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The Journal of Biological Chemistry Sep 2022Plasmepsin V (PM V) is a pepsin-like aspartic protease essential for growth of the malarial parasite Plasmodium falciparum. Previous work has shown PM V to be an...
Plasmepsin V (PM V) is a pepsin-like aspartic protease essential for growth of the malarial parasite Plasmodium falciparum. Previous work has shown PM V to be an endoplasmic reticulum-resident protease that processes parasite proteins destined for export into the host cell. Depletion or inhibition of the enzyme is lethal during asexual replication within red blood cells as well as during the formation of sexual stage gametocytes. The structure of the Plasmodium vivax PM V has been characterized by X-ray crystallography, revealing a canonical pepsin fold punctuated by structural features uncommon to secretory aspartic proteases; however, the function of this unique structure is unclear. Here, we used parasite genetics to probe these structural features by attempting to rescue lethal PM V depletion with various mutant enzymes. We found an unusual nepenthesin 1-type insert in the PM V gene to be essential for parasite growth and PM V activity. Mutagenesis of the nepenthesin insert suggests that both its amino acid sequence and one of the two disulfide bonds that undergird its structure are required for the insert's role in PM V function. Furthermore, molecular dynamics simulations paired with Markov state modeling suggest that mutations to the nepenthesin insert may allosterically affect PM V catalysis through multiple mechanisms. Taken together, these data provide further insights into the structure of the P. falciparum PM V protease.
Topics: Aspartic Acid Endopeptidases; Disulfides; Humans; Malaria, Falciparum; Pepsin A; Plasmodium falciparum; Protozoan Proteins
PubMed: 35952758
DOI: 10.1016/j.jbc.2022.102355 -
Microbiology Spectrum Aug 2022Acid tolerance is an important feature of probiotic development. It is one of the factors underlying the beneficial effects of probiotics in the intestine. However, the...
Acid tolerance is an important feature of probiotic development. It is one of the factors underlying the beneficial effects of probiotics in the intestine. However, the methods used by different researchers to test acid tolerance vary, causing confusion in the interpretation of the results. Therefore, in this study, we determine the optimal conditions for the acid tolerance test using response surface methodology. The factors of pH (2.5 to 3.5), exposure time (1 to 2 h), and pepsin (presence or absence) were used as independent variables, and the survival rates of seven strains (Lacticaseibacillus casei KACC 12413, Lactiplantibacillus plantarum KACC 15357, Limosilactobacillus fermentum KACC 11441, WCFS1, Lacticaseibacillus rhamnosus GG, KCTC 21024, and WiKim 0112) known to have probiotic properties were used as dependent variables. The results of the analysis of variance (ANOVA) indicated that the pH value and exposure time in acidic environments significantly affected the acid tolerance test model, and their interaction also had an effect ( < 0.05). Using the ANOVA results, the condition of the acid tolerance test was optimized with a target of an 85% survival rate for each strain. The optimized conditions of the acid tolerance test were as follows: pH 2.92, exposure time of 1.73 h, and presence of pepsin and pH 3, exposure time of 1.98 h, and absence of pepsin. These results can optimize strain selection with rigorous acid tolerance without confusion by unifying the conditions for the acid tolerance test. The acid tolerance test, which is the first step in selecting probiotics, is not standardized and can often cause confusion in the interpretation of results. Thus, in the present study, we optimized the conditions for the acid tolerance test using response surface methodology. These optimized conditions can be used to screen for strains with acid tolerance.
Topics: Intestines; Lactobacillaceae; Lactobacillus plantarum; Pepsin A; Probiotics
PubMed: 35876583
DOI: 10.1128/spectrum.01625-22 -
Food and Chemical Toxicology : An... Sep 2022The susceptibility of a novel food protein to digestion in the pepsin resistance test is widely used to inform the allergenicity risk assessment process. However, it... (Comparative Study)
Comparative Study
The susceptibility of a novel food protein to digestion in the pepsin resistance test is widely used to inform the allergenicity risk assessment process. However, it does not model the variation in the intragastric environment found in vivo. Consequently a 96-well plate format in vitro gastric digestion protocol has been developed with a high and low pepsin activity test executed at pH 1.2, 2.5, 5.5 and 6.5. It was used to analyse seven allergens (from milk, egg, peach and peanut) and two non-allergens (cytochrome c and zein). Digestion was monitored using SDS-PAGE and densitometry. In silico predictions were not confirmed experimentally for most of the proteins studied. Proteins were ranked according to half-life and showed susceptibility to digestion was related to the stability of protein structure and protein solubility rather than allergenicity per se. Highly digestible proteins, such as β-casein and Ara h 1, generated abundant resistant fragments Mr > 3.5 kDa in the low pepsin activity test which could be immunologically significant within the context of allergenicity risk assessment for susceptible groups such as infants. The high- and low pepsin activity tests used in this study provided complementary data to support allergenicity risk assessment and used only 10 mg protein.
