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Molecules (Basel, Switzerland) Aug 2023The lysozyme in the chicken egg white consists of various bioactive amino acids. However, these compounds are inactive when they are in the sequence of parent proteins....
The lysozyme in the chicken egg white consists of various bioactive amino acids. However, these compounds are inactive when they are in the sequence of parent proteins. They become active only when isolated from these proteins. The aim of this study was to modify lysozyme with proteolytic enzymes under specific conditions of the reaction environment so as to obtain active biopeptides. The physicochemical properties of the resulting preparations were also assessed. Our study showed that the modification of lysozyme with hydrolytic enzymes (pepsin and trypsin) under strictly specified conditions resulted in obtaining biopeptide preparations with new and valuable properties, as compared with native lysozyme. After the enzymatic modification of lysozyme, two structural fractions were distinguished in the composition of the resulting preparations-the monomeric fraction and the peptide fraction. The modified lysozyme exhibited high surface hydrophobicity and high total antibacterial activity despite the decrease in the hydrolytic activity. Modification of lysozyme with hydrolytic enzymes, especially pepsin, resulted in preparations with very good antioxidative properties.
Topics: Muramidase; Peptide Hydrolases; Pepsin A; Hydrolysis; Dermatologic Agents
PubMed: 37687089
DOI: 10.3390/molecules28176260 -
Scientific Reports Dec 2023Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells...
Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers a major drawback, namely reduced fluorescence signals caused by excessive proteolysis and swelling effects. This caveat results in a lower photon budget and disfavors fluorescence imaging over a large field of view that can cover an entire expanded cell, especially in 3D. In addition, the complex procedures and specialized reagents of expansion microscopy hinder its popularization. Here, we modify expansion microscopy by deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal for point-scanning-based imaging. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. TT-ExM displayed enhanced protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures. Importantly, TT-ExM-based lipid staining clearly revealed the complex 3D membrane structures in entire expanded cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. These high-quality 3D images demonstrate the practicality of TT-ExM. Thus, readily available reagents can be deployed in TT-ExM to significantly enhance fluorescence signals and generate high-quality and ultrafine-resolution images under confocal microscopy.
Topics: Trypsin; Imaging, Three-Dimensional; Proteins; Microscopy, Confocal; Indicators and Reagents; DNA; Lipids
PubMed: 38081848
DOI: 10.1038/s41598-023-48959-9 -
Frontiers in Immunology 2024Intrapancreatic activation of trypsinogen caused by alcohol or high-fat intake and the subsequent autodigestion of the pancreas tissues by trypsin are indispensable... (Review)
Review
INTRODUCTION
Intrapancreatic activation of trypsinogen caused by alcohol or high-fat intake and the subsequent autodigestion of the pancreas tissues by trypsin are indispensable events in the development of acute pancreatitis. In addition to this trypsin-centered paradigm, recent studies provide evidence that innate immune responses triggered by translocation of intestinal bacteria to the pancreas due to intestinal barrier dysfunction underlie the immunopathogenesis of acute pancreatitis. Although severe acute pancreatitis is often associated with pancreatic colonization by fungi, the molecular mechanisms linking fungus-induced immune responses to the development of severe acute pancreatitis are poorly understood. Leucine-rich repeat kinase 2 (LRRK2) is a multifunctional protein that mediates innate immune responses to fungi and bacteria. Mutations in is a risk factor for Parkinson's disease and Crohn's disease, both of which are driven by innate immune responses to gut organisms.
DISCUSSION
In this Minireview article, we discuss how activation of LRRK2 by the recognition of fungi induces severe acute pancreatitis.
Topics: Humans; Pancreatitis; Leucine; Acute Disease; Trypsin; Pancreas
PubMed: 38440723
DOI: 10.3389/fimmu.2024.1364839 -
Cell Discovery Jul 2023The bat coronaviruses (CoV) BANAL-20-52 and BANAL-20-236 are two newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) closely related...
The bat coronaviruses (CoV) BANAL-20-52 and BANAL-20-236 are two newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) closely related coronaviruses (SC2r-CoV) and the genome of BANAL-20-52 shares the highest homology with SARS-CoV-2. However, the risk of their potential zoonotic transmission has not been fully evaluated. Here, we determined their potential host susceptibility among 13 different bat species and 26 different animal species, and found that both might have extensive host ranges, indicating high zoonotic transmission potential. We also determined the cryo-EM structures of BANAL-20-52 and BANAL-20-236 S proteins at pH 5.5 and the complex of BANAL-20-236 S1 and Rhinolophus affinis ACE2, and found that both trimeric S proteins adopt all three receptor binding domains (RBDs) in "closed" conformation and are more compact than SARS-CoV-2. Strikingly, the unique sugar moiety at N370 of bat SC2r-CoVs acts like a "bolt" and crosses over two neighboring subunits, facilitating the S proteins in the locked conformation and underpinning the architecture stability. Removal of the glycosylation at N370 by a T372A substitution substantially enhances virus infectivity but becomes highly sensitive to trypsin digestion at pH 5.5, a condition roughly mimicking the insectivorous bat's stomach digestion. In contrast, WT S proteins of SC2r-CoVs showed considerable resistance to trypsin digestion at pH 5.5, indicating that the highly conserved T372 in bat CoVs might result from the selective advantages in stability during the fecal-oral transmission over A372. Moreover, the results of cross-immunogenicity among S proteins of SARS-CoV-2, BANAL-20-52, and BANAL-20-236 showed that A372 pseudoviruses are more sensitive to anti-S sera than T372, indicating that immune evasion might also play a role in the natural selection of T372 over A372 during evolution. Finally, residues 493 and 498 of the S protein affect host susceptibility, and residue 498 also influences the immunogenicity of the S protein. Together, our findings aid a better understanding of the molecular basis of CoV entry, selective evolution, and immunogenicity and highlight the importance of surveillance of susceptible hosts of these viruses to prevent potential outbreaks.
