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Molecular & Cellular Proteomics : MCP Jan 2023Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein...
Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein structure and function. To address this challenge, we developed a strategy that uses immobilized metal-affinity chromatography-enrichable phosphonate affinity tags, for efficient and selective enrichment of peptides bound to an activity-based probe, enabling the identification of the exact drug binding site. As a proof of concept, using this approach, termed PhosID-ABPP (activity-based protein profiling), over 500 unique binding sites were reproducibly identified of an alkynylated afatinib derivative (PF-06672131). As PhosID-ABPP is compatible with intact cell inhibitor treatment, we investigated the quantitative differences in approachable binding sites in intact cells and in lysates of the same cell line and observed and quantified substantial differences. Moreover, an alternative protease digestion approach was used to capture the previously reported binding site on the epidermal growth factor receptor, which turned out to remain elusive when using solely trypsin as protease. Overall, we find that PhosID-ABPP is highly complementary to biotin-based enrichment strategies in ABPP studies, with PhosID-ABPP providing the advantage of direct activity-based probe interaction site identification.
Topics: Organophosphonates; Proteins; Peptides; Cell Line; Trypsin
PubMed: 36435334
DOI: 10.1016/j.mcpro.2022.100455 -
PloS One 2015To explore the inhibitory mechanism of catechins for digestive enzymes, we investigated the binding mode of catechins to a typical digestive enzyme-trypsin and analyzed...
To explore the inhibitory mechanism of catechins for digestive enzymes, we investigated the binding mode of catechins to a typical digestive enzyme-trypsin and analyzed the structure-activity relationship of catechins, using an integration of molecular docking, molecular dynamics simulation and binding free energy calculation. We found that catechins with different structures bound to a conservative pocket S1 of trypsin, which is comprised of residues 189-195, 214-220 and 225-228. In the trypsin-catechin complexes, Asp189 by forming strong hydrogen bonding, and Gln192, Trp215 and Gly216 through hydrophobic interactions, all significantly contribute to the binding of catechins. The number and the position of hydroxyl and aromatic groups, the structure of stereoisomers, and the orientation of catechins in the binding pocket S1 of trypsin all affect the binding affinity. The binding affinity is in the order of Epigallocatechin gallate (EGCG) > Epicatechin gallate (ECG) > Epicatechin (EC) > Epigallocatechin (EGC), and 2R-3R EGCG shows the strongest binding affinity out of other stereoisomers. Meanwhile, the synergic conformational changes of residues and catechins were also analyzed. These findings will be helpful in understanding the knowledge of interactions between catechins and trypsin and referable for the design of novel polyphenol based functional food and nutriceutical formulas.
Topics: Amino Acids; Catechin; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Protein Binding; Structure-Activity Relationship; Tea; Trypsin
PubMed: 25938485
DOI: 10.1371/journal.pone.0125848 -
Ultrasonics Sonochemistry Sep 2004Encapsulating proteins in polymeric microspheres is a useful mode of drug delivery, but the proteins are subjected to damage in the process of ultrasound emulsion...
Encapsulating proteins in polymeric microspheres is a useful mode of drug delivery, but the proteins are subjected to damage in the process of ultrasound emulsion microencapsulation. The objective of this study was to investigate the effects of ultrasound power and duration on the function and structure of trypsin, and the reason of protein denaturation when it was irradiated by 20 kHz ultrasound. The relatively stable enzyme, trypsin, was dissolved in aqueous solution in the presence and absence of additives to study the stability of trypsin during the ultrasound irradiation. The damage of the molecular structure of trypsin was detected via combined high performance liquid chromatogram and electrospray ionization mass spectrometry (HPLC-ESI-MS). The results showed that the activity of trypsin decreased with increasing ultrasound power from 100 to 500 W or extending the irradiation time from 1 to 20 min. This effect could be enhanced via aerating the solution for a duration 10 min at 300 W. Fragments of trypsin were detected in the treatment (300 W, 10 min) by HPLC-ESI-MS. The additives, Tween 80 and mannitol, could protect trypsin against the inactivation caused by ultrasound. The reason of inactivation was partly from the alteration of the molecular conformation and partly from the modification or damage of trypsin's molecular structure.
Topics: Drug Compounding; Drug Delivery Systems; Excipients; Indicators and Reagents; Mass Spectrometry; Solutions; Spectrometry, Mass, Electrospray Ionization; Temperature; Trypsin; Ultrasonics
PubMed: 15302026
DOI: 10.1016/j.ultsonch.2003.09.004 -
Biophysical Journal Aug 2005Trypsin and chymotrypsin are both serine proteases with high sequence and structural similarities, but with different substrate specificity. Previous experiments have...
