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Food Chemistry Feb 2020This research aimed to explore the role of protein S-nitrosylation in regulating the tenderness of postmortem beef, from the perspective of μ-calpain autolysis and...
This research aimed to explore the role of protein S-nitrosylation in regulating the tenderness of postmortem beef, from the perspective of μ-calpain autolysis and protein proteolysis. Five bovine semimembranosus muscles were incubated with three treatments including S-nitrosoglutathione (GSNO, nitric oxide donor), normal saline and Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME, nitric oxide synthase inhibitor). The results showed that the level of protein S-nitrosylation was improved by GSNO treatment and reduced by L-NAME treatment (p < 0.05). Compared to the control, GSNO treatment had higher shear force while L-NAME treatment presented lower shear force at 7 d postmortem (p < 0.05). In addition, μ-calpain autolysis, myofibrillar protein and desmin degradation were reduced by GSNO treatment and accelerated by L-NAME treatment (p < 0.05). Therefore, it can be speculated that protein S-nitrosylation could affect beef tenderization by regulating the autolysis of μ-calpain and the degradation of myofibrillar proteins.
Topics: Animals; Arginine; Calpain; Cattle; Desmin; Protein S; Proteolysis; Red Meat
PubMed: 31622832
DOI: 10.1016/j.foodchem.2019.125616 -
International Journal of Molecular... Jun 2024Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an...
Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an outstanding contribution from the team of Prof. Greg Lemke. MerTK activity is implicated in a wide variety of functions involving the elimination of apoptotic cells and has recently been linked to cancers, auto-immune diseases, and atherosclerosis/stroke. In the retina, MerTK is required for the circadian phagocytosis of oxidized photoreceptor outer segments by the retinal-pigment epithelial cells, a function crucial for the long-term maintenance of vision. We previously showed that MerTK ligands carry the opposite role in vitro, with Gas6 inhibiting the internalization of photoreceptor outer segments while Protein S acts conversely. Using site-directed mutagenesis and ligand-stimulated phagocytosis assays on transfected cells, we presently demonstrate, for the first time, that Gas6 and Protein S recognize different amino acids on MerTK Ig-like domains. In addition, MerTK's function in retinal-pigment epithelial cells is rhythmic and might thus rely on the respective stoichiometry of both ligands at different times of the day. Accordingly, we show that ligand bioavailability varies during the circadian cycle using RT-qPCR and immunoblots on retinal and retinal-pigment epithelial samples from control and beta5 integrin knockout mice where retinal phagocytosis is arrhythmic. Taken together, our results suggest that Gas6 and Protein S might both contribute to refine the acute regulation of MerTK in time for the daily phagocytic peak.
Topics: Animals; Mice; c-Mer Tyrosine Kinase; Circadian Rhythm; Intercellular Signaling Peptides and Proteins; Ligands; Phagocytosis; Protein S; Receptor Protein-Tyrosine Kinases; Retina; Retinal Pigment Epithelium
PubMed: 38928335
DOI: 10.3390/ijms25126630 -
Cureus Jan 2023The dual coagulation disorder hereditary protein S deficiency and activated protein C (APC) resistance, which clinically manifests with recurrent venous thrombosis and...
The dual coagulation disorder hereditary protein S deficiency and activated protein C (APC) resistance, which clinically manifests with recurrent venous thrombosis and multifocal ischemic stroke, has only rarely been reported in the same patient. The patient is a 54-year-old male with a history of recurrent, asymptomatic ischemic stroke or transient ischemic attack (TIA) since age 14 and four episodes of deep vein thromboses (DVT), two complicated by pulmonary embolism, attributed to hereditary protein S deficiency and homozygous factor V Leiden mutation. In addition, the medical history was positive for obesity, previous chronic alcoholism, smoking, gynecomastia with left breast resection, arterial hypertension, hepatic steatosis, and cholecystolithiasis. Because of low compliance, long-term oral anticoagulation with phenprocoumon from the age of 38 was replaced by dabigatran (300 mg/d) after another stroke with bleeding at the age of 54. In summary, the simultaneous presence of two hereditary coagulation disorders can lead to multiple venous thromboses and recurrent ischemic stroke. An appealing therapeutic option in poorly compliant patients with these two hereditary clotting defects is the replacement of long-term anticoagulation with a vitamin K antagonist (VKA) by a direct oral anticoagulant.
PubMed: 36824536
DOI: 10.7759/cureus.34012 -
Journal of Thrombosis and Haemostasis :... Dec 2023For maximal TFPIα functionality, 2 synergistic cofactors, protein S and FV-short, are required. Both interact with TFPIα, protein S through Kunitz 3 residues...
