Abstract
Understanding the molecular mechanisms which regulate signal transduction is fundamental to the development of therapeutic molecules for the treatment of several diseases. In particular, signaling proteins, such as cyclic nucleotide dependent enzymes are the orchestrators of many tissue functions. As a consequence, their function and behaviour are complex and dynamic,
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with constant activation and deactivation through protein modification, allowing the system to quickly achieve equilibrium where cell function is optimal to face the environmental conditions. In particular cGMP and its related kinase are understudies compared to the cAMP and PKA, hence also the characterization of the direct PKG interactome and its downstream targets are not well known.
In Chapter 1 I review the discovery and function of these important cyclic nucleotide signaling proteins and the technologies used in mass spectrometry based proteomics.
Chapter 2 describes a competitive chemical proteomics approach for the identification of new GKAPs. Among the proteins enriched we were able to identify a known GKAP, IRAG, and a new GKAP, the Huntingtin associated protein (HAP1). To confirm the interaction between HAP1 and PKGIβ we performed binding affinity assay using fluorescence anisotropy and mutations on the HAP1 region that binds to PKG. Moreover we docked the proposed GKAP binding domain of HAP1 onto PKGIβ to compare it with the known GKAPs, demonstrating that the proteins not only contain a similar domain but interact in a similar manner with PKG.
The importance of PKG cerebellar signaling is evaluated in Chapter 3, where a quantitative (phospho)proteomics approach is applied in a PKG knock-out mouse model. After the identification of several interesting differentially regulated (phospho)proteins a more targeted validation was performed using western blot and immunohistochemistry. The main observation in this study is that the loss of PKG induces regulation of several proteins associated with cyclic nucleotide and Ca2+ signaling pathways. These are then connected with the impaired motility and memory, that these knock-out mice present.
Understanding how kinase levels change in a relevant disease context, such as heart failure, can be achieved with chemical proteomics. Chapter 4 illustrates the use of this technique, combined with label free mass spectrometry to identify the variation of PKA/AKAP levels and their localization during the evolution of cardiac remodeling in progression to heart failure. For this study we used a rat model of pressure overload.
Immobilized inhibitors on agarose beads are a good alternative for the use of cyclic nucleotides for the isolation of cAMP and cGMP regulated proteins other than PKA and PKG. Chapter 5 focuses on the use of IBMX, an unspecific phosphodiesterase inhibitor, to capture various phosphodiesterases and their interactomes. The combination of our pull down with the use of specific PDEs inhibitors shed more light on the interactome of PDEs in HeLa cells.
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