مجال التميز | تميز دراسي وبحثي |
البحوث المنشورة |
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البحث (1): | |
عنوان البحث: |
Myricetin, the Main Flavonoid in Syzygium cumini Leaf, Is a Novel Inhibitor of Platelet Thiol Isomerases PDI and ERp5 |
رابط إلى البحث: | |
تاريخ النشر: |
31/10/2020 |
موجز عن البحث: |
Background: Flavonoids have been characterized as a prominent class of compounds to treat thrombotic diseases through the inhibition of thiol isomerases. Syzygium cumini is a flavonoid-rich medicinal plant that contains myricetin and gallic acid. Little is known about the potential antiplatelet properties of S. cumini and its constituent flavonoids. Objective: To evaluate the antiplatelet effects and mechanism of action of a polyphenol-rich extract (PESc) from S. cumini leaf and its most prevalent polyphenols, myricetin and gallic acid. Methods: PESc, myricetin, and gallic acid were incubated with platelet-rich plasma and washed platelets to assess platelet aggregation and activation. In vitro platelet adhesion and thrombus formation as well as in vivo bleeding time were performed. Finally, myricetin was incubated with recombinant thiol isomerases to assess its potential to bind and inhibit these, while molecular docking studies predicted possible binding sites. Results: PESc decreased platelet activation and aggregation induced by different agonists. Myricetin exerted potent antiplatelet effects, whereas gallic acid did not. Myricetin reduced the ability of platelets to spread on collagen, form thrombi in vitro without affecting hemostasis in vivo. Fluorescence quenching studies suggested myricetin binds to different thiol isomerases with similar affinity, despite inhibiting only protein disulfide isomerase (PDI) and ERp5 reductase activities. Finally, molecular docking studies suggested myricetin formed non-covalent bonds with PDI and ERp5. Conclusions: PESc and its most abundant flavonoid myricetin strongly inhibit platelet function. Additionally, myricetin is a novel inhibitor of ERp5 and PDI, unveiling a new therapeutic perspective for the treatment of thrombotic disorders. |
المؤتمرات العلمية |
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المؤتمر (1): | |
عنوان المؤتمر: |
BSHT Annual Scientific meeting 2018 |
تاريخ الإنعقاد: |
8-9/11/2018 |
مكان الإنعقاد: |
Coventry. UK |
طبيعة المشاركة: |
Oral Presentation |
عنوان المشاركة: |
ERp44, a new thiol isomerase enzyme that is present in platelets |
ملخص المشاركة: |
Background: Thiol isomerases are endoplasmic reticulum (ER) resident proteins that modulate the formation, reduction and isomerisation of disulphide bonds, which are often rate-limiting reactions during protein folding. Recent evidence suggests that this family of proteins are surface presented in a number of different cell types where they are important in cell remodelling and recognition. Previous research has identified thiol isomerases within platelet surfaces including, PDI, ERp5, ERp57 and ERp72 which play pivotal roles in the regulation of platelet function. ERp44 is a thiol protein with one hydrophobic N-terminal thioredoxin domain containing a CRFS motif, followed by two thioredoxin inactive domains arranged in a clover-leaf-like structure. It is also acts as a multifunctional chaperone protein that regulates redox homeostasis and modulates thiol-mediated quality control and calcium and KDEL-receptor signalling. ERp44 is also implicated in the regulation and correct folding of various molecules including IgM within B lymphocytes, adiponectin, and formylglycinegenerating enzyme. Aims: The aim of this study is to investigate the expression and localisation of ERp44 in platelets to determine whether this thiol isomerase is also likely to be implicated in the regulation of platelet activation. Methods: The presence of ERp44 in human/mouse platelets was examined by immunoblotting. Immunofluorescence and sucrose density gradient subcellular platelet fractionation were used to determine ERp44 location in human platelets. Results: Immunoblotting and Immunofluorescence analysis confirmed that ERp44 is present in human and mouse platelets, as well as in the human megakaryocytic cell line (MEG-01). The location, levels and subcellular associations of ERp44 with different organelles were therefore examined. Immunofluorescence microscopy (IFM) of permeabilised platelets revealed ERp44 to be dispersed throughout the cytoplasm in a punctate arrangement. Following activation, ERp44 partially translocated to a ring-like staining pattern towards the plasma membrane in confocal images and colocalised with proteins known to be