Topics: Allergens; Arachis; Digestion; Food Hypersensitivity; Humans; Pepsin A; Proteins
PubMed: 35809717
DOI: 10.1016/j.fct.2022.113273 -
BMC Veterinary Research Jul 2022Toxocara cati, the cat roundworm, is a parasitic nematode that known to cause toxocariasis in intermediate hosts and humans. In this study, we characterized the dynamics...
BACKGROUND
Toxocara cati, the cat roundworm, is a parasitic nematode that known to cause toxocariasis in intermediate hosts and humans. In this study, we characterized the dynamics of T. cati larvae migration in BALB/c mice after inoculation with eggs and ensured the migration detecting the larval DNA by a PCR. To evaluate the dynamics of larval migration and distribution, twenty-four BALB/c mice were orally inoculated with 2500 T. cati infective eggs and the visceral organs of the infected animals were examined by pepsin digestion and microscopic parasite counts, followed by PCR at day 1 to 28 post-inoculation.
RESULTS
The PCR assays were successfully used for detection of T. cati larvae in tissue samples and T. cati larvae and the DNAs were found in the liver, lungs, heart, kidneys and the brain. We detected T. cati in 92.2% of tissue samples by PCR, 30% higher than the conventional pepsin digestion technique.
CONCLUSION
Our findings demonstrated that the PCR assay is a sensitive and specific for the detection of T. cati larvae. Therefore, it could become a useful tool for the investigation of the dynamics of larval migration and Toxocara infection in murine model.
Topics: Animals; Larva; Larva Migrans; Mice; Mice, Inbred BALB C; Ovum; Pepsin A; Polymerase Chain Reaction; Toxocara; Toxocariasis
PubMed: 35791007
DOI: 10.1186/s12917-022-03366-6 -
Scientific Reports Jun 2022Studies of microbiota reveal inter-relationships between the microbiomes of the gut and lungs. This relationship may influence the progression of lung disease,...
Studies of microbiota reveal inter-relationships between the microbiomes of the gut and lungs. This relationship may influence the progression of lung disease, particularly in patients with cystic fibrosis (CF), who often experience extraoesophageal reflux (EOR). Despite identifying this relationship, it is not well characterised. Our hypothesis is that the gastric and lung microbiomes in CF are related, with the potential for aerodigestive pathophysiology. We evaluated gastric and sputum bacterial communities by culture and 16S rRNA gene sequencing in 13 CF patients. Impacts of varying levels of bile acids, pepsin and pH on patient isolates of Pseudomonas aeruginosa (Pa) were evaluated. Clonally related strains of Pa and NTM were identified in gastric and sputum samples from patients with symptoms of EOR. Bacterial diversity was more pronounced in sputa compared to gastric juice. Gastric and lung bile and pepsin levels were associated with Pa biofilm formation. Analysis of the aerodigestive microbiomes of CF patients with negative sputa indicates that the gut can be a reservoir of Pa and NTM. This combined with the CF patient's symptoms of reflux and potential aspiration, highlights the possibility of communication between microorganisms of the gut and the lungs. This phenomenon merits further research.
Topics: Bacteria; Bile; Cystic Fibrosis; Gastric Juice; Gastroesophageal Reflux; Humans; Lung; Microbiota; Pepsin A; Pseudomonas aeruginosa; RNA, Ribosomal, 16S; Sputum
PubMed: 35773410
DOI: 10.1038/s41598-022-15375-4 -
EFSA Journal. European Food Safety... Jun 2022The food enzyme containing chymosin (EC 3.4.23.4) and pepsin A (EC 3.4.23.1) is prepared from the abomasum of calves by RENCO New Zealand. The food enzyme is intended to...
