Abstract
Mutations in human cationic trypsinogen (PRSS1) cause autosomal dominant hereditary pancreatitis. Increased intrapancreatic autoactivation of trypsinogen mutants has been hypothesized to initiate the disease. Autoactivation of cationic trypsinogen is proteolytically regulated by chymotrypsin C (CTRC), which mitigates the development of trypsin activity by promoting degradation of both trypsinogen and trypsin. Paradoxically, CTRC also increases the rate of autoactivation by processing the trypsinogen activation peptide to a shorter form. The aim of this study was to investigate the effect of CTRC on the autoactivation of clinically relevant trypsinogen mutants. We found that in the presence of CTRC, trypsinogen mutants associated with classic hereditary pancreatitis (N29I, N29T, V39A, R122C, and R122H) autoactivated at increased rates and reached markedly higher active trypsin levels compared with wild-type cationic trypsinogen. The A16V mutant, known for its variable disease penetrance, exhibited a smaller increase in autoactivation. The mechanistic basis of increased activation was mutation-specific and involved resistance to degradation (N29I, N29T, V39A, R122C, and R122H) and/or increased N-terminal processing by CTRC (A16V and N29I). These observations indicate that hereditary pancreatitis is caused by CTRC-dependent dysregulation of cationic trypsinogen autoactivation, which results in elevated trypsin levels in the pancreas.
Highlights
We found that 29% of all human promoters contain a good match to the 24-nt experimentally defined ZNF263 motif, whereas 86% of the promoters bound by ZNF263 contain this motif
To determine whether the motif predicted to be recognized by ZNF263 by the zinc finger code would have allowed a bioinformatically based identification of ZNF263 target promoters, we examined the prevalence of the predicted motif in the set of all human core promoters versus in the ZNF263 target promoters
The zinc finger family has arisen through duplication and diverexperimentally determined to be bound by ZNF263 that contain a good match to the predicted motif
Summary
Cell Culture—Human chronic myelogenous leukemia cells (K562, ATCC #CCL-243) were grown in RPMI supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin-streptomycin. The ChIPMotifs approach incorporates a statistical bootstrap re-sampling method to identify a number of motifs detected from the set of high stringent 1473 ZNF263 binding regions obtained from the Top 0.1% level using ab initio motif-finding programs such as Weeder (12), MaMF (13), and MEME (14). A total of seven biological samples from seven different institutions (from seven ENCODE consortium groups) were performed on this array platform, and the generated quantified Affymetrix image files (seven *.CEL files) were analyzed simultaneously and normalized using the RMA algorithm provided in the Expression Console software developed by Affymetrix Inc. Probe sets were annotated as supported by RefSeq and full-length GenBankTM Transcripts, resulting in 21,924 annotated probe sets representing ϳ18,000 annotated genes. The array data (GSE19146) has been deposited in the NCBI Gene Expression Omnibus
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