Trypsin and chymotrypsin are both serine proteases with high sequence and structural similarities, but with different substrate specificity. Previous experiments have demonstrated the critical role of the two loops outside the binding pocket in controlling the specificity of the two enzymes. To understand the mechanism of such a control of specificity by distant loops, we have used the Gaussian network model to study the dynamic properties of trypsin and chymotrypsin and the roles played by the two loops. A clustering method was introduced to analyze the correlated motions of residues. We have found that trypsin and chymotrypsin have distinct dynamic signatures in the two loop regions, which are in turn highly correlated with motions of certain residues in the binding pockets. Interestingly, replacing the two loops of trypsin with those of chymotrypsin changes the motion style of trypsin to chymotrypsin-like, whereas the same experimental replacement was shown necessary to make trypsin have chymotrypsin's enzyme specificity and activity. These results suggest that the cooperative motions of the two loops and the substrate-binding sites contribute to the activity and substrate specificity of trypsin and chymotrypsin.
Topics: Amino Acid Sequence; Binding Sites; Chymotrypsin; Computer Simulation; Enzyme Activation; Models, Chemical; Models, Molecular; Molecular Sequence Data; Motion; Protein Binding; Protein Conformation; Sequence Analysis, Protein; Sequence Homology, Amino Acid; Structure-Activity Relationship; Substrate Specificity; Trypsin
PubMed: 15923233
DOI: 10.1529/biophysj.104.057158 -
In Vitro Cellular & Developmental... Jun 2018Muscle stem cells, termed satellite cells (SC), and SC-derived myogenic progenitor cells (MPC) are involved in postnatal muscle growth, regeneration, and muscle...
Effects of trypsinization and of a combined trypsin, collagenase, and DNase digestion on liberation and in vitro function of satellite cells isolated from juvenile porcine muscles.
Muscle stem cells, termed satellite cells (SC), and SC-derived myogenic progenitor cells (MPC) are involved in postnatal muscle growth, regeneration, and muscle adaptability. They can be released from their natural environment by mechanical disruption and tissue digestion. The literature contains several isolation protocols for porcine SC/MPC including various digestion procedures, but comparative studies are missing. In this report, classic trypsinization and a more complex trypsin, collagenase, and DNase (TCD) digestion were performed with skeletal muscle tissue from 4- to 5-d-old piglets. The two digestion procedures were compared regarding cell yield, viability, myogenic purity, and in vitro cell function. The TCD digestion tended to result in higher cell yields than digestion with solely trypsin (statistical trend p = 0.096), whereas cell size and viability did not differ. Isolated myogenic cells from both digestion procedures showed comparable proliferation rates, expressed the myogenic marker Desmin, and initiated myogenic differentiation in vitro at similar levels. Thus, TCD digestion tended to liberate slightly more cells without changes in the tested in vitro properties of the isolated cells. Both procedures are adequate for the isolation of SC/MPC from juvenile porcine muscles but the developmental state of the animal should always be considered.
Topics: Animals; Animals, Newborn; Cell Separation; Cells, Cultured; Collagenases; Deoxyribonucleases; Muscle, Skeletal; Satellite Cells, Skeletal Muscle; Swine; Trypsin
PubMed: 29785535
DOI: 10.1007/s11626-018-0263-5 -
Analytical Chemistry Jul 2020Trypsin is the most used enzyme in proteomics. Nevertheless, proteases with complementary cleavage specificity have been applied in special circumstances. In this work,...
Trypsin is the most used enzyme in proteomics. Nevertheless, proteases with complementary cleavage specificity have been applied in special circumstances. In this work, we analyzed the characteristics of five protease alternatives to trypsin for protein identification and sequence coverage when applied to whole cell lysates. The specificity of the protease heavily impacted the number of proteins identified. Proteases with higher specificity led to the identification of more proteins than proteases with lower specificity. However, AspN, GluC, chymotrypsin, and proteinase K largely benefited from being paired with trypsin in sequential digestion, as had been shown by us for elastase before. In the most extreme case, predigesting with trypsin improves the number of identified proteins for proteinase K by 731%. Trypsin predigestion also improved the protein identifications of other proteases, AspN (+62%), GluC (+80%), and chymotrypsin (+21%). Interestingly, the sequential digest with trypsin and AspN yielded even a higher number of protein identifications than digesting with trypsin alone.
Topics: Proteins; Proteomics; Schizosaccharomyces; Trypsin
PubMed: 32628831
DOI: 10.1021/acs.analchem.0c00478 -
Molecular Vision 2013Aging of the vitreous body can result in sight-threatening pathology. One aspect of vitreous aging is liquefaction, which results from the vanishing of collagen fibrils....
PURPOSE
Aging of the vitreous body can result in sight-threatening pathology. One aspect of vitreous aging is liquefaction, which results from the vanishing of collagen fibrils. We investigated the possibility that trypsins are involved in vitreous type II collagen degradation.
METHODS
Immunohistochemistry and western blotting were used for detecting and locating trypsin isoforms in the vitreous and retina of human donor eyes. The capability of the retina to produce these trypsins was analyzed with polymerase chain reaction. Whether the different trypsins degraded type II collagen was tested in vitro. The sizes of the in vitro induced type II collagen degradation products were compared to those present in the vitreous of human eyes of different ages.
RESULTS
Trypsin-1 and trypsin-2 were detected in the vitreous. In the retina, messenger ribonucleic acid (mRNA) coding for trypsin-2, -3, and -4 was present. Using immunohistochemistry, trypsin-2 was detected in microglial cells located in the vitreous and the retina. All trypsin isoforms degraded type II collagen and produced degradation products of similar sizes as those present in the vitreous.
