{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T23:24:14Z","timestamp":1781652254915,"version":"3.54.5"},"reference-count":36,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2023,8,2]],"date-time":"2023-08-02T00:00:00Z","timestamp":1690934400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Russian Foundation for Basic Research","award":["20-54-56044 Iran_T"],"award-info":[{"award-number":["20-54-56044 Iran_T"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Roughly 1% of the global population is susceptible to celiac disease (CD)\u2014inheritable autoimmune inflammation of the small intestine caused by intolerance to gliadin proteins present in wheat, rye, and barley grains, and called gluten in wheat. Classical treatment is a life-long gluten-free diet, which is constraining and costly. An alternative approach is based upon the development and oral reception of effective peptidases that degrade in the stomach immunogenic proline- and glutamine-rich gliadin peptides, which are the cause of the severe reaction in the intestine. In previous research, we have established that the major digestive peptidase of an insect Tribolium castaneum\u2014cathepsin L\u2014hydrolyzes immunogenic prolamins after Gln residues but is unstable in the extremely acidic environment (pH 2\u20134) of the human stomach and cannot be used as a digestive aid. In this work, using molecular dynamics simulations, we discover the probable cause of the pH instability of cathepsin L\u2014loss of the catalytically competent rotameric state of one of the active site residues, His 275. To \u201cfix\u201d the correct orientation of this residue, we designed a V277A mutant variant, which extends the range of stability of the peptidase in the acidic environment while retaining most of its activity. We suggest this protein as a lead glutenase for the development of oral medical preparation that fights CD and gluten intolerance in susceptible people.<\/jats:p>","DOI":"10.3390\/ijms241512369","type":"journal-article","created":{"date-parts":[[2023,8,2]],"date-time":"2023-08-02T11:04:38Z","timestamp":1690974278000},"page":"12369","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Fighting Celiac Disease: Improvement of pH Stability of Cathepsin L In Vitro by Computational Design"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1331-3949","authenticated-orcid":false,"given":"Anton O.","family":"Chugunov","sequence":"first","affiliation":[{"name":"M.M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia"},{"name":"L.D. Landau School of Physics, Moscow Institute of Physics and Technology (State University), 141701 Dolgoprudny, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elena A.","family":"Dvoryakova","sequence":"additional","affiliation":[{"name":"A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria A.","family":"Dyuzheva","sequence":"additional","affiliation":[{"name":"M.M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia"},{"name":"Higher Chemical College of the Russian Academy of Sciences, D. Mendeleev University of Chemical Technology, 125047 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tatyana R.","family":"Simonyan","sequence":"additional","affiliation":[{"name":"Department of Chemistry, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Valeria F.","family":"Tereshchenkova","sequence":"additional","affiliation":[{"name":"Department of Chemistry, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Irina Yu.","family":"Filippova","sequence":"additional","affiliation":[{"name":"Department of Chemistry, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5474-4721","authenticated-orcid":false,"given":"Roman G.","family":"Efremov","sequence":"additional","affiliation":[{"name":"M.M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia"},{"name":"L.D. Landau School of Physics, Moscow Institute of Physics and Technology (State University), 141701 Dolgoprudny, Russia"},{"name":"Department of Applied Mathematics, National Research University Higher School of Economics, 101000 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4988-4842","authenticated-orcid":false,"given":"Elena N.","family":"Elpidina","sequence":"additional","affiliation":[{"name":"A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,8,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2413","DOI":"10.1016\/S0140-6736(22)00794-2","article-title":"Coeliac Disease","volume":"399","author":"Catassi","year":"2022","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"h4347","DOI":"10.1136\/bmj.h4347","article-title":"Celiac