PubMed: 37507385
DOI: 10.1038/s41421-023-00581-9 -
Frontiers in Nutrition 2023Chickpea ( L.), an annual plant of the family Fabaceae is mainly grown in semiarid and temperate regions. Among pulses, cultivated worldwide chickpeas are considered an... (Review)
Review
Chickpea ( L.), an annual plant of the family Fabaceae is mainly grown in semiarid and temperate regions. Among pulses, cultivated worldwide chickpeas are considered an inexpensive and rich source of protein. Chickpea is a good source of protein and carbohydrate, fiber, and important source of essential minerals and vitamins. The quality of protein is better among other pulses. Consumption of chickpeas is related to beneficial health outcomes. Dietary peptides from the protein of chickpeas gaining more attention. Peptides can be obtained through acid, alkali, and enzymatic hydrolysis. Among all these, enzymatic hydrolysis is considered safe. Various enzymes are used for the production of peptides, i.e., flavorzyme, chymotrypsin, pepsin, alcalase, papain, and trypsin either alone or in combinations. Chickpea hydrolysate and peptides have various bioactivity including angiotensin 1-converting enzyme inhibition, digestive diseases, hypocholesterolemic, CVD, antioxidant activity, type 2 diabetes, anti-inflammatory, antimicrobial, and anticarcinogenic activity. This review summarizes the nutritional composition and bioactivity of hydrolysate and peptides obtained from chickpea protein. The literature shows that chickpea peptides and hydrolysate have various functional activities. But due to the limited research and technology, the sequences of peptides are unknown, due to which it is difficult to conduct the mechanism studies that how these peptides interact. Therefore, emphasis must be given to the optimization of the production of chickpea bioactive peptides, studies of chickpea bioactivity, and conducting human study trials to check the bioactivity of these peptides and hydrolysate.
PubMed: 37854353
DOI: 10.3389/fnut.2023.1218468 -
Scientific Reports Aug 2023Periwinkle shells of Tympanotonus fuscatus, Pachymelania aurita, and Thais coronata were analyzed for their proximate composition, nutritionally significant minerals,...
Periwinkle shells of Tympanotonus fuscatus, Pachymelania aurita, and Thais coronata were analyzed for their proximate composition, nutritionally significant minerals, trypsin inhibitors, and carotenoids. The mean values obtained were compared using an ANOVA test. The results showed that T. fuscatus had the highest mean moisture content of 0.96 ± 0.14% and a mean value of 0.49 ± 0.13% for crude fibre but was not significantly different (P > 0.05) from P. auritus. The crude protein and fibre content of T. fuscatus was significantly higher (P < 0.05) than other periwinkle samples. T. coronata had the highest mean total ash content and was significantly different (p < 0.05) from other periwinkle samples. T. fuscatus had the highest mean value for Mg (0.32 ± 0.03 mg/kg) and differed significantly (P < 0.05). The mean Ca content of P. aurita was not significantly different (P > 0.05) from that of T. coronata. The mean values of CaCO in T. fuscatus, P. aurita, and T. coronata were 57.20 ± 2.46, 59.50 ± 3.23, and 62.36 ± 1.56 (mg/kg), respectively. T. coronata was significantly different (P < 0.05) from other periwinkle samples. The mean values of carotenoids in T. fuscatus, P. aurita, and T. coronata were 7.17 ± 2.14, 18.00 ± 5.27, and 11.20 ± 3.60 (mg/kg), respectively, and P. aurita was significantly different (P < 0.05) from other periwinkle samples. T. fuscatus and P. aurita had shells with significant amounts of trypsin inhibitor (23.30 ± 4.50 mg/kg and 22.90 ± 14.10 mg/kg, respectively), making them less suitable for livestock feed. In contrast, T. coronata had a lower mean value of 11.80 ± 7.19 mg/kg for trypsin inhibitor, making it an excellent addition to livestock feed. The low crude fibre and fat contents of the periwinkle samples in this study make them suitable for processing complementary foods, especially for hypertensive patients. The high percentage of CaCO in periwinkle shells makes them a probable source used in the production of slurry for chromatography. The findings suggest that periwinkle shells contain specific minerals that can be applied in numerous industries. Increased use of these gastropod shells will result in successful application in product creation and a sustainable bio-circular economy.