CONCLUSIONS
Trypsin-1 and trypsin-2 appear to have a function in the degradation of vitreous type II collagen. They could therefore have a role in the development of vitreous liquefaction.
Topics: Adult; Aged; Blotting, Western; Collagen Type II; Humans; Immunohistochemistry; Isoenzymes; Middle Aged; Proteolysis; RNA, Messenger; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Tissue Extracts; Trypsin; Trypsinogen; Vitreous Body
PubMed: 23882137
DOI: No ID Found -
Advanced Science (Weinheim,... Jun 2023Defects in cellular protein/enzyme encoding or even in organelles are responsible for many diseases. For instance, dysfunctional lysosome or macrophage activity results...
Defects in cellular protein/enzyme encoding or even in organelles are responsible for many diseases. For instance, dysfunctional lysosome or macrophage activity results in the unwanted accumulation of biomolecules and pathogens implicated in autoimmune, neurodegenerative, and metabolic disorders. Enzyme replacement therapy (ERT) is a medical treatment that replaces an enzyme that is deficient or absent in the body but suffers from short lifetime of the enzymes. Here, this work proposes the fabrication of two different pH-responsive and crosslinked trypsin-loaded polymersomes as protecting enzyme carriers mimicking artificial organelles (AOs). They allow the enzymatic degradation of biomolecules to mimic simplified lysosomal function at acidic pH and macrophage functions at physiological pH. For optimal working of digesting AOs in different environments, pH and salt composition are considered the key parameters, since they define the permeability of the membrane of the polymersomes and the access of model pathogens to the loaded trypsin. Thus, this work demonstrates environmentally controlled biomolecule digestion by trypsin-loaded polymersomes also under simulated physiological fluids, allowing a prolonged therapeutic window due to protection of the enzyme in the AOs. This enables the application of AOs in the fields of biomimetic therapeutics, specifically in ERT for dysfunctional lysosomal diseases.
Topics: Artificial Cells; Trypsin; Lysosomes; Organelles; Proteins
PubMed: 37076948
DOI: 10.1002/advs.202207214 -
Food Chemistry Jul 2017This work presents an inexpensive, simple and fast procedure to purify trypsin based on affinity binding with ferromagnetic particles of azocasein composite (mAzo)....
This work presents an inexpensive, simple and fast procedure to purify trypsin based on affinity binding with ferromagnetic particles of azocasein composite (mAzo). Crude extract was obtained from intestines of fish Nile tilapia (Oreochromis niloticus) homogenized in buffer (01g tissue/ml). This extract was exposed to 100mg of mAzo and washed to remove unbound proteins by magnetic field. Trypsin was leached off under high ionic strength (3M NaCl). Preparation was achieved containing specific activity about 60 times higher than that of the crude extract. SDS-PAGE showed that the purified protein had molecular weight (24kDa) in concordance with the literature for the Nile tilapia trypsin. The mAzo composite can be reused and applied to purify trypsin from other sources.
Topics: Animals; Caseins; Chemical Fractionation; Cichlids; Fish Proteins; Intestines; Iron; Magnetite Nanoparticles; Molecular Weight; Trypsin
PubMed: 28254021
DOI: 10.1016/j.foodchem.2016.12.094 -
The Journal of Biological Chemistry Mar 2020Mesotrypsin is an unusual human trypsin isoform with inhibitor resistance and the ability to degrade trypsin inhibitors. Degradation of the protective serine protease...
Mesotrypsin is an unusual human trypsin isoform with inhibitor resistance and the ability to degrade trypsin inhibitors. Degradation of the protective serine protease inhibitor Kazal type 1 (SPINK1) by mesotrypsin in the pancreas may contribute to the pathogenesis of pancreatitis. Here we tested the hypothesis that the regulatory digestive protease chymotrypsin C (CTRC) mitigates the harmful effects of mesotrypsin by cleaving the autolysis loop. As human trypsins are post-translationally sulfated in the autolysis loop, we also assessed the effect of this modification. We found that mesotrypsin cleaved in the autolysis loop by CTRC exhibited catalytic impairment on short peptides due to a 10-fold increase in , it digested β-casein poorly and bound soybean trypsin inhibitor with 10-fold decreased affinity. Importantly, CTRC-cleaved mesotrypsin degraded SPINK1 with markedly reduced efficiency. Sulfation increased mesotrypsin activity but accelerated CTRC-mediated cleavage of the autolysis loop and did not protect against the detrimental effect of CTRC cleavage. The observations indicate that CTRC-mediated cleavage of the autolysis loop in mesotrypsin decreases protease activity and thereby protects the pancreas against unwanted SPINK1 degradation. The findings expand the role of CTRC as a key defense mechanism against pancreatitis through regulation of intrapancreatic trypsin activity.
Topics: Biocatalysis; Caseins; Chymotrypsin; HEK293 Cells; Humans; Kinetics; Mutation; Proteolysis; Trypsin; Trypsin Inhibitor, Kazal Pancreatic; Trypsin Inhibitors
PubMed: 32014997
DOI: 10.1074/jbc.RA120.012526