Summary
Within the last years it was proven that stress of the endoplasmatic reticulum (ER-stress) can play a role in the development of several diseases. ER-stress is caused by deficient production or misfolding of proteins followed by their accumulation in the cell. To prevent cell damages these "stored" proteins need to be degraded or disposed, which is done by specific ER-stress disposal mechanisms of the cell. Errors in protein folding or in the ER-stress disposal mechanisms have been confirmed for some hereditary storage diseases like for example liver diseases and forms of pancreatitis (inflammation of the pancreas). Permanent ER-stress will lead to the development of local inflammation, tissue decline and to chronic organ damages.
The project uniquely combines expertise of the leading research groups in Mecklenburg-Vorpommern in the field of protein misfolding and thereby caused ER-stress, producing severe diseases of the liver and pancreas. The participating clinical scientists from Greifswald and Rostock will not only investigate the mechanisms behind hereditary liver and pancreas diseases, but at the same time will develop and test new pharmaceutic active ingredients together with partners from fundamental research. The newly developed pharmaceutic active ingredients are supposed to help preventing ER-stress or, respectively, to support or stimulate the degradation of misfolded proteins in the cell.
Besides translational and fundamental research the project strongly emphasizes the promotion of young researchers to foster their scientific careers: Within the project duration two junior research group leaders, six postdoctorates, ten Ph.D. students and 17 M.A. students and medical doctoral candidates, respectively, will be funded to reach a higher qualification level in their scientific careers.
Subproject 4 - SPINK1 and antibody formation
The main issue of this project is to investigate the protein SPINK1 (Serin Protease Inhibitor Kazal-Type 1) and its interaction with antibodies. The physiological function of SPINK1 is to inhibit the digestion enzyme trypsin within the pancreas, where trypsin is stored as an inactive premature enzyme. Otherwise the pancreas would be subject to damage by the digestive effect of trypsin and subsequent development of pancreatitis. Thus, defects of SPINK1, for example triggered by SPINK1 mutations, are associated with chronic pancreatitis (CP). Defect and misfolded SPINK1 accumulated in the endoplasmic reticulum (ER) tends to result in pancreatic diseases. Up to 25 % of CP patients show N34S SPINK1 mutation. Yet, this mutation occurs also in 2.5 % of the healthy population. Additionally, both wild type and mutant SPINK1 behave similarly concerning for instance trypsin binding and inhibitory effects. Thus, the presence of N34S SPINK1 mutation alone cannot be the cause of CP. Rather it is a risk indicator for developing CP. There must be a second trigger involved. In addition, autoantibodies against SPINK1 were found in up to 43 % of CP patients. The relationship between these factors is to be investigated in this project. For this purpose, one step is to study the structure and conformation of both the wild type SPINK1 and the N34S SPINK1 mutation. Found conformational changes in the beta sheet structure are examined in the next step concerning their relevance to antibody binding. Therefore, the binding affinity between the autoantibodies and SPINK1 is determined under various experimental conditions. This is done using single-molecule force microscopy, with which the differences between wild type and mutant can be clearly identified. This results in the possibility to develop a test to detect and quantify with much higher sensitivity than previously possible SPINK1 autoantibodies. Thus a widespread problem of clinical autoantibody tests for studies in humans could be solved and this would allow to optimize an assay for the detection of SPINK1 autoantibodies.
Publications
Nagel, F., Susemihl, A., Eulberg, T., Delcea, M., "Identification of Kazal Inhibitor Scaffolds with Identical Canonical Binding Loops and Their Effects on Binding Properties" (2023), Biochemistry, doi: 10.1021/acs.biochem.2c00573
Nagel, F., Susemihl, A., Geist, N., Möhlis, K., Palm, G. J., Lammers, M., Delcea, M., "Structural Basis of the Pancreatitis-Associated Autoproteolytic Failsafe Mechanism in Human Anionic Trypsin" (2022), Journal of Inflammation Research, doi: 10.2147/jir.s367699
Nagel, F., Palm, G. J., Geist, N., McDonnell, T. C. R., Susemihl, A., Girbardt, B., Mayerle, J., Lerch, M. M., Lammers, M., Delcea, M., "Structural and Biophysical Insights into SPINK1 Bound to Human Cationic Trypsin" (2022), International Journal of Molecular Sciences, doi: 10.3390/ijms23073468
Kulke M, Nagel F, Schulig L, Geist N, Gabor M, Mayerle J, Lerch MM, Link A, Delcea M. A Hypothesized Mechanism for Chronic Pancreatitis Caused by the N34S Mutation of Serine Protease Inhibitor Kazal-Type 1 Based on Conformational Studies. J Inflamm Res. 2021;14:2111-2119
- Lukas, J., Pospech, J., Oppermann, C., Hund, C., Iwanov, K., Pantoom, S., Petters, J., Frech, M., Seemann, S., Thiel, F-G., Modenbach, J-M., Bolsmann, R., Chama, L. d. F., Kraatz, F., El-Hage, F., Gronbach, M., Klein, A., Müller, R., Salloch, S., Weiss, F-U., Simon, P., Wagh, P., Klemenz, A., Krüger, E., Mayerle, J., Delcea, M., Kragl, U., Beller, M., Rolfs, A., Lerch, M. M., Sendler, M., "Role of endoplasmic reticulum stress and protein misfolding in disorders of the liver and pancreas" (2019), Advances in Medical Sciences, doi: 10.1016/j.advms.2019.03.004
- Buchholz, I., Nagel, F., Klein, A., Wagh, P., Mahajan, U., Greinacher, A., Lerch, M., Mayerle, J., Delcea, M., "The impact of physiological stress conditions on protein structure and trypsin inhibition of serine protease inhibitor Kazal type 1 (SPINK1) and its N34S variant" (2019), BBA, doi: 10.1016/j.bbapap.2019.140281
Achievements
- Master Thesis Award from the (German) Society for Biochemistry and Molecular Biology (Gesellschaft für Biochemie und Molekularbiologie, GBM) for Felix Nagel's Master Thesis with the subject „Kinetic and Thermodynamic Analysis of Serine Protease Interactions with the ICP associated SPINK1 N34S mutant“ (more)
Collaborators
- Prof. Markus Lerch (University Medicine Greifswald, Germany) Link
Members
Annelie Klein, Dr. Martin Kulke, Felix Nagel
M.Sc. Students: Kevin Möhlis
B.Sc. Students: Marcel Gabor