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.