Chronic pancreatitis is a persistent inflammatory disease of the pancreas. The digestive protease trypsin plays a fundamental role in the pathogenesis. Here we analyzed the gene encoding the trypsindegrading enzyme chymotrypsin C (CTRC) in German subjects with idiopathic or hereditary chronic pancreatitis. Two alterations, p.R254W and p.K247_R254del, were significantly overrepresented in the pancreatitis group and were present in 30/901 (3.3%) affected individuals but only in 21/2,804 (0.7%) controls (OR=4.6; CI=2.6−8.0; P=1.3×10 −7 ). A replication study identified these two variants in 10/348 (2.9%) individuals with alcoholic chronic pancreatitis but only in 3/432 (0.7%) subjects with alcoholic liver disease (OR=4.2; CI=1.2−15.5; P=0.02). CTRC variants were also found in 10/71 (14.1%) Indian subjects with tropical pancreatitis but only in 1/84 (1.2%) control (OR=13.6; CI=1.7 −109.2; P=0.0028). Functional analysis of the CTRC variants revealed impaired activity and/or reduced secretion. The results indicate that loss-of-function alterations in CTRC predispose to pancreatitis by diminishing its protective trypsin-degrading activity.Chronic pancreatitis is a continuing inflammatory disorder characterized by permanent destruction of the pancreatic parenchyma leading to maldigestion and diabetes mellitus due to exocrine and endocrine insufficiency. Penetrating insight into the pathomechanism came from relatively recent studies investigating the genes encoding cationic trypsinogen (PRSS1; OMIM 276000), anionic trypsinogen (PRSS2; OMIM 601564), and the pancreatic secretory trypsin inhibitor (SPINK1; OMIM 167790). Gain-of-function variants in PRSS1 have been linked to autosomal dominant hereditary pancreatitis and subsequently also to idiopathic chronic pancreatitis 1-4 . Recently, triplication of the PRSS1 locus has been observed in a subset of families with hereditary pancreatitis 5 . In vitro biochemical studies revealed that the majority of disease predisposing PRSS1 variants increase autocatalytic conversion of trypsinogen to active trypsin and probably promote premature intrapancreatic trypsin activation in vivo 6,7 . Consistent with the central pathophysiological role of trypsin, p.N34S and other loss-offunction alterations in the trypsin inhibitor SPINK1 predispose to idiopathic, tropical, and alcoholic chronic pancreatitis 8-15 . In contrast to pathogenic PRSS1 and SPINK1 variations, the p.G191R PRSS2 variant affords protection against chronic pancreatitis due to rapid autodegradation 16 . Taken together, genetic and biochemical evidence defines a pathological pathway in which a sustained imbalance between intrapancreatic trypsinogen activation and trypsin inactivation results in the development of chronic pancreatitis ( Supplementary Fig. 1).Because trypsin degradation serves as a protective mechanism against pancreatitis, we hypothesized that loss of function in trypsin degrading enzymes increases the risk for pancreatitis. We recently demonstrated that chymotrypsin C (CTRC) degrades all human tryps...

1 The abbreviations used are: SBTI, soybean trypsin inhibitor; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

Chronic pancreatitis is an inflammatory disorder of the pancreas. We analyzed CPA1 encoding carboxypeptidase A1 in subjects with non-alcoholic chronic pancreatitis and controls in a German discovery cohort and three replication cohorts. Functionally impaired variants were present in 29/944 (3.1%) German patients and in 5/3,938 (0.1%) controls (odds ratio [OR] = 24.9; P = 1.5 × 10-16). The association was strongest in subjects aged ≤10 years (9.7%; OR = 84.0; P = 4.1 × 10-24). In the replication cohorts, defective CPA1 variants were observed in 8/600 (1.3%) patients and in 9/2,432 (0.4%) controls from Europe (P = 0.01), in 5/230 (2.2%) patients and 0/264 controls from India (P = 0.02), and in 5/247 (2.0%) patients but 0/341 controls from Japan (P = 0.013). The mechanism of increased pancreatitis risk by CPA1 variants may involve misfolding-induced endoplasmic reticulum stress rather than elevated trypsin activity as seen with other genetic risk factors.