BACKGROUND
For maximal TFPIα functionality, 2 synergistic cofactors, protein S and FV-short, are required. Both interact with TFPIα, protein S through Kunitz 3 residues Arg199/Glu226 and FV-short with the C-terminus. How these interactions impact the synergistic enhancement remains unclear.
OBJECTIVES
To determine the importance of the TFPIα-protein S and TFPIα-FV-short interactions for TFPIα enhancement.
METHODS
TFPIα variants unable to bind protein S (K3m [R199Q/E226Q]) or FV-short (ΔCT [aa 1-249]) were generated. TFPIα-FV-short binding was studied by plate-binding and co-immunoprecipitation assays; functional TFPIα enhancement by FXa inhibition and prothrombin activation.
RESULTS
While WT TFPIα and TFPIα K3m bound FV-short with high affinity (K∼2nM), TFPIα ΔCT did not. K3m, in contrast to WT, did not incorporate protein S in a TFPIα-FV-short-protein S complex while TFPIα ΔCT bound neither FV-short nor protein S. Protein S enhanced WT TFPIα-mediated FXa inhibition, but not K3m, in the absence of FV-short. However, once FV-short was present, protein S efficiently enhanced TFPIα K3m (EC50: 4.7nM vs 2.0nM for WT). FXa inhibition by ΔCT was not enhanced by protein S alone or combined with FV-short. In FXa-catalyzed prothrombin activation assays, FV-short enhanced TFPIα K3m function in the presence of protein S (5.5 vs 10.4-fold enhancement of WT) whereas ΔCT showed reduced or lack of enhancement by FV-short and protein S, respectively.
CONCLUSION
Full TFPIα function requires the presence of both cofactors. While synergistic enhancement can be achieved in the absence of TFPIα-protein S interaction, only TFPIα with an intact C-terminus can be synergistically enhanced by protein S and FV-short.
Topics: Humans; Blood Coagulation; Blood Coagulation Tests; Factor V; Factor Xa; Prothrombin
PubMed: 37739040
DOI: 10.1016/j.jtha.2023.09.003 -
Acta Histochemica Jul 2022Tail regression is a remarkable process where a complex organ like the tail is completely resorbed by cell death during anuran metamorphosis. Nitric oxide is a...
Tail regression is a remarkable process where a complex organ like the tail is completely resorbed by cell death during anuran metamorphosis. Nitric oxide is a signalling molecule involved in various physiological processes and along with reactive nitrogen species induces apoptosis. The present study describes the contribution of nitric oxide and reactive nitrogen species (nitrosative stress) during tail regression in the tadpoles of Indian tree frog, Polypedates maculatus. Spectrophotometric estimation revealed significantly higher levels of nitrite, nitrate and peroxynitrite in the regressing tails of the late climactic stages as compared to the early climactic stages and pre-regressing tails. S-nitrosylated proteins were detected in the apoptotic cells of epidermis and muscle, denervated and fragmented myofibres, outer notochordal sheath of the degenerating notochord, endothelium of blood vessels, blood cells and spinal cord of the regressing tail of the late climactic stages using fluorescent detection methods. Thus, a higher level of nitrosative stress in the late climactic stages is suggested to cause S-nitrosylation of proteins and subsequent apoptosis in the tail tissues. Macrophages were found engulfing the apoptotic cells and cell debris at the distal end of the regressing tail. Interestingly, macrophages were always found to be associated with melanocytes suggesting a close association for clearing cell debris by phagocytosis.
Topics: Animals; Anura; Larva; Metamorphosis, Biological; Nitric Oxide; Protein S
PubMed: 35580443
DOI: 10.1016/j.acthis.2022.151899 -
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 -
The Journal of Applied Laboratory... May 2022One of the most complex risk factors for the laboratory assessment of thrombophilia is Protein S (PS). The testing algorithm for PS employs the plasma-based assays of...
BACKGROUND
One of the most complex risk factors for the laboratory assessment of thrombophilia is Protein S (PS). The testing algorithm for PS employs the plasma-based assays of free PS antigen, total PS antigen, and PS activity creating a complex diagnostic scheme that can lead to misdiagnosis if incorrectly used, and a potential waste of resources and money.
CONTENT
This paper compares the recently published evidence-based algorithm from the International Society for Hemostasis and Thrombosis (ISTH) with several commonly performed nonevidence-based testing schemes, to demonstrate the efficiency of the evidence-based algorithm for diagnostic efficiency with improved patient care and increased cost savings for the laboratory.
