-
Frontiers in Cardiovascular Medicine 2021Binge drinking has become the most common and deadly pattern of excessive alcohol use in the United States, especially among younger adults. It is closely related to...
Binge drinking has become the most common and deadly pattern of excessive alcohol use in the United States, especially among younger adults. It is closely related to the increased risk of cardiovascular disease. Oxidative stress as a result of ethanol metabolism is the primary pathogenic factor for alcohol-induced end organ injury, but the role of protein S-glutathionylation-a reversible oxidative modification of protein cysteine thiol groups that mediates cellular actions by oxidants-in binge drinking-associated cardiovascular disease has not been explored. The present study defines the effect of alcohol binge drinking on the formation of protein S-glutathionylation in a mouse model of atherosclerosis. To mimic the weekend binge drinking pattern in humans, ApoE deficient ( ) mice on the Lieber-DeCarli liquid diet received ethanol or isocaloric maltose (as a control) gavages (5 g/kg/day, 2 consecutive days/week) for 6 weeks. The primary alcohol-targeted organs (liver, brain), and cardiovascular system (heart, aorta, lung) of these two groups of the mice were determined by measuring the protein S-glutathionylation levels and its regulatory enzymes including [Glutaredoxin1(Grx1), glutathione reductase (GR), glutathione-S-transferase Pi (GST-π)], as well as by assessing aortic endothelial function and liver lipid levels. Our results showed that binge drinking selectively stimulated protein S-glutathionylation in aorta, liver, and brain, which coincided with altered glutathionylation regulatory enzyme expression that is downregulated Grx1 and upregulated GST-π in aorta, massive upregulation of GST-π in liver, and no changes in Grx1 and GST-π in brain. Functionally, binge drinking induced aortic endothelial cell function, as reflected by increased aortic permeability and reduced flow-mediated vasodilation. This study is the first to provide evidence for differential effects of binge drinking on formation of protein S-glutathionylation and its enzymatic regulation system in major alcohol-target organs and cardiovascular system. The selective induction of protein S-glutathionylation in aorta and liver is associated with aortic endothelial dysfunction and fatty liver, which may be a potential redox mechanism for the increased risk of vascular disease in human binge-drinkers.
PubMed: 33796575
DOI: 10.3389/fcvm.2021.649813 -
Blood Coagulation & Fibrinolysis : An... Dec 2019: Protein S Tokushima (p.Lys196Glu) and two protein C gene variants (p.Arg189Trp, p.Lys193del) are hereditary thrombophilia in Japanese and Chinese populations,...
: Protein S Tokushima (p.Lys196Glu) and two protein C gene variants (p.Arg189Trp, p.Lys193del) are hereditary thrombophilia in Japanese and Chinese populations, respectively; however, their diagnosis by plasma analyses is difficult because of the type II deficiency phenotype. Three gene variant genotypes were examined in young Japanese women (n = 231). Plasma total protein S activity and total protein S antigen levels were measured using a total protein S assay system, protein C and protein S activities by clot-based methods, and protein C and free protein S antigen levels by latex agglutination methods. protein S Tokushima (p.Lys196Glu) and protein C p.Lys193del variants were prevalent among participants with allele frequencies of 1.08 and 0.86%, respectively, whereas any carrier of protein C p.Arg189Trp variant was not identified. The plasma phenotype of the type II deficiency of protein S Tokushima heterozygotes was demonstrated by decreased total protein S activity with a normal total protein S antigen level; however, the protein C activities of protein C p.Lys193del heterozygotes were within reference intervals, whereas their protein C antigen levels were elevated. We compared the diagnostic accuracy of the total protein S activity/total protein S antigen ratio for identifying protein S Tokushima heterozygotes with that of the clot-based protein S activity/free protein S antigen ratio and found that sensitivity and specificity of 100% each was only achieved by the former. Protein S Tokushima and protein C p.Lys193del are prevalent among young Japanese women, and a plasma analysis using the total protein S assay system is more accurate than the clot-based protein S activity/free protein S antigen ratio for diagnosing protein S Tokushima carriers.
Topics: Adult; Antigens; Asian People; Female; Gene Frequency; Heterozygote; Humans; Japan; Mutation; Phenotype; Plasma; Protein C; Protein S; Thrombophilia; Young Adult
PubMed: 31490209
DOI: 10.1097/MBC.0000000000000854 -
Journal of Visualized Experiments : JoVE Apr 2020Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty acids via a labile...
Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty acids via a labile thioester bond. S-acylation, which is emerging as a widespread regulatory mechanism, can modulate almost all aspects of the biological activity of proteins, from complex formation to protein trafficking and protein stability. The recent progress in understanding of the biological function of protein S-acylation was achieved largely due to the development of novel biochemical tools allowing robust and sensitive detection of protein S-acylation in a variety of biological samples. Here, we describe acyl resin-assisted capture (Acyl-RAC), a recently developed method based on selective capture of endogenously S-acylated proteins by thiol-reactive Sepharose beads. Compared to existing approaches, Acyl-RAC requires fewer steps and can yield more reliable results when coupled with mass spectrometry for identification of novel S-acylation targets. A major limitation in this technique is the lack of ability to discriminate between fatty acid species attached to cysteines via the same thioester bond.
Topics: Acylation; Protein S
PubMed: 32338654
DOI: 10.3791/61016 -
FASEB Journal : Official Publication of... Apr 2020Reactive oxygen species (ROS) increase during adipogenesis and in obesity. Oxidants react with cysteine residues of proteins to form glutathione (GSH) adducts,...
Reactive oxygen species (ROS) increase during adipogenesis and in obesity. Oxidants react with cysteine residues of proteins to form glutathione (GSH) adducts, S-glutathionylation, that are selectively removed by glutaredoxin-1 (Glrx). We have previously reported that Glrx knockout mice had increased protein S-glutathionylation and developed obesity by an unknown mechanism. In this study, we demonstrated that 3T3L1 adipocytes differentiation increased ROS and protein S-glutathionylation. Glrx ablation elevated protein S-glutathionylation and lipid content in 3T3L1 cells. Glrx replenishment decreased the lipid content of Glrx KO 3T3L1 cells. Glrx KO also increased protein expression and protein S-glutathionylation of the adipogenic transcription factor CCAAT enhancer-binding protein (C/EBP) β. Protein S-glutathionylation decreased the interaction of C/EBPβ and protein inhibitor of activated STAT (PIAS) 1, a small ubiquitin-related modifier E3 ligase that facilitates C/EBPβ degradation. Experiments with truncated mutant C/EBPβ demonstrated that PIAS1 interacted with the liver-enriched inhibitory protein (LIP) region of C/EBPβ. Furthermore, mass spectrometry analysis identified protein S-glutathionylation of Cys201 and Cys296 in the LIP region of C/EBPβ. The C201S, C296S double-mutant C/EBPβ prevented protein S-glutathionylation and preserved the interaction with PIAS1. In summary, Glrx ablation stimulated 3T3L1 cell differentiation and adipogenesis via increased protein S-glutathionylation of C/EBPβ, stabilizing and increasing C/EBPβ protein levels.
Topics: 3T3-L1 Cells; Adipocytes; Adipogenesis; Animals; CCAAT-Enhancer-Binding Protein-beta; Gene Expression Regulation; Glutaredoxins; Glutathione; Mice; Mice, Knockout; Protein Processing, Post-Translational; Protein S
PubMed: 32141127
DOI: 10.1096/fj.201902575R -
Cureus Dec 2021Introduction Bleeding and thrombotic events are known to occur in beta-thalassemia major (BTM) patients and have been attributed to hepatic iron overload associated with...
Introduction Bleeding and thrombotic events are known to occur in beta-thalassemia major (BTM) patients and have been attributed to hepatic iron overload associated with multiple blood transfusions. We evaluated hemostatic parameters in children with BTM who had no previous history of bleeding or thrombotic episodes. Materials and Methods Hemostatic parameters including prothrombin time (PT), activated partial thromboplastin time (APTT), platelet aggregation, protein C and S, iron profile, and liver function tests were evaluated in 54 children (median age = 12 months, age range = 4-144 months) with BTM and 15 age and sex-matched controls. Results The mean PT and APTT of patients were significantly higher (=0.016 and <.001) than that of controls. Mean protein C, protein S activity and platelet aggregability with adenosine 5-diphosphate (ADP) as an agonist in patients were significantly lower (<.001, <.001 and =0.007, respectively) than that in controls. Mean serum ferritin in BTM children was not significantly elevated to be associated with hepatic dysfunction. Conclusion Deranged hemostatic parameters indicative of bleeding and thrombotic tendencies are observed in BTM children from an early age and may not be solely due to hyperferritinemia-associated hepatic dysfunction. Despite the presence of deranged hemostatic parameters, a state of balance exists between bleeding and thrombosis, and an imbalance may lead to bleeding or thrombotic events at a later age.