Topics: Animals; Humans; Gastropoda; Trypsin Inhibitors; Exoskeleton Device; Minerals; Carotenoids; Seafood
PubMed: 37567917
DOI: 10.1038/s41598-023-38345-w -
Journal of Agricultural and Food... Mar 2024Coffee is one of the most popular beverages around the world and its consumption contributes to the daily intake of dietary melanoidins. Despite the emerging...
Coffee is one of the most popular beverages around the world and its consumption contributes to the daily intake of dietary melanoidins. Despite the emerging physiological role of food melanoidins, their effect on digestive processes has not been studied so far. In this study, the activity of the gastrointestinal enzymes pepsin and trypsin was investigated in the presence of water-soluble coffee melanoidins. The gastric enzyme pepsin is only slightly affected, whereas the intestinal enzyme trypsin is severely inhibited by coffee melanoidins. The intestinal digestibility of casein was significantly inhibited by coffee melanoidins at a concentration achievable by regular coffee consumption. The inhibition of proteolytic enzymes by coffee melanoidins might decrease the nutritional value of dietary proteins.
Topics: Coffee; Pepsin A; Peptide Hydrolases; Trypsin; Dietary Proteins; Polymers
PubMed: 38456211
DOI: 10.1021/acs.jafc.3c09654 -
Journal of Experimental & Clinical... Aug 2023The pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations...
BACKGROUND
The pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations in the equilibrium between proteases and their inhibitors. The identification of proteolytic events, targets and pathways would set the basis for the design of new therapeutic approaches.
METHODS AND RESULTS
Here we demonstrate that spheroids isolated from human and murine healthy pancreas and co-transplanted orthotopically with pancreatic ductal adenocarcinoma (PDAC) in mouse pancreas inhibited tumor growth. The effect was mediated by trypsin-generated fibronectin (FN) fragments released by pancreatic spheroids. Tumor inhibition was observed also in a model of acute pancreatitis associated with trypsin activation. Mass spectrometry proteomic analysis of fragments and mAb against different FN epitopes identified the FN type III domain as responsible for the activity. By inhibiting integrin α5β1, FAK and FGFR1 signaling, the fragments induced tumor cell detachment and reduced cell proliferation. Consistent with the mutual relationship between the two pathways, FGF2 restored both FGFR1 and FAK signaling and promoted PDAC cell adhesion and proliferation. FAK and FGFR inhibitors additively inhibited PDAC growth in vitro and in orthotopic in vivo models.
CONCLUSIONS
This study identifies a novel role for pancreatic trypsin and fibronectin cleavage as a mechanism of protection against cancer by the pancreatic microenvironment. The finding of a FAK-FGFR cross-talk in PDAC support the combination of FAK and FGFR inhibitors for PDAC treatment to emulate the protective effect of the normal pancreas against cancer.
Topics: Animals; Humans; Mice; Acute Disease; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Fibronectins; Pancreas; Pancreatic Neoplasms; Pancreatitis; Proteomics; Trypsin; Tumor Microenvironment
PubMed: 37559126
DOI: 10.1186/s13046-023-02778-y -
Journal of Mass Spectrometry and... Nov 2023LC-MS-based methods for protein quantification have a stigma of being relatively expensive and low-throughput. This is partly due to the cost and speed of trypsin...
INTRODUCTION
LC-MS-based methods for protein quantification have a stigma of being relatively expensive and low-throughput. This is partly due to the cost and speed of trypsin digestion, which has primarily focused on advancements in research-based biomarker discovery applications that rely on protein/peptide identifications rather than clinical biomarker quantification. However, there is a need for simple, fast, and reproducibly efficient surrogate peptide recovery in clinical biomarker quantification.
METHODS
Multiple methodologies were evaluated to enhance tryptic digestion for the analysis of thyroglobulin, a prototypical serum protein biomarker. The main criteria for assessment were the yield and speed of formation of surrogate peptides. Various factors such as different additives, types of trypsin, microwave- and pressure-assisted systems, and enzyme concentration were considered as key variables, in addition to digestion time.
RESULTS
It was observed that digestion additives/denaturants had a significant impact on the speed and yield of digestion for each surrogate peptide. Increasing the concentration of trypsin alone was found to accelerate digestions appreciably for most surrogate peptides, without affecting the yield. However, the use of sequencing-grade trypsins and microwave/pressure-assisted systems did not offer significant advantages over the use of 'standard-grade' TPCK-treated trypsin in combination with a conventional incubator, once digestion time and additive had been optimized.
CONCLUSION
We have dispelled the notion that trypsin digestion is inherently slow and expensive for targeted quantification of serum proteins. Additionally, we have established a groundwork for experimentation that can pave the way for the creation of efficient trypsin digestion protocols, aiming to optimize yield, speed, and cost. It is our hope that these advancements will promote the wider incorporation of such assays in clinical laboratories.
PubMed: 38093969
DOI: 10.1016/j.jmsacl.2023.11.002 -
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