92 research outputs found

    Genetic predisposition for sudden cardiac death in myocardial ischaemia: the Arrhythmia Genetics in the NEtherlandS study

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    Sudden cardiac death from ventricular fibrillation during myocardial infarction is a leading cause of total and cardiovascular mortality. This multifactorial, complex condition clusters in families, suggesting a substantial genetic cause. We carried out a genomewide association study (GWAS) for sudden cardiac death, in the AGNES (Arrhythmia Genetics in the Netherlands) population, consisting of patients with (cases) and without (controls) ventricular fibrillation during a first ST-elevation myocardial infarction. The most significant association was found at chromosome 21q21 (rs2824292; odds ratio = 1.78, 95% CI 1.47–2.13, P = 3.3 × 10−10), 98 kb proximal of the CXADR gene, encoding the Coxsackie and adenovirus receptor. This locus has not previously been implicated in arrhythmia susceptibility. Further research on the mechanism of this locus will ultimately provide novel insight into arrhythmia mechanisms in this condition

    Regulation of proteasome assembly and activity in health and disease

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    Tyrosine Nitration of PA700 Links Proteasome Activation to Endothelial Dysfunction in Mouse Models with Cardiovascular Risk Factors

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    Oxidative stress is believed to cause endothelial dysfunction, an early event and a hallmark in cardiovascular diseases (CVD) including hypertension, diabetes, and dyslipidemia. However, the targets for oxidative stress-mediated endothelial dysfunction in CVD have not been completely elucidated. Here we report that 26S proteasome activation by peroxynitrite (ONOO−) is a common pathway for endothelial dysfunction in mouse models of diabetes, hypertension, and dyslipidemia. Endothelial function, assayed by acetylcholine-induced vasorelaxation, was impaired in parallel with significantly increased 26S proteasome activity in aortic homogenates from streptozotocin (STZ)-induced type I diabetic mice, angiotensin-infused hypertensive mice, and high fat-diets -fed LDL receptor knockout (LDLr−/−) mice. The elevated 26S proteasome activities were accompanied by ONOO−-mediated PA700/S10B nitration and increased 26S proteasome assembly and caused accelerated degradation of molecules (such as GTPCH I and thioredoxin) essential to endothelial homeostasis. Pharmacological (administration of MG132) or genetic inhibition (siRNA knockdown of PA700/S10B) of the 26S proteasome blocked the degradation of the vascular protective molecules and ablated endothelial dysfunction induced by diabetes, hypertension, and western diet feeding. Taken together, these results suggest that 26S proteasome activation by ONOO−-induced PA700/S10B tyrosine nitration is a common route for endothelial dysfunction seen in mouse models of hypertension, diabetes, and dyslipidemia

    On the Reproducibility of Label-Free Quantitative Cross-Linking/Mass Spectrometry

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    Quantitative cross-linking/mass spectrometry (QCLMS) is an emerging approach to study conformational changes of proteins and multi-subunit complexes. Distinguishing protein conformations requires reproducibly identifying and quantifying cross-linked peptides. Here we analyzed the variation between multiple cross-linking reactions using bis[sulfosuccinimidyl] suberate (BS3)-cross-linked human serum albumin (HSA) and evaluated how reproducible cross-linked peptides can be identified and quantified by LC-MS analysis. To make QCLMS accessible to a broader research community, we developed a workflow that integrates the established software tools MaxQuant for spectra preprocessing, Xi for cross-linked peptide identification, and finally Skyline for quantification (MS1 filtering). Out of the 221 unique residue pairs identified in our sample, 124 were subsequently quantified across 10 analyses with coefficient of variation (CV) values of 14% (injection replica) and 32% (reaction replica). Thus our results demonstrate that the reproducibility of QCLMS is in line with the reproducibility of general quantitative proteomics and we establish a robust workflow for MS1-based quantitation of cross-linked peptides

    Genetic code expansion for multiprotein complex engineering

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    We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, \u2018MultiBacTATAG\u2019, (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure\u2013function studies of novel eukaryotic complexes using single-molecule F\uf6rster resonance energy transfer as well as site-specific crosslinking strategies