PubMed: 34877233
DOI: 10.7759/cureus.20192 -
Saudi Journal of Biological Sciences Oct 2023The "A" protein plays an essential role in the pathogenicity and virulence of this bacterial species. To gain deeper insights into the protein's characteristics, we...
BACKGROUND
The "A" protein plays an essential role in the pathogenicity and virulence of this bacterial species. To gain deeper insights into the protein's characteristics, we conducted an in-depth analysis of its sequence and structure.
OBJECTIVE
This study aimed to unravel the underlying genetic and structural components that contribute to the protein's functional properties.
RESULTS
Utilizing various bioinformatics tools and techniques, we first examined the protein's primary sequence, identifying key amino acid residues and potential functional domains. Additionally, we employed computational modeling and simulation approaches to determine the tertiary structure of the "A" protein. Through this comprehensive analysis, we discovered novel features and interactions within the protein's structure, shedding light on its potential mechanisms of action. Furthermore, we investigated the protein's evolutionary conservation and compared it with related proteins from other bacterial species.
CONCLUSIONS
Overall, our findings provide valuable insights into the sequence and structure of the Staphylococcus aureus "A" protein, which may have implications for understanding its role in pathogenicity and guiding the development of novel therapeutic strategies.
PubMed: 37766889
DOI: 10.1016/j.sjbs.2023.103812 -
ACS Chemical Biology Aug 2021Protein -acylation is a dynamic lipid post-translational modification that can modulate the localization and activity of target proteins. In humans, the installation of...
Protein -acylation is a dynamic lipid post-translational modification that can modulate the localization and activity of target proteins. In humans, the installation of the lipid onto target proteins is catalyzed by a family of 23 Asp-His-His-Cys domain-containing protein acyltransferases (DHHC-PATs). DHHCs are increasingly recognized as critical players in cellular signaling events and in human disease. However, progress elucidating the functions and mechanisms of DHHC "writers" has been hampered by a lack of chemical tools to perturb their activity in live cells. Herein, we report the synthesis and characterization of cyano-myracrylamide (), a broad-spectrum DHHC family inhibitor with similar potency to 2-bromopalmitate (2BP), the most commonly used DHHC inhibitor in the field. Possessing an acrylamide warhead instead of 2BP's α-halo fatty acid, inhibits DHHC family proteins while demonstrating decreased toxicity and avoiding inhibition of the -acylation eraser enzymes, two of the major weaknesses of 2BP. Our studies show that engages with DHHC family proteins in cells, inhibits protein -acylation, and disrupts DHHC-regulated cellular events. represents an improved chemical scaffold for untangling the complexities of DHHC-mediated cell signaling by protein -acylation.
Topics: Acrylamides; Acylation; Acyltransferases; Animals; CD36 Antigens; Cell Line, Tumor; Enzyme Inhibitors; ErbB Receptors; Humans; Lipoylation; Mice; Protein Processing, Post-Translational
PubMed: 34309372
DOI: 10.1021/acschembio.1c00405 -
The Journal of Cell Biology Aug 2023As eukaryotic cells progress through cell division, the nuclear envelope (NE) membrane must expand to accommodate the formation of progeny nuclei. In Saccharomyces...
As eukaryotic cells progress through cell division, the nuclear envelope (NE) membrane must expand to accommodate the formation of progeny nuclei. In Saccharomyces cerevisiae, closed mitosis allows visualization of NE biogenesis during mitosis. During this period, the SUMO E3 ligase Siz2 binds the inner nuclear membrane (INM) and initiates a wave of INM protein SUMOylation. Here, we show these events increase INM levels of phosphatidic acid (PA), an intermediate of phospholipid biogenesis, and are necessary for normal mitotic NE membrane expansion. The increase in INM PA is driven by the Siz2-mediated inhibition of the PA phosphatase Pah1. During mitosis, this results from the binding of Siz2 to the INM and dissociation of Spo7 and Nem1, a complex required for the activation of Pah1. As cells enter interphase, the process is then reversed by the deSUMOylase Ulp1. This work further establishes a central role for temporally controlled INM SUMOylation in coordinating processes, including membrane expansion, that regulate NE biogenesis during mitosis.