The food enzyme containing chymosin (EC 3.4.23.4) and pepsin A (EC 3.4.23.1) is prepared from the abomasum of calves by RENCO New Zealand. The food enzyme is intended to be used in milk processing for cheese production. As no concerns arise from the animal source of the food enzyme or from its manufacture and based on the history of safe use and consumption, the Panel considered that toxicological data were not required, and the estimation of dietary exposure was unnecessary. On the basis of literature data, the Panel considered that the risk of allergic sensitisation and elicitation reactions by dietary exposure could not be excluded, but the likelihood for this to occur was considered to be low. The Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.
PubMed: 35765379
DOI: 10.2903/j.efsa.2022.7361 -
Marine Drugs Jun 2022Fish collagen has been widely used in tissue engineering (TE) applications as an implant, which is generally transplanted into target tissue with stem cells for better...
Fish collagen has been widely used in tissue engineering (TE) applications as an implant, which is generally transplanted into target tissue with stem cells for better regeneration ability. In this case, the success rate of this research depends on the fundamental components of fish collagen such as amino acid composition, structural and rheological properties. Therefore, researchers have been trying to find an innovative raw material from marine origins for tissue engineering applications. Based on this concept, collagens such as acid-soluble (ASC) and pepsin-soluble (PSC) were extracted from a new type of cartilaginous fish, the blacktip reef shark, for the first time, and were further investigated for physicochemical, protein pattern, microstructural and peptide mapping. The study results confirmed that the extracted collagens resemble the protein pattern of type-I collagen comprising the α, α, β and γ chains. The hydrophobic amino acids were dominant in both collagens with glycine and hydroxyproline as major amino acids. From the FTIR spectra, α helix (27.72 and 26.32%), β-sheet (22.24 and 23.35%), β-turn (21.34 and 22.08%), triple helix (14.11 and 14.13%) and random coil (14.59 and 14.12%) structures of ASC and PSC were confirmed, respectively. Collagens retained their triple helical and secondary structure well. Both collagens had maximum solubility at 3% NaCl and pH 4, and had absorbance maxima at 234 nm, respectively. The peptide mapping was almost similar for ASC and PSC at pH 2, generating peptides ranging from 15 to 200 kDa, with 23 kDa as a major peptide fragment. The microstructural analysis confirmed the homogenous fibrillar nature of collagens with more interconnected networks. Overall, the preset study concluded that collagen can be extracted more efficiently without disturbing the secondary structure by pepsin treatment. Therefore, the blacktip reef shark skin could serve as a potential source for collagen extraction for the pharmaceutical and biomedical applications.
Topics: Acids; Amino Acids; Animals; Collagen; Collagen Type I; Fishes; Pepsin A; Sharks; Skin; Solubility
PubMed: 35736179
DOI: 10.3390/md20060376 -
Journal of Food and Drug Analysis Jun 2021This study aims to clarify the effects of chicken liver hydrolysates (CLHs) on long-term high-fat diet (HFD)-induced insulin resistance (IR) and hepatosteatosis in mice....
This study aims to clarify the effects of chicken liver hydrolysates (CLHs) on long-term high-fat diet (HFD)-induced insulin resistance (IR) and hepatosteatosis in mice. In vitro, the 400 μM oleic acid (OA)-added medium successfully stimulated the cellular steatosis on FL83B cells, and the cellular steatosis was attenuated ( p < 0.05) by supplementing with CLHs (4 mg/L). In vivo, the effects of CLHs on IR and hepatosteatosis development were tested in 20-week HFD-fed mice. HFD-induced increases in final body weight, but body weight gains of mice were decreased ( p < 0.05) by supplementing CLHs. Elevated ( p < 0.05) serum aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), free fatty acids (FFAs), triglyceride (TG), total cholesterol (TC), and fasted glucose values in HFD-fed mice decreased ( p < 0.05) by supplementing CLHs. Both results of hepatic steatosis and fibrotic scores also indicated the retardation ( p < 0.05) of the hepatosteatosis in cotreated groups. Moreover, the CLH supplementation sustained ( p < 0.05) hepatic and peripheral insulin signal sensitivity in HFD-fed mice. CLH supplementation could ameliorate hepatic lipid deposition, hepatic/peripheral IR in a long-term high-fat dietary habit, and also improve the universal glucose homeostasis by upregulating hepatic and peripheral insulin sensitivities.
