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Molecules (Basel, Switzerland) Oct 2018Trypsin is the protease of choice for protein sample digestion in proteomics. The most typical active forms are the single-chain β-trypsin and the two-chain α-trypsin,... (Review)
Review
Trypsin is the protease of choice for protein sample digestion in proteomics. The most typical active forms are the single-chain β-trypsin and the two-chain α-trypsin, which is produced by a limited autolysis of β-trypsin. An additional intra-chain split leads to pseudotrypsin (ψ-trypsin) with three chains interconnected by disulfide bonds, which can be isolated from the autolyzate by ion-exchange chromatography. Based on experimental data with artificial substrates, peptides, and protein standards, ψ-trypsin shows altered kinetic properties, thermodynamic stability and cleavage site preference (and partly also cleavage specificity) compared to the above-mentioned proteoforms. In our laboratory, we have analyzed the performance of bovine ψ-trypsin in the digestion of protein samples with a different complexity. It cleaves predominantly at the characteristic trypsin cleavage sites. However, in a comparison with common tryptic digestion, non-specific cleavages occur more frequently (mostly after the aromatic residues of Tyr and Phe) and more missed cleavages are generated. Because of the preferential cleavages after the basic residues and more developed side specificity, which is not expected to occur for the major trypsin forms (but may appear anyway because of their autolysis), ψ-trypsin produces valuable information, which is complementary in part to data based on a strictly specific trypsin digestion and thus can be unnoticed following common proteomics protocols.
Topics: Animals; Autolysis; Cattle; Enzyme Stability; Kinetics; Protein Isoforms; Proteolysis; Trypsin
PubMed: 30322187
DOI: 10.3390/molecules23102637 -
Molecules (Basel, Switzerland) Jul 2021Understanding the interaction between proteins and polyphenols is of significance to food industries. The aim of this research was to investigate the mode of aggregation...
Understanding the interaction between proteins and polyphenols is of significance to food industries. The aim of this research was to investigate the mode of aggregation for trypsin-EGCG (Epigallocatechin-3-gallate) complexes. For this, the complex was characterized by fluorescence spectroscopy, circular dichroism (CD) spectra, small-angel X-ray scattering (SAXS), and atomic force microscope (AFM) techniques. The results showed that the fluorescence intensity of trypsin-EGCG complexes decreased with increasing the concentration of EGCG, indicating that the interaction between trypsin and EGCG resulted in changes in the microenvironment around fluorescent amino acid residues. The results of CD analysis showed conformational changes in trypsin after binding with EGCG. The results from SAXS analysis showed that the addition of EGCG results in the formation of aggregates of trypsin-EGCG complexes, and increasing the concentration of EGCG resulted in larger aggregates. AFM images showed that the trypsin-EGCG complex formed aggregates of irregular ellipsoidal shapes with the size of about 200 × 400 × 200 nm, with EGCG interconnecting the trypsin particles. Overall, according to these results, it was concluded that the large aggregates of trypsin-EGCG complexes are formed from several small aggregates that are interconnected. The results of this study shed some light on the interaction between digestive enzymes and EGCG.
Topics: Catechin; Humans; Microscopy, Atomic Force; Models, Molecular; Protein Aggregates; Protein Conformation; Solutions; Spectrometry, Fluorescence; Trypsin
PubMed: 34361715
DOI: 10.3390/molecules26154567 -
JCI Insight Nov 2022Pancreatitis, the inflammatory disorder of the pancreas, has no specific therapy. Genetic, biochemical, and animal model studies revealed that trypsin plays a central...
