title: Characterising the phosphorylation and SUMOylation of cardiac troponin I in heart failure
Creators: name: Fertig, Bracy Andrea
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: troponin, SUMOylation, phosphodiesterase.

description: Troponin I (TnI) is the inhibitory subunit of the troponin (Tn) complex of the myofilament. TnI’s function is regulated by phosphorylation at a number of sites by different kinases. As a result of β-adrenergic stimulation, protein kinase A (PKA) phosphorylates TnI at Ser23/24, which causes positive lusitropy and inotropy. The PKA-mediated phosphorylation events on cardiac excitation contraction coupling proteins are often controlled by phosphodiesterase (PDE) enzymes which degrade cyclic 3’,5’-adenosine monophosphate (cAMP), reducing local activation of PKA. This is accomplished by the integration of PDEs into macromolecular complexes, or signalosomes, containing PKA, PKA substrate proteins, anchoring proteins and often other kinases and phosphatases. Research published in another thesis from the Baillie lab indicated that PDE4D9 binds directly to TnI, thereby regulating the cAMP dynamics at the myofilament and the PKA phosphorylation events. Little is known about other post-translational modifications of TnI, but the identification of other modifications could lead to better understanding of the regulation of TnI function within the myofilament and how this is altered in disease states. SUMOylation is a post-translational modification in which a small ubiquitin-like modifier (SUMO) is covalently attached to a substrate protein by an enzymatic cascade similar to the ubiquitination cascade. This thesis began with the testing of a disruptor peptide which was designed to interrupt the proposed interaction between PDE4D9 and TnI. It was hypothesised that disruptor peptides would ‘unhook’ the PDE4D9-TnI complex, allowing enhanced cAMP dynamics at the myofilament and enhanced PKA phosphorylation of TnI. However, using fluorescence resonance energy transfer (FRET) and immunoblotting for phosphorylation levels, it was shown that disruption of the proposed interaction did not significantly affect the outcomes of β-adrenergic signalling at the myofilament. Subsequent attempts to confirm the existence of the proposed interaction were unsuccessful suggesting that PDE4D9 may not be a TnI binding partner after all. Further study is necessary to determine the mechanisms by which PDEs regulate signalling at the myofilament. In silico analysis of TnI revealed a high probability SUMOylation site (K177) suggesting that TnI could be a SUMO substrate. This hypothesis was tested in the second part of this thesis. For the first time, it was shown that TnI can be SUMOylated using a number of biochemical techniques. Furthermore, detection of SUMOylated TnI was facilitated by the successful development of a SUMO-TnI site-specific antibody, the first of its kind to detect a SUMOylated substrate protein. Interestingly, the levels of SUMOylated TnI were significantly enhanced in human heart disease, suggesting a role in the pathophysiology of disease. Functional analyses of the role of TnI SUMOylation in protein stability and myofilament dynamics were then carried out. Viral overexpression of mutant TnI in which the SUMO acceptor lysine was mutated did not have any effect on TnI stability nor the contractility of neonatal rat ventricular myocytes (NRVM). However, functional differences were discovered when mutant TnI was overexpressed in adult rabbit ventricular myocytes (ARVM) and SUMOylation was globally upregulated. The data suggested that TnI SUMOylation may have a scavenging effect, sequestering SUMO proteins from other myofilament SUMO targets. The present work provides a major contribution to the field, showing for the first time that TnI can be modified by SUMOylation and that the modified protein alters myofilament function. Understanding the way post-translational modifications affect the function of cardiac excitation-contraction coupling proteins is necessary for a full understanding of pathophysiology, especially when the modification has been shown to be altered in disease states. Further work elucidating the molecular mechanisms by which TnI SUMOylation alters myofilament dynamics may reveal potential therapeutic targets for heart disease.