Cellular clearance and protein binding partners of pathogenic CEL-HYB

Abstract

The CEL gene encodes the digestive enzyme carboxyl ester lipase, which is mainly expressed in the acinar cells of the pancreas. In 2015, our research group discovered a CEL hybrid gene, named CEL-HYB, resulting from non-allelic homologous recombination between CEL and its pseudogene CELP. Interestingly, the CEL-HYB allele was found to be a genetic risk factor for chronic pancreatitis. In cellular studies performed by us and others, CEL-HYB showed reduced secretion, intracellular aggregation as well as induced cell stress and autophagy. Based on these findings, CEL-HYB allele is likely to belong to the misfolding-dependent pathway of genetic risk in chronic pancreatitis. With this master’s project, we wanted to study the cellular fate and to reveal potential protein binding partners of CEL-HYB, to learn more about its disease mechanism. To do so, we used both cellular and mouse model systems. Our results showed that CEL-HYB is less secreted and tends to aggregate in the insoluble pellet fraction of transfected HEK293 cells. Moreover, Cel-HYB proteins accumulated on the inside of the apical cell membrane, forming a tubular-like expression pattern in the pancreatic acinar cells of mice. We also found the autophagy marker LC3B to be upregulated in the pancreas of Cel-HYB expressing mice, but not in control mice. By co-immunoprecipitation and mass spectrometry we discovered possible protein binding partners of CEL-HYB, including a cluster of nine proteins related to protein folding. In summary, we have shown that CEL-HYB aggregation takes place both in the cell and at the organ level, strengthening the hypothesis that protein misfolding is involved in the disease mechanism. Misfolded CEL-HYB is then most likely cleared in the cell by induced autophagy. Interestingly, in our search for CEL-HYB binding partners we found proteins that facilitate the folding of other proteins. These results will be followed up and further analyzed in future studies.