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Simultaneously, prematurely activated trypsin is inhibited by the serine protease inhibitor Kazal-type 1 (SPINK1) or can be degraded and cleared by autoproteolysis. Enterokinase, which activates trypsinogen by cleavage of the propeptide, is spatially restricted to the duodenum. Premature autoactivation is prevented by expression as zymogens with the propeptide inhibiting trypsin. 4 In order to prevent pancreatitis, the tryptic activity within zymogen granules of pancreatic acinar cells has to be tightly regulated. 1–3 The underlying mechanisms for the development of the disease are diverse and are primarily caused by alcohol abuse or genetic risk factors but can also include smoking, autoimmunity and pancreatic injury. Keywords: pancreas, pancreatitis, serine protease, crystal structure, autoproteolysis, R122HĬhronic pancreatitis (CP) is a progressive inflammatory disorder of the pancreas that leads to fibrosis and exocrine and endocrine insufficiencies. A proposed autoinhibition mode was confirmed and the structural basis of the autoproteolytic failsafe mechanism elucidated. The TRY2–TRY2 dimer confirms a previously hypothesized autoinhibitory state with an unexpectedly large binding interface.Ĭonclusion: We provide a structure of TRY2, which is the predominant trypsin isoform in chronic pancreatitis and pancreatic cancer. The crystal structure of TRY2 reveals that the enzyme crystallized in the autoproteolytic state with Arg122 placed in the S1 binding pocket and the corresponding loop cleaved. Results: All trypsin isoforms display similar kinetic properties. Equilibration molecular dynamics simulations were used to generate the corresponding TRY1–TRY1 model. The structure was solved by molecular replacement and refined to a resolution of 1.7 Å. Enzymatic activities of all trypsin isoforms were determined and crystals of TRY2 were grown using the vapor-diffusion method. Methods: Trypsin isoforms were recombinantly expressed in E. Here, we aim to elucidate the molecular structure of human anionic trypsin and obtain insights into the autoproteolytic regulation of tryptic activity. However, exacerbated pancreatitis in TRY2 overexpressing mice was recently demonstrated. The involvement of TRY2 in pancreatitis is considered limited due to the absence of disease-causing mutations and its increased prevalence for autoproteolysis. This ratio is reversed during chronic alcohol abuse, pancreatic cancer, or pancreatitis due to selectively upregulated expression of TRY2, causing anionic trypsin to become the predominant isoform. Typically, they exist in a molar ratio of 2:1 (cationic:anionic). Human cationic (TRY1) and anionic (TRY2) trypsins are the two major trypsin isoforms and their activities are tightly regulated within pancreatic acinar cells. Objective: The pathophysiological mechanisms underlying chronic pancreatitis (CP) are still poorly understood. Felix Nagel, 1 Anne Susemihl, 1, 2 Norman Geist, 1 Kevin Möhlis, 1, 3 Gottfried J Palm, 4 Michael Lammers, 4 Mihaela Delcea 1ġBiophysical Chemistry, Institute of Biochemistry, University of Greifswald, Greifswald, Germany 2Department of Hematology and Oncology, Internal Medicine C, University of Greifswald, Greifswald, Germany 3Helmholtz Institute for Metabolic, Obesity and Vascular Research, Leipzig, Germany 4Synthetic and Structural Biochemistry, Institute of Biochemistry, University of Greifswald, Greifswald, GermanyĬorrespondence: Mihaela Delcea, Biophysical Chemistry, Institute of Biochemistry, University of Greifswald, Greifswald, Germany, Tel +49 38, Fax +49 38, Email