    TEST-RESTEST RELIABILITY OF BIOIMPEDANCE SPECTROSCOPY FOR THE ANALYSIS OF BODY COMPOSITION IN PHYSICALLY ACTIVE MALES

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    Tyler W.D. Muddle1,2, Patrick M. Tomko1,2, Ryan J. Colquhoun1,2, Mitchel A. Magrini1, Nile F. Banks1,2, Nathaniel D.M. Jenkins1,2 1Applied Neuromuscular Physiology Laboratory 2Laboratory for Applied Nutrition and Exercise Science Oklahoma State University, Stillwater OK No previous studies, to our knowledge, have examined the reliability of bioimpedance spectroscopy (BIS) for the evaluation of body composition. PURPOSE: To evaluate the test-retest reliability of BIS for the assessment of total body water (TBW), extracellular water (ECW), and intracellular water (ICW) content, as well as fat mass (FM), fat-free mass (FFM), and body fat percentage (BF%) in physically active males. METHODS: Sixteen males (Mean ± SD, 25 ± 3 y, 90 ± 11 kg, 176 ± 6 cm) were assessed at two visits, separated by 2 – 7 days. During each visit, participants rested quietly for 3 – 5 min in a supine position with their arms abducted ≥ 30° away from their torso and legs separated prior to their assessment. Two single-tab electrodes were placed on the right side of the body 5 cm apart on both the dorsal surface of the wrist and dorsal surface of the ankle, respectively. The BIS device was used to estimate TBW, ECW, and ICW (liters; L) based on Cole modelling with Hanai mixture theory, which were then used to calculate FM (kg), FFM (kg), and BF%. Reliability was examined by calculating the intraclass correlation coefficient (ICC; model 2,1) and standard error of measurement (SEM). The coefficient of variation (CV) was calculated by expressing the SEM relative to the grand mean (%). The 95% confidence interval (CI) for each ICCwas calculated and used to test the null hypothesis that each ICC was equal to zero. Systematic variability was assessed for each variable via a paired t-test. RESULTS: Reliability statistics are displayed in Table 1. None of the dependent variables displayed systematic variability (p \u3e 0.05). ‘Excellent’ relative and absolute reliability was observed among all body water (ICC = 0.91 – 0.99; CVs = 1.08 – 3.50%) and body mass (ICC = 0.95 – 0.99; CVs = 1.10 – 6.99%) measurements. CONCLUSION: These results indicate that the BIS device used in this study allows for the reliable assessment of TBW, ECW, ICW, FM, FFM, and BF% in physically active men

    Independent mechanistic inhibition of Cdc25 phosphatases by a natural product caulibugulone

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    Caulibugulones are novel but poorly characterized cytotoxic isoquinoline quinones and iminoquinones identified in extracts from the marine bryozoan Caulibugula intermis. We now report that the caulibugulones are selective in vitro inhibitors of the Cdc25 family of cell cycle-controlling protein phosphatases compared with either human vaccinia H1-related phosphatase (VHR) or tyrosine phosphatase 1B (PTP1B). The in vitro inhibition of Cdc25B by caulibugulone A was irreversible and attenuated by reducing agents or catalase, consistent with direct oxidation of the enzyme by reactive oxygen species. Mechanistically, caulibugulone A directly inhibited cellular Cdc25B activity, generated intracellular reactive oxygen species and arrested cells in both G1 and G2/M phases of the cell cycle. Caulibugulone A also caused the selective degradation of Cdc25A protein by a process that was independent of reactive oxygen species production, proteasome activity, and the Chk1 signaling pathway. Instead, caulibugulone A stimulated the phosphorylation and subsequent activation of p38 stress kinase, leading to Cdc25A degradation. Thus, caulibugulone inhibition of cellular Cdc25A and B phosphatases occurred through at least two different mechanisms, leading to pronounced cell cycle arrest. Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics

    Reconfiguration of the proteasome during chaperone-mediated assembly

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    The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric α ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt C-terminal tails inserting into pockets of the α ring[superscript 1-4]. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit[superscript 5-10]. We report that the base subassembly of the proteasome, which includes the Rpt ring, forms a high affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6, and Rpn14. Chaperone-mediated dissociation was abrogated by a nonhydrolyzable ATP analog, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3 pocket. Although the Rpt6 tail is not visualized within an α pocket in mature proteasomes[superscript 2-4], it inserts into the α2/α3 pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme

    Recombinant Expression, Unnatural Amino Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes

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    The 26S proteasome is the major regulated protease in eukaryotes and is responsible for degrading ubiquitinated substrates. It consists of a barrel-shaped 20S core peptidase and one or two 19S regulatory particles, which recognize, unfold, and translocate substrates into the core. The regulatory particle can be further divided into two multi-subunit complexes: the base and the lid. Here we present protocols for expressing the Saccharomyces cerevisiae base and lid recombinantly in Escherichia coli and purifying the assembled subcomplexes using a tandem affinity purification method. The purified complexes can then be reconstituted with 20S core to form fully functional proteasomes. Furthermore, we describe a method for incorporating the unnatural amino acid p-azido-L-phenylalanine into the recombinant complexes at any residue position, allowing for non-disruptive site-specific modifications of these large assemblies. The use of recombinant proteins allows for complete mutational control over the proteasome regulatory particle, enabling detailed studies of the mechanism by which the proteasome processes its substrates. The ability to then specifically modify residues in the regulatory particle opens the door to a wide range of previously impossible biochemical and biophysical studies. The techniques described below for incorporating unnatural amino acids into the proteasomal subcomplexes should be widely transferable to other recombinant proteins, whether individually purified or in larger multi-subunit assemblies
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