present in the membrane proximal dense tubular system. Subcellular fraction of platelets by sucrose density gradient centrifugation revealed ERp44 to be principally located in low density fractions, consistent with interactions with the plasma membrane, and suggested that, like other platelet thiol isomerases, this enzyme is not located in -granules. Indeed, cell surface localisation of ERp44 was detected on resting platelets by flow cytometry and found to increase in detectable levels following stimulation with 1U/mL thrombin. The mobilisation of ERp44 to the platelet surface was found to be dependent on actin polymerisation. It is possible that surface bound ERp44 may result from co-secretion with transmembrane proteins to which is it bound, or through binding to cell surface molecules following release as a soluble protein. ERp44 was detected in the supernatant of activated platelets suggesting that surface localisation is due, at least in part to protein release. Conclusion: These properties are shared with other functionally important platelet thiol isomerases, raising the possibility that ERp44 may also contribute to platelet regulation. We have generated antibodies to ERp44 and are currently exploring this question. This study has aided our understanding of subcellular DTS association of ERp44 and its release and mobilisation upon platelet activation. Therefore, affinity-purified function blocking anti-ERp44 antibodies will be used to determine the protein’s role and its surface substrates. Further research will be undertaken to explore co-precipitation of ERp44 and investigate whether there is a direct association between ERP44 and other platelet surface proteins. |
المؤتمر (2): | |
عنوان المؤتمر: | The Platelet Society 1st Early Career Research Meeting (ECM) |
تاريخ الإنعقاد: |
13/07/2018 |
مكان الإنعقاد: |
Manchester, UK |
طبيعة المشاركة: |
Poster presentation |
عنوان المشاركة: |
A role for the thiol isomerase protein ERp44 in platelet function |
ملخص المشاركة: |
Background: Thiol isomerases are endoplasmic reticulum (ER) resident proteins that modulate formation, reduction and isomerization of disulphide bonds, which are often rate-limiting reactions during protein folding. Recent evidence suggests that this family of proteins are surface presented in a number of different cell types where they are important in cell remodelling and recognition. Previous research has identified thiol isomerases within platelet surfaces including, PDI, ERp5 and ERp57, where they play pivotal roles in platelet function regulation. ERp44 is a thiol protein with one hydrophobic N-terminal thioredoxin domain containing a CRFS motif, followed by two thioredoxin inactive domains arranged in a clover-leaf-like structure. It has been proposed that since it lacks the second reactive cysteine, ERp44 functions mainly as an isomerase rather than an oxidase or reductase. ERp44 is also implicated in the regulation and correct folding of various molecules including IgM within B lymphocytes, adiponectin, and formylglycine-generating enzyme (FGE). The aim of this study is to investigate the role of ERp44 platelet-surface thiol isomerase in human platelets and identify its role in the regulation of platelet function. Method: This study has shown that ERp44 is present in human and mouse platelets, as well as in the human megakaryocytic cell line (MEG-01). In this study, the location, levels and subcellular associations of ERp44 with different organelles were examined. The localisation of ERp44 human platelets was investigating using immunofluorescence (IMF) microscopy. In permeabilised resting platelets, ERp44 was found dispersed throughout the cytoplasm in a punctate arrangement. Following activation, ERp44 partially translocated to a ring-like staining pattern towards the membrane periphery in confocal images. This was corroborated with partly present ERp44 in the low-density fractions, comprised mostly of cytoplasmic and plasma membrane proteins. ERp44 was detected on the surface of resting platelets using Fluorescence-activated cell sorting (FACS). Following stimulation with 1U/mL thrombin, an increase in surface expression of this protein was detected. The mobilisation of ERp44 to the surface was shown to be dependent on actin polymerisation. ERp44 was found to be released from human platelets which may be recycled to enable constant soluble secretion. 6 This study suggested that ERP44 might be involved in platelet function as well as in the regulation of coagulation due to its presence in platelet-derived microparticle fractions. This study has aided our understanding of subcellular DTS association of ERp44 and its release and mobilisation upon platelet activation. Therefore, an affinity-purified function blocking anti-ERp44 antibody should be used to determine the protein’s role and its surface substrates. Further research should be undertaken to explore co-precipitation of ERp44 and investigate whether there is a direct association between ERP44 and other proteins. |