Digestive trypsins undergo proteolytic breakdown during their transit in the human alimentary tract, which has been assumed to occur through trypsin-mediated cleavages, termed autolysis. Autolysis was also postulated to play a protective role against pancreatitis by eliminating prematurely activated intrapancreatic trypsin. However, autolysis of human cationic trypsin is very slow in vitro, which is inconsistent with the documented intestinal trypsin degradation or a putative protective role. Here we report that degradation of human cationic trypsin is triggered by chymotrypsin C, which selectively cleaves the Leu 81 -Glu 82 peptide bond within the Ca 2؉ binding loop. Further degradation and inactivation of cationic trypsin is then achieved through tryptic cleavage of the Arg 122 -Val 123 peptide bond. Consequently, mutation of either Leu 81 or Arg 122 blocks chymotrypsin C-mediated trypsin degradation. Calcium affords protection against chymotrypsin C-mediated cleavage, with complete stabilization observed at 1 mM concentration. Chymotrypsin C is highly specific in promoting trypsin degradation, because chymotrypsin B1, chymotrypsin B2, elastase 2A, elastase 3A, or elastase 3B are ineffective. Chymotrypsin C also rapidly degrades all three human trypsinogen isoforms and appears identical to enzyme Y, the enigmatic trypsinogen-degrading activity described by Heinrich Rinderknecht in 1988. Taken together with previous observations, the results identify chymotrypsin C as a key regulator of activation and degradation of cationic trypsin. Thus, in the high Ca 2؉ environment of the duodenum, chymotrypsin C facilitates trypsinogen activation, whereas in the lower intestines, chymotrypsin C promotes trypsin degradation as a function of decreasing luminal Ca 2؉ concentrations. chronic pancreatitis ͉ digestive enzymes ͉ trypsinogen degradation

We investigated the biochemical properties and cellular expression of the c.346C>T (p.R116C) human cationic trypsinogen (PRSS1) mutant, which we identified in a German family with autosomal dominant hereditary pancreatitis. This mutation leads to an unpaired Cys residue with the potential to interfere with protein folding via incorrect disulfide bond formation. Recombinantly expressed p.R116C trypsinogen exhibited a tendency for misfolding in vitro. Biochemical analysis of the correctly folded, purified p.R116C mutant revealed unchanged activation and degradation characteristics compared to wild type trypsinogen. Secretion of mutant p.R116C from transfected 293T cells was reduced to ~20% of wild type. A similar secretion defect was observed with another rare PRSS1 variant, p.C139S, whereas mutants p.A16V, p.N29I, p.N29T, p.E79K, p.R122C, and p.R122H were secreted normally. All mutants were detected in cell extracts at comparable levels but a large portion of mutant p.R116C was present in an insoluble, protease-sensitive form. Consistent with intracellular retention of misfolded trypsinogen, the endoplasmic reticulum (ER) stress markers BiP and XBP1s were elevated in cells expressing mutant p.R116C. The results indicate that mutation induced misfolding and intracellular retention of human cationic trypsinogen causes hereditary pancreatitis in carriers of the p.R116C mutation. ER stress triggered by trypsinogen misfolding represents a new potential disease mechanism for chronic pancreatitis.

Objective-Chronic pancreatitis is a progressive inflammatory disorder of the pancreas characterized by permanent destruction of acinar cells. Mutations in the chymotrypsinogen C (CTRC) gene have been linked to the development of chronic pancreatitis. The aim of the present study was to explore whether CTRC mutants induce endoplasmic reticulum (ER) stress in pancreatic acinar cells.Design-Dexamethasone-differentiated AR42J rat acinar cells and freshly isolated mouse acini were transfected with recombinant adenovirus carrying wild type CTRC or the p.A73T pancreatitisassociated mutant. ER stress markers were assessed by reverse transcription-PCR and western blotting. Apoptosis was characterized by caspase-3/7 activity and the TUNEL assay.Results-Acinar cells transfected with the p.A73T mutant, but not those with wild-type CTRC, developed significant ER stress, as judged by elevated mRNA and protein levels of the ER chaperone immunoglobulin-binding protein (BiP), increased splicing of the X-box binding protein-1 (XBP1) mRNA and marked induction of the transcription factor C/EBP-homologous protein (CHOP), a mediator of ER stress-associated apoptosis. Consistent with higher CHOP expression, AR42J cells expressing the p.A73T mutant became detached over time and showed considerably increased caspase-3/7 activity and TUNEL staining.Conclusions-Pancreatitis-associated CTRC mutations can markedly increase the propensity of chymotrypsinogen C to elicit ER stress in pancreatic acinar cells. Thus, carriers of CTRC mutations may be at a higher risk of developing ER stress in the exocrine pancreas, which may contribute to parenchymal damage through acinar cell apoptosis.Keywords chronic pancreatitis; protein misfolding; endoplasmic reticulum stress; unfolded protein response; apoptosis Chronic pancreatitis is a persistent inflammatory disorder characterized by destruction of the pancreatic parenchyma, maldigestion, chronic pain and diabetes mellitus.[1] Identification of genetic risk factors and elucidation of their mechanism of action has allowed the formulation *Correspondence to Miklós Sahin-Tóth, 72 East Concord Street, Evans-433; Boston, MA 02118; Tel: (617) 414-1070; Fax: (617) 414-1041; miklos@bu.edu . Competing interest: None to declare. COPYRIGHT LICENCE STATEMENT"The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd, and its Licensees to permit this article (if accepted) to be published in GUT and any other BMJPGL products and to exploit all subsidiary rights, as set out in our licence." NIH Public Access Author ManuscriptGut. Author manuscript; available in PMC 2010 September 1. The trypsin-dependent disease model described above assumes that gain or loss of catalytic activity of the participant proteins is critical in disease pathogenesis. We hypothesized; however, that mutations in digestive enzymes might increase the risk of ch...