Disease and Non-Celiac Gluten Sensitivity","volume":"351","author":"Lebwohl","year":"2015","journal-title":"BMJ"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1631","DOI":"10.12688\/f1000research.15849.1","article-title":"Recent Advances in Understanding Non-Celiac Gluten Sensitivity","volume":"7","author":"Barbaro","year":"2018","journal-title":"F1000Research"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Dunaevsky, Y.E., Tereshchenkova, V.F., Belozersky, M.A., Filippova, I.Y., Oppert, B., and Elpidina, E.N. (2021). Effective Degradation of Gluten and Its Fragments by Gluten-Specific Peptidases: A Review on Application for the Treatment of Patients with Gluten Sensitivity. Pharmaceutics, 13.","DOI":"10.3390\/pharmaceutics13101603"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Wei, G., Helmerhorst, E.J., Darwish, G., Blumenkranz, G., and Schuppan, D. (2020). Gluten Degrading Enzymes for Treatment of Celiac Disease. Nutrients, 12.","DOI":"10.3390\/nu12072095"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"7505","DOI":"10.1038\/s41598-019-43837-9","article-title":"Pharmaceutically Modified Subtilisins Withstand Acidic Conditions and Effectively Degrade Gluten in Vivo","volume":"9","author":"Darwish","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"20513","DOI":"10.1021\/ja3094795","article-title":"Computational Design of an \u03b1-Gliadin Peptidase","volume":"134","author":"Gordon","year":"2012","journal-title":"J. Am. Chem. Soc."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"13106","DOI":"10.1021\/jacs.5b08325","article-title":"Engineering of Kuma030: A Gliadin Peptidase That Rapidly Degrades Immunogenic Gliadin Peptides in Gastric Conditions","volume":"137","author":"Wolf","year":"2015","journal-title":"J. Am. Chem. Soc."},{"key":"ref_9","unstructured":"(2023, June 22). CTG Labs\u2014NCBI, Available online: https:\/\/www.clinicaltrials.gov\/study\/NCT03701555."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"103679","DOI":"10.1016\/j.ibmb.2021.103679","article-title":"Primary Digestive Cathepsins L of Tribolium castaneum Larvae: Proteomic Identification, Properties, Comparison with Human Lysosomal Cathepsin L","volume":"140","author":"Dvoryakova","year":"2022","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Dvoryakova, E.A., Klimova, M.A., Simonyan, T.R., Dombrovsky, I.A., Serebryakova, M.V., Tereshchenkova, V.F., Dunaevsky, Y.E., Belozersky, M.A., Filippova, I.Y., and Elpidina, E.N. (2022). Recombinant Cathepsin L of Tribolium castaneum and Its Potential in the Hydrolysis of Immunogenic Gliadin Peptides. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23137001"},{"key":"ref_12","unstructured":"Helmenstine, A.M. (2023, June 23). What Is the pH of the Stomach?. Available online: https:\/\/www.thoughtco.com\/ph-of-the-stomach-608195."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"6148","DOI":"10.1021\/acs.jctc.2c00516","article-title":"Scalable constant pH molecular dynamics in GROMACS","volume":"18","author":"Aho","year":"2022","journal-title":"J. Chem. Theory Comp."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2414","DOI":"10.1063\/1.472109","article-title":"\u03bb-dynamics: A new approach to free energy calculations","volume":"105","author":"Kong","year":"1996","journal-title":"J. Chem. Phys."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"9968","DOI":"10.1021\/bi9705974","article-title":"A classical enzyme active center motif lacks catalytic competence until modulated electrostatically","volume":"36","author":"Pinitglang","year":"1997","journal-title":"Biochemistry"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1080\/15216540211468","article-title":"Structural basis of perturbed pKa values of catalytic groups in enzyme active sites","volume":"53","author":"Harris","year":"2002","journal-title":"IUBMB life"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2170","DOI":"10.1021\/acs.jcim.3c00004","article-title":"Benchmarking In Silico Tools for Cysteine pKa Prediction","volume":"63","author":"Golosov","year":"2023","journal-title":"J. Chem. Inf. Model."},{"key":"ref_18","unstructured":"Rawlings, N.D., and Barrett, A.J. (2013). Handbook of Proteolytic Enzymes, Academic Press."