Topics: Cell Nucleus; Mitosis; Nuclear Envelope; Nuclear Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sumoylation; Organelle Biogenesis
PubMed: 37398994
DOI: 10.1083/jcb.202208137 -
TH Open : Companion Journal To... Oct 2021Protein S (PS) deficiency is a risk factor for venous thromboembolism (VTE) and can be caused by variants of the gene encoding PS ( ). This study aimed to evaluate the...
Protein S (PS) deficiency is a risk factor for venous thromboembolism (VTE) and can be caused by variants of the gene encoding PS ( ). This study aimed to evaluate the clinical value of molecular analysis of the gene in PS-deficient participants. We performed Sanger sequencing of the coding region of the gene and multiplex ligation-dependent probe amplification to exclude large structural rearrangements. Free PS was measured by a particle-enhanced immunoassay, while PS activity was assessed by a clotting method. A total of 87 PS-deficient participants and family members were included. In 22 index participants, we identified 13 coding variants. Five variants were novel. In 21 index participants, no coding sequence variants or structural rearrangements were identified. The free PS level was lower in index participants carrying a variant compared with index participants with no variant (0.51 [0.32-0.61] vs. 0.62 [0.57-0.73] × 10 IU/L; < 0.05). The p.(Thr78Met) variant was associated with only slightly decreased free PS levels (0.59 [0.53-0.66] × 10 IU/L) compared with the p.(Glu390Lys) variant (0.27 [0.24-0.37] × 10 IU/L, < 0.01). The frequency of VTE in participants with a coding variant was 43 and 17% in the group with normal gene ( = 0.05). In conclusion, we report 13 coding variants including five novel variants. PS levels differ by variant and the frequency of VTE was higher when a coding variant was present. Hence, molecular analysis of the gene may add clinical value in the diagnostic work-up of PS deficiency.
PubMed: 34729451
DOI: 10.1055/s-0041-1736636 -
ERJ Open Research Oct 2021COPD patients have an increased risk of cardiovascular disease and venous thromboembolism.
BACKGROUND
COPD patients have an increased risk of cardiovascular disease and venous thromboembolism.
METHODS
This study aimed to investigate whether patients with stable COPD have a prothrombotic state compared to COPD-free smokers. We conducted an observational study comparing levels of: D-dimers, INR, aPTT, coagulation factors; fibrinogen, FII, FV, FVII, FVIII, FIX, FX and coagulation inhibitors; protein S, proteins C and antithrombin between stable COPD patients and control subjects.
RESULTS
A total of 103 COPD patients and 42 controls with similar age, sex, current smoking status, comorbidity burden and cardiovascular risk met the inclusion criteria. Compared to controls, COPD patients had higher levels of D-dimers (median (interquartile range): 360 (230-600) ng·mL 240 (180-400) ng·mL, p=0.001), fibrinogen (mean±sd: 399±82 mg·dL 346±65 mg·dL, p<0.001), FII (122±22% 109±19%, p=0.004), FV (131±25% 121±19%, p=0.015), FVIII (143±32% 122±20%, p<0.001) and FX (111 (94-134)% 98 (88-107)%, p=0.002), and lower levels of protein S (95 (85-105)% 116 (98-121)%, p<0.001) and antithrombin (94.4±11.5% 102.3±13.2%, p=0.001). In the COPD group, patients with more severe airflow limitation and frequent exacerbations had significantly higher levels of FII, FV and FX, whereas patients with higher COPD assessment test score had significantly higher levels of FX and lower levels of protein S.
CONCLUSION
Patients with stable COPD exhibited increased levels of key coagulation factors and decreased levels of coagulation inhibitors, namely protein S and antithrombin, compared to COPD-free smokers. Among COPD patients, increased levels of FII, FV and FX and decreased levels of protein S were found in patients with more severe disease.
PubMed: 34729369
DOI: 10.1183/23120541.00297-2021