Human digestive carboxypeptidases CPA1, CPA2, and CPB1 are secreted by the pancreas as inactive proenzymes containing a 94 -96-amino acid-long propeptide. Activation of procarboxypeptidases is initiated by proteolytic cleavage at the C-terminal end of the propeptide by trypsin. Here, we demonstrate that subsequent cleavage of the propeptide by chymotrypsin C (CTRC) induces a nearly 10-fold increase in the activity of trypsin-activated CPA1 and CPA2, whereas CPB1 activity is unaffected. Other human pancreatic proteases such as chymotrypsin B1, chymotrypsin B2, chymotrypsin-like enzyme-1, elastase 2A, elastase 3A, or elastase 3B are inactive or markedly less effective at promoting procarboxypeptidase activation. On the basis of these observations, we propose that CTRC is a physiological co-activator of proCPA1 and proCPA2. Furthermore, the results confirm and extend the notion that CTRC is a key regulator of digestive zymogen activation.

A heveny hasnyálmirigy-gyulladás, mint az egyik leggyakoribb akut emésztőszervi kórkép, amelyet szignifi káns morbiditás és mortalitás jellemez, korszerű és bizonyítékokon alapuló kezelési útmutatót igényel. A Magyar Hasnyálmirigy Munkacsoport célul tűzte ki, hogy a jelenleg elérhető nemzetközi irányvonalakat, illetve evidenciákat alapul véve az akut pancreatitis konzervatív és sebészi kezelésének kulcskérdései vonatkozásában bizonyítékalapú irányelveket fogalmazzon meg. A Magyar Hasnyálmirigy Munkacsoport által kijelölt előkészítő és konzulens munkacsoport lefordította, és ahol szükségesnek találta, kiegészítette és/vagy módosította a nemzetközi irányelveket. Összesen 11 témakörben (Diagnózis és etiológia, Prognózis, Képalkotók, Folyadékterápia, Intenzív kezelés, Fertőzéses szövődmények megelő-zése, Táplálás, Epeúti beavatkozások, Endoszkópos retrográd cholangiopancreatographiát követő pancreatitis, Intervenció indikációi, időzítése és stratégiája nekrotizáló pancreatitisben, Cholecystectomia [vagy endoszkópos sphincterotomia] időzítése) 42 releváns kérdést állított össze. Az evidencia osztályozását az UpToDate ® rendszere alapján határozta meg. Az irányelvek a 2014. szeptember 12-ei konszenzustalálkozón kerültek bemutatásra és megvitatásra. A résztvevők 25 kérdést csaknem teljes egyetértéssel (95% felett), míg 17 kérdést erős egyetértéssel (70% felett) fogadtak el. A Magyar Hasnyálmirigy Munkacsoport jelen kezelési irányelve az első, bizonyítékon alapuló akut pancreatitiskezelési útmutató hazánkban. Az irányelv komoly segítséget nyújt az akut pancreatitis oktatásához, a mindennapi betegellátáshoz és a megfelelő fi nanszírozás kialakításához, ezért a szerzők bíznak abban, hogy ezen irányelvek minél széle-sebb körben alapreferenciaként szolgálnak majd majd Magyarországon. Orv. Hetil., 2015, 156(7), 244-261.Kulcsszavak: bizonyítékon alapuló orvoslás, kezelési irányelvek, akut pancreatitis Acute pancreatitis Evidence based management guidelines of the Hungarian Pancreatic Study Group 2014Acute pancreatitis is one of the most common diseases of the gastrointestinal tract associated with signifi cant morbidity and mortality that requires up-to-date and evidence based treatment guidelines. The Hungarian Pancreatic Study Group proposed to prepare evidence based guideline for the medical and surgical management of acute pancreatitis