SUMMARY
Significant savings (31%-60%) can be realized when the evidence-based algorithm is used in place of other testing modalities of initial PS activity testing (31%) or testing with all 3 assays simultaneously (60%). This study utilizing the PS testing evidence-based algorithm as part of a thrombophilia evaluation demonstrates that the appropriate testing methods can be used to limit wasteful practices while achieving the maximum level of information in this time of limited resources and need for increase monetary savings.
Topics: Algorithms; Cost-Benefit Analysis; Humans; Protein S; Risk Factors; Thrombophilia
PubMed: 35060606
DOI: 10.1093/jalm/jfab175 -
Journal of Thrombosis and Haemostasis :... May 2022Factor V-short (FV756-1458) is a natural splice variant functioning in synergy with protein S as tissue factor pathway inhibitor alpha (TFPIα)-cofactor in inhibition of...
A hydrophobic patch (PLVIVG; 1481-1486) in the B-domain of factor V-short is crucial for its synergistic TFPIα-cofactor activity with protein S and for the formation of the FXa-inhibitory complex comprising FV-short, TFPIα, and protein S.
BACKGROUND
Factor V-short (FV756-1458) is a natural splice variant functioning in synergy with protein S as tissue factor pathway inhibitor alpha (TFPIα)-cofactor in inhibition of factor Xa (FXa). An exposed acid region (AR2; 1493-1537) in the B domain binds TFPIα. The preAR2 (1458-1492) is crucial for the synergistic TFPIα-cofactor activity between FV-short and protein S and for assembly of a trimolecular FXa-inhibitory complex among FV-short, protein S, and TFPIα.
OBJECTIVE
To identify which part of preAR2 is required for the synergistic TFPIα-cofactor activity between FV-short and protein S.
METHODS
A FXa-inhibition assay was used to test the synergistic TFPIα cofactor activity between protein S and new FV-short variants FV709-1476, FV712-1478, FV712-1481, FV712-1484, FV712-1487, and FV712-1490. A microtiter-based assay analyzed binding among FV-short variants, protein S, and TFPIα.
RESULTS
FV709-1476, FV712-1478, and FV712-1481 were fully active as synergistic TFPIα cofactors with protein S; FV712-1484 showed intermediate activity; and FV712-1487 and FV712-1490 were inactive. TFPIα interacted with all variants in the absence of protein S but FV712-1478 and FV712-1481 bound TFPIα with highest affinity. None of the FV-short variants bound directly to protein S in the absence of TFPIα. In the presence of TFPIα, efficient cooperative binding was demonstrated between protein S, TFPIα, and FV709-1476, FV712-1478, or FV712-1481. In contrast, no cooperativity among TFPIα, protein S, and FV712-1484, FV712-1487, or FV712-1490 was seen.
CONCLUSION
A short hydrophobic patch in preAR2 (PLVIVG, 1481-1486) in FV-short is crucial for the synergistic TFPIα-cofactor activity between FV-short and protein S and for the assembly of a trimolecular FXa-inhibitory complex among FV-short, protein S, and TFPIα.
Topics: Blood Coagulation; Factor V; Factor Xa; Humans; Hydrophobic and Hydrophilic Interactions; Lipoproteins; Protein S; Thrombin
PubMed: 35247027
DOI: 10.1111/jth.15690 -
Cureus Jul 2023Protein S deficiency is a form of inherited thrombophilia that occurs due to low levels of or improper function of protein S. The role of protein S is to inactivate...
Protein S deficiency is a form of inherited thrombophilia that occurs due to low levels of or improper function of protein S. The role of protein S is to inactivate procoagulant factors, and a deficiency results in an increased risk of thrombotic events. The coronavirus disease 2019 (COVID-19) infection has also been studied to increase the risk of venous thromboembolism (VTE) due to an interplay of several mechanisms. However, the risk of VTE in patients affected by both of these disease processes simultaneously has not been thoroughly studied, and so recommendations regarding routine screening and prophylaxis of VTE have also not been established. We discuss the case of a 46-year-old woman with a past history of protein S deficiency and a recent COVID-19 infection who presented with complaints of shortness of breath. Upon examination, she was found to be hypoxic and tachycardic. A computed tomography angiography of the chest was done and revealed acute submassive bilateral pulmonary embolism with right heart strain and pulmonary infarcts. She was initially treated with intravenous heparin and later transitioned to oral anticoagulation for a minimum of six months.
PubMed: 37554606
DOI: 10.7759/cureus.41560