JOHANSON-BLIZZARD-SYNDROM - UBR-1 TARGET IDENTIFICATION BY PROTEOME ANALYSIS AND PATHOPHYSIOLOGICAL RELEVANCE

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Introduction: Johanson-Blizzard syndrome (JBS; OMIM 243800), an autosomal recessive multisystem disorder, is characterized by congenital abnormalies including prenatal pancreatic inflammation and later exocrine pancreatic insufficiency. JBS is associated with mutations in the UBR1 gene on chromosome 15q14-21.1, which encodes one of the E3 ubiquitin ligases of the N-end rule pathway. UBR1 is part of the ubiquitin-dependent proteolytic pathway whose substrates include proteins with destabilizing N-terminal residues. The UBR1-dependent mechanism that causes pancreatic damage in JBS is unknown and was studied here. Methods: Ubr-1-deficient mice generated by targeted disruption of the Ubr-1 gene (Mol Cell Biol. 2001; 21:8007-21) were obtained from Y.T. Kwon, Univ. of Pittburgh, USA. Acute pancreatitis was induced by caerulein (50 μg/h, i.p.). Proteome analysis of pancreatic tissue from caerulein treated WT and UBR-1 knock-out animals was performed to identify disease associated targets and those candidates were further characterised in functional assays. Results: Proteome analysis in UBR-1-ko and WT animals after caerulein treatment with a focus on destabilizing N-terminal residues revealed a significant accumulation of pancreatic proteases such as chymotrypsin B, anionic trypsin and pancreatic elastase. Furthermore, we found an up-regulation of ER-stress proteins and inflammation related proteins. Phenotypic characterisation of pancreatitis after 8h revealed significantly increased lipase levels in UBR-1-ko animals, a significantly increased histology score (WT 2.04+/−0,78; UBR-1 ko 2.79 +/−0.72, p = 0.023) and significantly increased elastase activity 8h after the onset of pancreatitis. In isolated pancreatic acini we found a significant increase in intracellular elastase activation upon supramaximal CCK stimulation in UBR-1 ko animals associated with a significant rise in the rate of necrosis. Conclusion: Experimental pancreatic injury in UBR-1 ko-animals was followed by significantly greater local and systemic inflammation suggesting a vital function of UBR1 in the defense against pathologic damage - and JBS as an inflammatory disorder due to an inadequate UBR1 defense. Proteom analysis and functional data from isolated pancreatic acini showed an accumulation of activated proteases and hereby support a crucial role of proteases as underlying pathomechanism for the pancreatic phenotype in JBS.