Elucidating novel mechanisms for pancreatitis through calcineurin signaling - PROJECT SUMMARY. Acute pancreatitis (AP) is an exquisitely painful, life-threatening, public health problem that has substantial morbidity and limited treatment modalities. Our long-term goal is to develop focused therapies to treat AP and mitigate disease severity. The premise is based on our discovery (1) that the calcium-activated phosphatase calcineurin (CN) is a potent mediator of AP in the primary parenchymal cell of the pancreas, the acinar cell, and (2) that the distinct molecular signatures underlying this potent effect can be leveraged to develop targeted therapies for AP. The two major objectives of the current proposal are (1) to elucidate the CN-modulated signaling pathways in AP and (2) to identify novel and potentially therapeutically targetable, direct CN substrates within these pathways. The three Specific Aims are to: (1) Define the phosphosignaling networks, including signatures regulated by CN activity in AP through phosphoproteomics coupled with powerful bioinformatics; (2) characterize the role of CN in the identified pathways of mTOR and autophagy during AP; and (3) evaluate novel CN substrate candidates that are integral to the identified pathways. Our hypothesis, which was generated from compelling preliminary phosphoproteomic data, is that CN mediates AP by impairing autophagy, and the mechanism for the impairment is through both (1) activation of the upstream autophagy inhibitor mTOR and (2) direct inhibition of CN substrates in the autophagy pathway itself. The design of the approach is that Aim 1 is an unbiased phosphoproteomic screen, using clinically and biologically relevant AP conditions, for phosphosignals that will provide clues to CN-modulated pathways in AP. Aim 2 is to conduct independent, empirical testing of CN modulation in AP of the identified pathways of autophagy and mTOR, by examining canonical components and phosphosites, including the ones that were not necessarily detected in the unbiased phosphoproteomic data. Aim 3 is a systematic identification, followed by biochemical validation, of novel CN substrates that are integral to the identified pathways in AP. Here, we will also probe the molecular mechanisms by which CN modulates novel substrate activity and function. Incorporating a highly multidisciplinary team of investigators and an ideally suited environment, the proposed studies are technologically and conceptually innovative since they utilize (1) advanced computational methods to identify CN-regulated pathways and substrates, (2) human pancreas specimens for ex vivo culture, (3) an innovative in vivo pressure-induced pancreatitis (PIP) model, and (4) cutting edge phosphoproteomic and biochemical tools including BioID, which captures in cell transient low-affinity interactions between CN and its substrate candidates. The significance of the proposal is that it creates a discovery pipeline to identify novel CN-modulated phosphosignaling networks in AP and will provide a valuable resource to the pancreas community that will aid in devising targeted AP therapies.
