period of ten weeks. Fish were sampled every two weeks for analysis. The kinetics of Se bioaccumulation and the effects on fish selenoprotein expression was determined in different tissues combining chemical and bimolecular techniques.ResultsThe Sel-Plex enriched diets did not have any effect on survival and growth performance. The highest Se levels were found in liver and kidney followed by muscle and blood cells. Analysis of the Se concentration factor showed that liver is able to initially regulate the amount of Se accumulated. However, with higher dietary Se level (4.8 and 8.9 g Kg-1) and longer times of exposure (10 weeks), regulation is ineffective and the Se tissue concentration increases. The expression of the selected trout selenoprotein transcripts showed an inverse correlation with Sel-Plex augmentation in most cases. In liver, kidney and blood cells the highest up-regulation of the trout selenoprotein genes was seen mostly in the group fed the diet enriched with the lowest concentration of Sel-Plex (0.5 g Kg-1) for 10 weeks.ConclusionSel-Plex may represent an excellent Se supplement to deliver a high level of Se without provoking harm to the fish and to guarantee the maximal absorption of the element. According to our results, a dietary supplementation of Sel-Plex between 0.5 and 4 g Kg-1 may allow maximal benefits, whereas 8 g Kg-1 may be excessive for the purpose of supplementation.
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Project description:BACKGROUND:Mass spectra are usually acquired from the Liquid Chromatography-Mass Spectrometry (LC-MS) analysis for isotope labeled proteomics experiments. In such experiments, the mass profiles of labeled (heavy) and unlabeled (light) peptide pairs are represented by isotope clusters (2D or 3D) that provide valuable information about the studied biological samples in different conditions. The core task of quality control in quantitative LC-MS experiment is to filter out low-quality peptides with questionable profiles. The commonly used methods for this problem are the classification approaches. However, the data imbalance problems in previous control methods are often ignored or mishandled. In this study, we introduced a quality control framework based on the extreme gradient boosting machine (XGBoost), and carefully addressed the imbalanced data problem in this framework. RESULTS:In the XGBoost based framework, we suggest the application of the Synthetic minority over-sampling technique (SMOTE) to re-balance data and use the balanced data to train the boosted trees as the classifier. Then the classifier is applied to other data for the peptide quality assessment. Experimental results show that our proposed framework increases the reliability of peptide heavy-light ratio estimation significantly. CONCLUSIONS:Our results indicate that this framework is a powerful method for the peptide quality assessment. For the feature extraction part, the extracted ion chromatogram (XIC) based features contribute to the peptide quality assessment. To solve the imbalanced data problem, SMOTE brings a much better classification performance. Finally, the XGBoost is capable for the peptide quality control. Overall, our proposed framework provides reliable results for the further proteomics studies.
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