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Title: Defining the molecular basis of H2S-mediated sensing and signalling
Author: Stubbert, Daniel David
ISNI:       0000 0004 6349 3076
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2015
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Hydrogen sulfide (H2S) is a well-known vasorelaxant. However, as is true of many of its biological actions, the molecular basis for this blood-pressure-lowering action is incompletely understood. A common first thought is that H2S functions as a reducing agent. Thus, it may reduce oxidised proteins (e.g., disulfides) back to a thiol state, thereby changing enzyme activity to achieve regulation. However, this thesis provides experimental evidence suggesting that H2S reacts with biological oxidants to generate polysulfide species that mediate H2S signalling. Once formed, polysulfides induced protein oxidation, such as S-sulfhydration or disulfide. Thus, the biological reducing agent H2S may counterintuitively induce protein oxidation. H2S may perform this function due to its acid dissociation constant (pKa) of ~6.9, meaning that it is ionised at physiological pH levels and will readily react with available oxidants to generate end products, including polysulfides. Polysulfides, including those formed from H2S, were shown to induce oxidation of glutathione and oxidant-sensitive kinases, such as cyclic GMP-dependent protein kinase (PKG) Iα or cyclic AMP-dependent protein kinase (PKA) regulatory RIα subunits. The physiological significance of the ability of H2S to oxidise PKG was investigated using a Cys42Ser PKG knock-in (KI) mouse line that expresses only a mutant form of kinase that cannot undergo disulfide activation. Tissue excised from KI mice showed impaired relaxation to H2S compared with that from their wild-type littermates. These findings demonstrate an oxygen sensing and signalling role for H2S that allows it to mediate vasorelaxation by directly oxidising PKG. A broader role for H2S as a general mediator of protein oxidation was investigated using a transgenic KI mouse expressing cardiac-restricted Cys35Ser thioredoxin (Trx). These KI mice express a ‘trapping mutant’ form of Trx that becomes covalently linked to proteins with oxidised thiol after H2O2 or H2S treatment. These covalent protein complexes were isolated and constituent proteins identified by mass spectrometry or western immunoblotting for candidate proteins. Several proteins were found to be oxidised after H2S treatment using this mouse model, including some involved in vasorelaxation, most notably PKG.
Supervisor: Eaton, Philip Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available