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly Accurate Protein Structure Prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"W296","DOI":"10.1093\/nar\/gky427","article-title":"SWISS-MODEL: Homology Modelling of Protein Structures and Complexes","volume":"46","author":"Waterhouse","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"655","DOI":"10.1016\/j.ibmb.2012.04.010","article-title":"The 3D Structure and Function of Digestive Cathepsin L-like Proteinases of Tenebrio molitor Larval Midgut","volume":"42","author":"Beton","year":"2012","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1016\/S0076-6879(03)74020-8","article-title":"Modeller: Generation and Refinement of Homology-Based Protein Structure Models","volume":"374","author":"Fiser","year":"2003","journal-title":"Methods Enzymol."},{"key":"ref_23","unstructured":"(2023). The PyMOL Molecular Graphics System, Schr\u00f6dinger, LLC. Version 2.0."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"GROMACS: High Performance Molecular Simulations through Multi-Level Parallelism from Laptops to Supercomputers","volume":"1\u20132","author":"Abraham","year":"2015","journal-title":"SoftwareX"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2135","DOI":"10.1002\/jcc.23354","article-title":"CHARMM36 All-Atom Additive Protein Force Field: Validation Based on Comparison to NMR Data","volume":"34","author":"Huang","year":"2013","journal-title":"J. Comput. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1950","DOI":"10.1002\/prot.22711","article-title":"Improved Side-Chain Torsion Potentials for the Amber ff99SB Protein Force Field","volume":"78","author":"Piana","year":"2010","journal-title":"Proteins"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"6269","DOI":"10.1021\/j100308a038","article-title":"The Missing Term in Effective Pair Potentials","volume":"91","author":"Berendsen","year":"1987","journal-title":"J. Phys. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"9050","DOI":"10.1063\/1.466711","article-title":"Finite Representation of an Infinite Bulk System: Solvent Boundary Potential for Computer Simulations","volume":"100","author":"Beglov","year":"1994","journal-title":"J. Chem. Phys."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"014101","DOI":"10.1063\/1.2408420","article-title":"Canonical Sampling through Velocity Rescaling","volume":"126","author":"Bussi","year":"2007","journal-title":"J. Chem. Phys."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"7182","DOI":"10.1063\/1.328693","article-title":"Polymorphic Transitions in Single Crystals: A New Molecular Dynamics Method","volume":"52","author":"Parrinello","year":"1981","journal-title":"J. Appl. Phys."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3684","DOI":"10.1063\/1.448118","article-title":"Molecular dynamics with coupling to an external bath","volume":"81","author":"Berendsen","year":"1984","journal-title":"J. Chem. Phys."},{"key":"ref_32","unstructured":"(2015). QuikChange II XL Site-Directed Mutagenesis Kit; Catalog #200521 and #200522, Agilent Technologies Inc."},{"key":"ref_33","first-page":"403","article-title":"Tampone Universale Di Britton E Robinson a Forza Ionica Costante","volume":"87","author":"Frugoni","year":"1957","journal-title":"Gazz. Chim. Ital."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1016\/j.cbpb.2006.05.004","article-title":"Fractionation of Digestive Proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) Larvae and Role in Protein Digestion","volume":"145","author":"Vinokurov","year":"2006","journal-title":"Comp. Biochem. Physiol. B Biochem. Mol. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"578758","DOI":"10.3389\/fmolb.2020.578758","article-title":"New Glutamine-Containing Substrates for the Assay of Cysteine Peptidases from the C1 Papain Family","volume":"7","author":"Filippova","year":"2020","journal-title":"Front Mol Biosci"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/j.ab.2013.12.032","article-title":"Selective Chromogenic and Fluorogenic Peptide Substrates for the Assay of Cysteine Peptidases in Complex Mixtures","volume":"449","author":"Semashko","year":"2014","journal-title":"Anal. Biochem."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/15\/12369\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:24:41Z","timestamp":1760127881000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/15\/12369"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,8,2]]},"references-count":36,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2023,8]]}},"alternative-id":["ijms241512369"],"URL":"https:\/\/doi.org\/10.3390\/ijms241512369","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,8,2]]}}}