المؤتمر (3): | |
عنوان المؤتمر: |
British Pharmacological Society, Pharmacology 2018 Conference |
تاريخ الإنعقاد: |
18-20/12/2018 |
مكان الإنعقاد: |
London, UK |
طبيعة المشاركة: |
Poster Presentation |
عنوان المشاركة: |
ERp44, a new thiol isomerase enzyme that is present in platelets |
ملخص المشاركة: |
Background: Thiol isomerases are endoplasmic reticulum (ER) resident proteins that modulate the formation, reduction and isomerisation of disulphide bonds, which are often rate-limiting reactions during protein folding. Recent evidence suggests that this family of proteins are surface presented in a number of different cell types where they are important in cell remodelling and recognition. Previous research has identified thiol isomerases within platelet surfaces including, PDI, ERp5, ERp57 and ERp72 which play pivotal roles in the regulation of platelet function. ERp44 is a thiol protein with one hydrophobic N-terminal thioredoxin domain containing a CRFS motif, followed by two thioredoxin inactive domains arranged in a clover-leaf-like structure. It is also acts as a multifunctional chaperone protein that regulates redox homeostasis and modulates thiol-mediated quality control and calcium and KDEL-receptor signalling. ERp44 is also implicated in the regulation and correct folding of various molecules including IgM within B lymphocytes, adiponectin, and formylglycinegenerating enzyme. Aims: The aim of this study is to investigate the expression and localisation of ERp44 in platelets to determine whether this thiol isomerase is also likely to be implicated in the regulation of platelet activation. Methods: The presence of ERp44 in human/mouse platelets was examined by immunoblotting. Immunofluorescence and sucrose density gradient subcellular platelet fractionation were used to determine ERp44 location in human platelets. Results: Immunoblotting and Immunofluorescence analysis confirmed that ERp44 is present in human and mouse platelets, as well as in the human megakaryocytic cell line (MEG-01). The location, levels and subcellular associations of ERp44 with different organelles were therefore examined. Immunofluorescence microscopy (IFM) of permeabilised platelets revealed ERp44 to be dispersed throughout the cytoplasm in a punctate arrangement. Following activation, ERp44 partially translocated to a ring-like staining pattern towards the plasma membrane in confocal images and colocalised with proteins known to be present in the membrane proximal dense tubular system. Subcellular fraction of platelets by sucrose density gradient centrifugation revealed ERp44 to be principally located in low density fractions, consistent with interactions with the plasma membrane, and suggested that, like other platelet thiol isomerases, this enzyme is not located in -granules. Indeed, cell surface localisation of ERp44 was detected on resting platelets by flow cytometry and found to increase in detectable levels following stimulation with 1U/mL thrombin. The mobilisation of ERp44 to the platelet surface was found to be dependent on actin polymerisation. It is possible that surface bound ERp44 may result from co-secretion with transmembrane proteins to which is it bound, or through binding to cell surface molecules following release as a soluble protein. ERp44 was detected in the supernatant of activated platelets suggesting that surface localisation is due, at least in part to protein release. Conclusion: These properties are shared with other functionally important platelet thiol isomerases, raising the possibility that ERp44 may also contribute to platelet regulation. We have generated antibodies to ERp44 and are currently exploring this question. This study has aided our understanding of subcellular DTS association of ERp44 and its release and mobilisation upon platelet activation. Therefore, affinity-purified function blocking anti-ERp44 antibodies will be used to determine the protein’s role and its surface substrates. Further research will be undertaken to explore co-precipitation of ERp44 and investigate whether there is a direct association between ERP44 and other platelet surface proteins. |
شروق عيظة عوض السفياني
دكتوراه
الطب والخدمات الصحية
University of Reading