Pancreatitis, the inflammatory disorder of the pancreas, has no specific therapy. Genetic, biochemical, and animal model studies revealed that trypsin plays a central role in the onset and progression of pancreatitis. Here, we performed biochemical and preclinical mouse experiments to offer proof of concept that orally administered dabigatran etexilate can inhibit pancreatic trypsins and shows therapeutic efficacy in trypsin-dependent pancreatitis. We found that dabigatran competitively inhibited all human and mouse trypsin isoforms (Ki range 10-79 nM) and dabigatran plasma concentrations in mice given oral dabigatran etexilate well exceeded the Ki of trypsin inhibition. In the T7K24R trypsinogen mutant mouse model, a single oral gavage of dabigatran etexilate was effective against cerulein-induced progressive pancreatitis, with a high degree of histological normalization. In contrast, spontaneous pancreatitis in T7D23A mice, which carry a more aggressive trypsinogen mutation, was not ameliorated by dabigatran etexilate, given either as daily gavages or by mixing it with solid chow. Taken together, our observations showed that benzamidine derivatives such as dabigatran are potent trypsin inhibitors and show therapeutic activity against trypsin-dependent pancreatitis in T7K24R mice. Lack of efficacy in T7D23A mice is probably related to the more severe pathology and insufficient drug concentrations in the pancreas.
Topics: Animals; Humans; Mice; Dabigatran; Disease Models, Animal; Pancreas; Pancreatitis; Trypsin; Trypsinogen
PubMed: 36136430
DOI: 10.1172/jci.insight.161145 -
Bioorganic & Medicinal Chemistry Aug 2020A range of guanidine-based pyridines, and related compounds, have been prepared (19 examples). These compounds were evaluated in relation to their competitive inhibition...
A range of guanidine-based pyridines, and related compounds, have been prepared (19 examples). These compounds were evaluated in relation to their competitive inhibition of bovine pancreatic trypsin. Results demonstrate that compounds in which the guanidinyl substituent can form an intramolecular hydrogen bond (IMHB) with the pyridinyl nitrogen atom (6a-p) are better trypsin inhibitors than their counterparts (10-13) that are unable to form an IMHB. Among the compounds 6a-p, examples containing a 5-halo substituent were, generally, found to be better trypsin inhibitors. This trend was inversely related to electronegativity, thus, 1-(5-iodopyridin-2-yl)guanidinium ion 6e (K = 0.0151 mM) was the optimum inhibitor in the 5-halo series. Amongst the isomeric methyl substituted compounds, 1-(3-methylpyridin-2-yl)guanidinium ion 6h demonstrated optimum levels of trypsin inhibition (K = 0.0140 mM). In order to rationalise the measured enzyme inhibition, selected compounds were docked with bovine and human trypsin with a view to understanding active site occupancy and taken together with the K values the order of inhibitory ability suggests that the 5-halo 2-guanidinyl pyridine inhibitors form a halogen bond with the catalytically active serine hydroxy group.
Topics: Animals; Cattle; Guanidine; Hydrogen Bonding; Molecular Docking Simulation; Pyridines; Trypsin; Trypsin Inhibitors
PubMed: 32690267
DOI: 10.1016/j.bmc.2020.115612 -
Journal of Computational Chemistry Aug 2009We have calculated the binding free energies of a series of benzamidine-like inhibitors to trypsin with a polarizable force field using both explicit and implicit...
We have calculated the binding free energies of a series of benzamidine-like inhibitors to trypsin with a polarizable force field using both explicit and implicit solvent approaches. Free energy perturbation has been performed for the ligands in bulk water and in protein complex with molecular dynamics simulations. The binding free energies calculated from explicit solvent simulations are well within the accuracy of experimental measurement and the direction of change is predicted correctly in all cases. We analyzed the molecular dipole moments of the ligands in gas, water and protein environments. Neither binding affinity nor ligand solvation free energy in bulk water shows much dependence on the molecular dipole moments of the ligands. Substitution of the aromatic or the charged group in the ligand results in considerable change in the solvation energy in bulk water and protein whereas the binding affinity varies insignificantly due to cancellation. The effect of chemical modification on ligand charge distribution is mostly local. Replacing benzene with diazine has minimal impact on the atomic multipoles at the amidinium group. We have also utilized an implicit solvent based end-state approach to evaluate the binding free energies of these inhibitors. In this approach, the polarizable multipole model combined with Poisson-Boltzmann/surface area (PMPB/SA) provides the electrostatic interaction energy and the polar solvation free energy. Overall the relative binding free energies obtained from the MM-PMPB/SA model are in good agreement with the experimental data.
