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Title: Modulation of transglutaminase 2 by the A1 adenosine receptor and β2-adrenoceptor in cardiomyoblasts : a role in cell survival
Author: Vyas, F. S.
ISNI:       0000 0004 6498 8505
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2017
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Transglutaminase 2 (TG2) is a multifunctional enzyme possessing transamidating, GTPase and protein disulphide isomerase activity. Due to its diverse nature and ubiquitous distribution TG2 is implicated in the regulation of many physiological processes, includeing cell adhesion, migration, growth, differentiation, survival and apoptosis. Recently, TG2 has been shown to be modulated by PKC and PKA and to play a role in cardioprotection. However, the precise mechanism(s) of its modulation by G-protein coupled receptors coupled to PKC and PKA activation are not fully understood. The present study investigated the molecular mechanisms underlying TG2 modulation by GPCRs (A1 adenosine receptor (A1R) and β2-adrenoceptor (β2-AR)) and its role in cardioprotection. Transglutaminase transamidation activity was determined using amine-incorporating (in vitro and in situ) and protein cross-linking assays. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1R- or β2-AR-induced cytoprotection was investigated by monitoring hypoxia- and hypoxia/reoxygenation-induced cell death. Novel TG2 substrates/interacting proteins were identified using SWATH MS. Stimulation of the A1R (with the selective agonist CPA and endogenous agonist adenosine) and β2-AR (with the selective agonist formoterol and non-selective agonist isoprenaline) resulted in activation of TG2 in a time- and concentration-dependent manner, which was attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA and adenosine were blocked by pharmacological inhibition of PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) and by removal of extracellular Ca2+. CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Responses to formoterol, whilst insensitive to pertussis toxin (PTX; Gi-protein inactivator), were abolished by PKA (Rp-cAMPs), MEK1/2, and PI-3K (AS 605240) inhibitors. Responses to isoprenaline were blocked by PTX and inhibitors of MEK1/2 and PI-3K. Formoterol triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKA, MEK1/2 PI-3K inhibitors and by removal of extracellular Ca2+. Furthermore, the A1R- or β2-AR-mediated TG2 activation (with the exception of formoterol-induced TG2 activation) was protective against hypoxia- and hypoxia/reoxygenation-induced cell death. Finally, identification of novel TG2 substrates/interacting proteins using SWATH MS illustrated differential modulation of substrates by TG2 upon stimulation of the A1R- or β2-AR which may contribute to differences in their cellular/physiological responses. The present study provides the first detailed characterisation of the molecular mechanisms underlying the modulation of TG2 by the A1R- or β2-AR and describes a role for TG2 in cardioprotection, along with identifying novel substrates/interacting proteins which may be involved in mediating this protection.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available