Topics: Animals; Body Weight; Chickens; Fatty Liver; Feeding Behavior; Glucose; Insulin; Insulin Resistance; Mice; Pepsin A
PubMed: 35696203
DOI: 10.38212/2224-6614.3351 -
The Laryngoscope Jan 2023More than 20% of the US population suffers from laryngopharyngeal reflux. Although dietary/lifestyle modifications and alginates provide benefit to some, there is no...
OBJECTIVE
More than 20% of the US population suffers from laryngopharyngeal reflux. Although dietary/lifestyle modifications and alginates provide benefit to some, there is no gold standard medical therapy. Increasing evidence suggests that pepsin is partly, if not wholly, responsible for damage and inflammation caused by laryngopharyngeal reflux. A treatment specifically targeting pepsin would be amenable to local, inhaled delivery, and could prove effective for endoscopic signs and symptoms associated with nonacid reflux. The aim herein was to identify small molecule inhibitors of pepsin and test their efficacy to prevent pepsin-mediated laryngeal damage in vivo.
METHODS
Drug and pepsin binding and inhibition were screened by high-throughput assays and crystallography. A mouse model of laryngopharyngeal reflux (mechanical laryngeal injury once weekly for 2 weeks and pH 7 solvent/pepsin instillation 3 days/week for 4 weeks) was provided inhibitor by gavage or aerosol (fosamprenavir or darunavir; 5 days/week for 4 weeks; n = 3). Larynges were collected for histopathologic analysis.
RESULTS
HIV protease inhibitors amprenavir, ritonavir, saquinavir, and darunavir bound and inhibited pepsin with IC in the low micromolar range. Gavage and aerosol fosamprenavir prevented pepsin-mediated laryngeal damage (i.e., reactive epithelia, increased intraepithelial inflammatory cells, and cell apoptosis). Darunavir gavage elicited mild reactivity and no discernable protection; aerosol protected against apoptosis.
CONCLUSIONS
Fosamprenavir and darunavir, FDA-approved therapies for HIV/AIDS, bind and inhibit pepsin, abrogating pepsin-mediated laryngeal damage in a laryngopharyngeal reflux mouse model. These drugs target a foreign virus, making them ideal to repurpose. Reformulation for local inhaled delivery could further improve outcomes and limit side effects.
LEVEL OF EVIDENCE
NA. Laryngoscope, 133:S1-S11, 2023.
Topics: Animals; Mice; Laryngopharyngeal Reflux; Larynx; Pepsin A; Sulfonamides; Carbamates; Furans
PubMed: 35678265
DOI: 10.1002/lary.30242 -
International Journal of Biological... Jun 2022Pepsin is a protease used in many different applications, and in many instances, it is utilized in an immobilized form to prevent contamination of the reaction product.... (Review)
Review
Pepsin is a protease used in many different applications, and in many instances, it is utilized in an immobilized form to prevent contamination of the reaction product. This enzyme has two peculiarities that make its immobilization complex. The first one is related to the poor presence of primary amino groups on its surface (just one Lys and the terminal amino group). The second one is its poor stability at alkaline pH values. Both features make the immobilization of this enzyme to be considered a complicated goal, as most of the immobilization protocols utilize primary amino groups for immobilization. This review presents some of the attempts to get immobilized pepsin biocatalyst and their applications. The high density of anionic groups (Asp and Glu) make the anion exchange of the enzyme simpler, but this makes many of the strategies utilized to immobilize the enzyme (e.g., amino-glutaraldehyde supports) more related to a mixed ion exchange/hydrophobic adsorption than to real covalent immobilization. Finally, we propose some possibilities that can permit not only the covalent immobilization of this enzyme, but also their stabilization via multipoint covalent attachment.
Topics: Enzyme Stability; Enzymes, Immobilized; Glutaral; Hydrogen-Ion Concentration; Pepsin A
PubMed: 35508226
DOI: 10.1016/j.ijbiomac.2022.04.224