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Structural Basis of the Pancreatitis-Associated Autoproteolytic Failsafe Mechanism in Human Anionic Trypsin

OBJECTIVE: The pathophysiological mechanisms underlying chronic pancreatitis (CP) are still poorly understood. Human cationic (TRY1) and anionic (TRY2) trypsins are the two major trypsin isoforms and their activities are tightly regulated within pancreatic acinar cells. Typically, they exist in a mo...

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Autores principales: Nagel, Felix, Susemihl, Anne, Geist, Norman, Möhlis, Kevin, Palm, Gottfried J, Lammers, Michael, Delcea, Mihaela
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Dove 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9239388/
https://www.ncbi.nlm.nih.gov/pubmed/35775010
http://dx.doi.org/10.2147/JIR.S367699
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author Nagel, Felix
Susemihl, Anne
Geist, Norman
Möhlis, Kevin
Palm, Gottfried J
Lammers, Michael
Delcea, Mihaela
author_facet Nagel, Felix
Susemihl, Anne
Geist, Norman
Möhlis, Kevin
Palm, Gottfried J
Lammers, Michael
Delcea, Mihaela
author_sort Nagel, Felix
collection PubMed
description OBJECTIVE: The pathophysiological mechanisms underlying chronic pancreatitis (CP) are still poorly understood. Human cationic (TRY1) and anionic (TRY2) trypsins are the two major trypsin isoforms and their activities are tightly regulated within pancreatic acinar cells. Typically, they exist in a molar ratio of 2:1 (cationic:anionic). 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. The involvement of TRY2 in pancreatitis is considered limited due to the absence of disease-causing mutations and its increased prevalence for autoproteolysis. However, exacerbated pancreatitis in TRY2 overexpressing mice was recently demonstrated. Here, we aim to elucidate the molecular structure of human anionic trypsin and obtain insights into the autoproteolytic regulation of tryptic activity. METHODS: Trypsin isoforms were recombinantly expressed in E. coli, purified and refolded. Enzymatic activities of all trypsin isoforms were determined and crystals of TRY2 were grown using the vapor-diffusion method. The structure was solved by molecular replacement and refined to a resolution of 1.7 Å. Equilibration molecular dynamics simulations were used to generate the corresponding TRY1–TRY1 model. RESULTS: All trypsin isoforms display similar kinetic properties. 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. The TRY2–TRY2 dimer confirms a previously hypothesized autoinhibitory state with an unexpectedly large binding interface. CONCLUSION: We provide a structure of TRY2, which is the predominant trypsin isoform in chronic pancreatitis and pancreatic cancer. A proposed autoinhibition mode was confirmed and the structural basis of the autoproteolytic failsafe mechanism elucidated.
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spelling pubmed-92393882022-06-29 Structural Basis of the Pancreatitis-Associated Autoproteolytic Failsafe Mechanism in Human Anionic Trypsin Nagel, Felix Susemihl, Anne Geist, Norman Möhlis, Kevin Palm, Gottfried J Lammers, Michael Delcea, Mihaela J Inflamm Res Original Research OBJECTIVE: The pathophysiological mechanisms underlying chronic pancreatitis (CP) are still poorly understood. Human cationic (TRY1) and anionic (TRY2) trypsins are the two major trypsin isoforms and their activities are tightly regulated within pancreatic acinar cells. Typically, they exist in a molar ratio of 2:1 (cationic:anionic). 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. The involvement of TRY2 in pancreatitis is considered limited due to the absence of disease-causing mutations and its increased prevalence for autoproteolysis. However, exacerbated pancreatitis in TRY2 overexpressing mice was recently demonstrated. Here, we aim to elucidate the molecular structure of human anionic trypsin and obtain insights into the autoproteolytic regulation of tryptic activity. METHODS: Trypsin isoforms were recombinantly expressed in E. coli, purified and refolded. Enzymatic activities of all trypsin isoforms were determined and crystals of TRY2 were grown using the vapor-diffusion method. The structure was solved by molecular replacement and refined to a resolution of 1.7 Å. Equilibration molecular dynamics simulations were used to generate the corresponding TRY1–TRY1 model. RESULTS: All trypsin isoforms display similar kinetic properties. 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. The TRY2–TRY2 dimer confirms a previously hypothesized autoinhibitory state with an unexpectedly large binding interface. CONCLUSION: We provide a structure of TRY2, which is the predominant trypsin isoform in chronic pancreatitis and pancreatic cancer. A proposed autoinhibition mode was confirmed and the structural basis of the autoproteolytic failsafe mechanism elucidated. Dove 2022-06-24 /pmc/articles/PMC9239388/ /pubmed/35775010 http://dx.doi.org/10.2147/JIR.S367699 Text en © 2022 Nagel et al. https://creativecommons.org/licenses/by-nc/3.0/This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/ (https://creativecommons.org/licenses/by-nc/3.0/) ). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php).
spellingShingle Original Research
Nagel, Felix
Susemihl, Anne
Geist, Norman
Möhlis, Kevin
Palm, Gottfried J
Lammers, Michael
Delcea, Mihaela
Structural Basis of the Pancreatitis-Associated Autoproteolytic Failsafe Mechanism in Human Anionic Trypsin
title Structural Basis of the Pancreatitis-Associated Autoproteolytic Failsafe Mechanism in Human Anionic Trypsin
title_full Structural Basis of the Pancreatitis-Associated Autoproteolytic Failsafe Mechanism in Human Anionic Trypsin
title_fullStr Structural Basis of the Pancreatitis-Associated Autoproteolytic Failsafe Mechanism in Human Anionic Trypsin
title_full_unstemmed Structural Basis of the Pancreatitis-Associated Autoproteolytic Failsafe Mechanism in Human Anionic Trypsin
title_short Structural Basis of the Pancreatitis-Associated Autoproteolytic Failsafe Mechanism in Human Anionic Trypsin
title_sort structural basis of the pancreatitis-associated autoproteolytic failsafe mechanism in human anionic trypsin
topic Original Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9239388/
https://www.ncbi.nlm.nih.gov/pubmed/35775010
http://dx.doi.org/10.2147/JIR.S367699
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