The development of a novel intracellular Ca2+-buffering agent and its impact on cell function
Intracellular Ca2+ signaling plays a central role in several physiological processes. As such, adequate regulation of Ca2+ signaling is crucial for proper functioning of cell systems, while its dysregulation has been implicated in several diseases, including cancer and neurodegenerative disorders. Moreover, insights into the molecular mechanisms underlying Ca2+ signaling and its contribution to disease have stimulated the development of novel therapeutic strategies and tools. In the context of fundamental research focused on the role of intracellular Ca2+ in various diseases, chemical, cell-permeable Ca2+ buffers, including BAPTA-AM, are often used to unravel the importance of intracellular Ca2+ signaling in a particular biological process. However, recent studies show that the universally used intracellular Ca2+ buffer BAPTA-AM also targets other cellular components, such as the Na+/K+-ATPase and PFKFB3. This means that the universally used Ca2+ buffer BAPTA-AM can no longer be considered a reliable tool, as it can affect numerous other signaling pathways. These findings now spur the development of new Ca2+ chelators for the assessment of intracellular Ca2+ signaling in fundamental research. Furthermore, exploiting all the direct binding partners of intracellular BAPTA will be usefull to unravel the mechanism behind BAPTA-induced cell death. The aim of this project is four-folded: 1. Development of BAPTA variants with reduced PFKFBP3 inhibition with the aim to create a variant that can efficiently chelate Ca2+ but does not display the off-target effects observed with BAPTA. 2. Development and testing of photo-activatable BAPTA probes on PFKFB3 and the cellular proteome via a photo-affinity labeling (PAL) experiment to identify direct binding partners of BAPTA. 3. Exploit known BAPTA variants (Fura-2, Fluo-3, Fluo-4, Fluo-8, Indo-1, Cal 590/520) with fast Ca2+-binding kinetics and their impact on PFKFB3 and downstream signaling (mTORC1/MCL-1). 4. Explore the impact of BAPTA on metabolism & autophagy.