Topics: Animals; Benzamidines; Cattle; Computer Simulation; Hydrogen Bonding; Ligands; Models, Molecular; Molecular Structure; Protein Binding; Solvents; Thermodynamics; Trypsin
PubMed: 19399779
DOI: 10.1002/jcc.21268 -
Pancreatology : Official Journal of the... Jun 2023Serine protease inhibitor Kazal type 1 (SPINK1) is a trypsin-selective inhibitor protein secreted by the exocrine pancreas. Loss-of-function SPINK1 mutations predispose...
Serine protease inhibitor Kazal type 1 (SPINK1) is a trypsin-selective inhibitor protein secreted by the exocrine pancreas. Loss-of-function SPINK1 mutations predispose to chronic pancreatitis through either reduced expression, secretion, or impaired trypsin inhibition. In this study, we aimed to characterize the inhibitory activity of mouse SPINK1 against cationic (T7) and anionic (T8, T9, T20) mouse trypsin isoforms. Kinetic measurements with a peptide substrate, and digestion experiments with β-casein indicated that the catalytic activity of all mouse trypsins is comparable. Human SPINK1 and its mouse ortholog inhibited mouse trypsins with comparable efficiency (K range 0.7-2.2 pM), with the sole exception of T7 trypsin, which was inhibited less effectively by the human inhibitor (K 21.9 pM). Characterization of four chronic pancreatitis-associated human SPINK1 mutations in the context of the mouse inhibitor revealed that the reactive-loop mutations R42N (human K41N) and I43M (human I42M) impaired SPINK1 binding to trypsin (K 60 nM and 47.5 pM, respectively), whereas mutations D35S (human N34S) and A56S (human P55S) had no impact on trypsin inhibition. Our results confirmed that high-affinity trypsin inhibition by SPINK1 is conserved in the mouse, and the functional consequences of human pancreatitis-associated SPINK1 mutations can be replicated in the mouse inhibitor.
Topics: Humans; Animals; Mice; Trypsin Inhibitor, Kazal Pancreatic; Trypsin; Chronic Disease; Mutation; Pancreatitis, Chronic; Protein Isoforms; Genetic Predisposition to Disease
PubMed: 37149461
DOI: 10.1016/j.pan.2023.04.043 -
American Journal of Physiology.... Mar 2014Variations in the serine protease 1 (PRSS1) gene encoding human cationic trypsinogen have been conclusively associated with autosomal dominant hereditary pancreatitis... (Review)
Review
Variations in the serine protease 1 (PRSS1) gene encoding human cationic trypsinogen have been conclusively associated with autosomal dominant hereditary pancreatitis and sporadic nonalcoholic chronic pancreatitis. Most high-penetrance PRSS1 variants increase intrapancreatic trypsin activity by stimulating trypsinogen autoactivation and/or by inhibiting chymotrypsin C-dependent trypsinogen degradation. Alternatively, some PRSS1 variants can cause trypsinogen misfolding, which results in intracellular retention and degradation with consequent endoplasmic reticulum stress. However, not all PRSS1 variants are pathogenic, and clinical relevance of rare variants is often difficult to ascertain. Here we review the PRSS1 variants published since 1996 and discuss their functional properties and role in chronic pancreatitis.
Topics: Animals; Humans; Models, Animal; Pancreatitis, Chronic; Polymorphism, Genetic; Trypsin
PubMed: 24458023
DOI: 10.1152/ajpgi.00419.2013 -
IUBMB Life Aug 2003Earlier investigations of the proteases that digest the blood meal in mosquitoes identified the midgut epithelial cells as the source of the main blood digesting... (Review)
Review
Earlier investigations of the proteases that digest the blood meal in mosquitoes identified the midgut epithelial cells as the source of the main blood digesting protease, trypsin. Surgical manipulations and cytoimmunochemical studies indicated that trypsin in the mosquito is not stored in the epithelial cells as an inactive enzyme, but is synthesized, activated and released into the gut lumen after the blood meal. Using molecular biology techniques the major trypsin genes have been sequenced and some information on the transcription factors that regulate the induction of the genes have been reported. Although all the hormone(s) and the factors that are responsible for the induction of trypsin biosynthesis are not known, the termination of trypsin biosynthesis is controlled by a decapeptide hormone; trypsin modulating oostatic factor (TMOF) that is secreted by the mosquito ovary. Genetic engineering and expression of TMOF in bacteria, yeast and algae is emerging as a potential future larval control of mosquitoes in the field.
Topics: Aedes; Animals; Anopheles; Biological Transport; Digestive System; Endopeptidases; Female; Genetic Engineering; Juvenile Hormones; Models, Biological; Models, Molecular; Neurons; Oligopeptides; Osmosis; Trypsin
PubMed: 14609198
DOI: 10.1080/15216540310001597721 -
BioMed Research International 2017Protease encapsulation and its targeted release in thrombi may contribute to the reduction of haemorrhagic complications of thrombolysis. We aimed to prepare sterically...
Protease encapsulation and its targeted release in thrombi may contribute to the reduction of haemorrhagic complications of thrombolysis. We aimed to prepare sterically stabilized trypsin-loaded liposomes (SSL) and characterize their structure and fibrinolytic efficiency. Hydrogenated soybean phosphatidylcholine-based SSL were prepared and their structure was studied by transmission electron microscopy combined with freeze fracture (FF-TEM), Fourier transform infrared spectroscopy (FT-IR), and small-angle X-ray scattering (SAXS). Fibrinolytic activity was examined at 45, 37, or 24°C on fibrin or plasma clots with turbidimetric and permeation-driven lysis assays. Trypsin was shown to be attached to the inner surface of vesicles (SAXS and FF-TEM) close to the lipid hydrophilic/hydrophobic interface (FT-IR). The thermosensitivity of SSL was evidenced by enhanced fibrinolysis at 45°C: time to reduce the maximal turbidity to 20% decreased by 8.6% compared to 37°C and fibrin degradation product concentration in the permeation lysis assay was 2-fold to 5-fold higher than that at 24°C. SSL exerted its fibrinolytic action on fibrin clots under both static and dynamic conditions, whereas plasma clot dissolution was observed only in the permeation-driven assay. The improved fibrinolytic efficiency of SSL under dynamic conditions suggests that they may serve as a novel therapeutic candidate for dissolution of intravascular thrombi, which are typically exposed to permeation forces.
Topics: Fibrinolysis; Fibrinolytic Agents; Humans; Liposomes; Structure-Activity Relationship; Thrombolytic Therapy; Trypsin
PubMed: 28758116
DOI: 10.1155/2017/5130495 -
Journal of Thrombosis and Haemostasis :... Apr 2024The residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question of whether...
BACKGROUND
The residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question of whether thrombin is the physiological activator of protein C across vertebrates.
OBJECTIVES
To establish if thrombin can cleave at Trp residues.
METHODS
The activity of wild-type thrombin and mutant D189S was tested with a library of chromogenic substrates and toward wild-type protein C and mutants carrying substitutions at the site of cleavage.
RESULTS
Thrombin has trypsin-like and chymotrypsin-like specificity and cleaves substrates at Arg or Trp residues. Cleavage at Arg is preferred, but cleavage at Trp is significant and comparable with that of chymotrypsin. The D189S mutant of thrombin has broad specificity and cleaves at basic and aromatic residues without significant preference. Thrombin also cleaves natural substrates at Arg or Trp residues, showing activity toward protein C across vertebrates, including the ray-finned fish. The rate of activation of protein C in the ray-finned fish is affected by the sequence preceding Trp at the scissile bond.
CONCLUSION
The results provide a possible solution for the paradoxical presence of a Trp residue at the site of cleavage of protein C in ray-finned fish and support thrombin as the physiological activator of protein C in all vertebrates. The dual trypsin-like and chymotrypsin-like specificity of thrombin suggests that the spectrum of physiological substrates of this enzyme is broader currently assumed.
Topics: Animals; Trypsin; Thrombin; Chymotrypsin; Protein C; Substrate Specificity; Kinetics; Binding Sites
PubMed: 38160728
DOI: 10.1016/j.jtha.2023.12.026