Inactivation Of Protease Research Articles

In vitro digestion for assessing micronutrient bioavailability: the importance of digestion duration

Static digestion in vitro is a commonly used technique for investigating micronutrient availability which allows the nutrients or foods of interest to be exposed to conditions that simulate those found within the stomach and small intestine. The activity of these digestive enzymes throughout their respective simulated digestion phases has been reported to decline due to the autolytic activity of the proteases and therefore incomplete digestion may result. The degree of protease inactivation under commonly simulated digestion conditions requires further quantification. Pepsin and pancreatic protease activities were assessed throughout a simulated digestion protocol in vitro over multiple time points using stop-rate spectroscopy. The protease activity of both pepsin and pancreatin decreased significantly during their respective digestion phases. Results suggest that gastric and intestinal proteases are destroyed or inactivated during their respective digestive phase. For this reason, prolonged digestion protocols may require protease supplementation throughout digestion to correctly simulate physiological conditions.

Optimization of Conditions for Blood Plasma Peptidome Analysis

The conditions for peptidome analysis of blood plasma were optimized and the efficacy of the proposed approach was compared with the methods described in the literature. The method implies solution of two main problems: inactivation of blood plasma proteases and dissociation of peptides from major blood plasma proteins, which they are quantitatively associated with. To solve these problems, we proposed a new method of sample preparation. The essence of the method is simultaneous denaturation of plasma proteins plus reduction and alkylation of thiol groups of Cys, which is achieved by heating a blood plasma sample (95°C) in the presence of sodium deoxycholate, tris(2-carboxyethyl)phosphine, and 2-chloroacetamide. After separation of peptides from proteins by ultrafiltration on microcentrifuge filters and removal of sodium deoxycholate, the peptides are identified by LC-MS/MS using a Q Exactive HF (Thermo Scientific) mass spectrometer. As a result of one LC-MS/MS run of the peptide mixture obtained from ~15 μL of blood plasma, 2257 peptide fragments of 867 proteins were identified, which is 1.5 times higher than the values achieved by using the generally accepted method of differential solubilization. Our immediate plans include the use of our approach for cataloguing human blood plasma peptides, as well as establishing the magnitude of individual variability and the features of the peptidome that are related to gender and age.

Non-protease native allergens partially purified from bodies of eight domestic mites using p-aminobenzamidine ligand

BackgroundOptimised purification steps for concentrating trace target native antigens are needed. Combining the p-aminobenzamidine ligand with protease inactivation enables partial purification of mite non-protease allergens lacking proteases. ObjectiveWe sought to analyse in detail proteins obtained using this method from eight species of synanthropic acaridid mites and tested IgE reactivity using pooled human sera. Materials and methodsProteins affinity bound to p-aminobenzamidine as a ligand were identified by MALDI TOF/TOF. After electroblotting, the proteins were visualised using the fluorescent SYPRO-Ruby protein blot stain, and IgE reactivity was further analysed using pooled human sera collected from patients allergic to house dust mites. ResultsMS/MS identification confirmed previous results that no proteases were purified. Protein patterns corresponding to the allergens Der f 7, Der f 30 and actins indicated that these proteins are purified using p-aminobenzamidine and are present across a wide spectrum of acaridid mites. When using Dermatophagoides farinae, apolipophorins (Der f 14), chitinase-like Der f 15 and 18, 70-kDa heat shock protein, and a Der f Alt a10 allergen homolog (gi|37958173) were also detected. The target antigens tropomyosins and paramyosins showed similar IgE binding among the mite species tested. IgE reactivity with miscellaneous D. farinae antigen was also observed. ConclusionsPartial purification of mite non-protease antigens using a strategy combining p-aminobenzamidine with protease inactivation was verified by 1D-E and 2D-E analyses. IgE binding to p-aminobenzamidine-purified native non-protease mite antigens was tested using pooled sera. This preliminary study allows for further work on individual serum samples, allowing confirmation of immunoreactivity.

A novel antithrombin domain dictates the journey's end of a proteinase

Antithrombin (AT) is an anticoagulant serpin that irreversibly inactivates the clotting proteinases factor Xa and thrombin by forming covalent complexes with them. Mutations in its critical domains, such as those that impair the conformational rearrangement required for proteinase inactivation, increase the risk of venous thrombosis. Águila et al. characterize for the first time the destabilizing effects of mutations in the region of AT that makes contact with the proteinase in the final acyl-enzyme complex. Their work adds new insight into the unique structural intricacies of the inhibitory mechanism.

Inactivation of proteolytic enzymes by cestodes.

Inhibitor activity of cestodes from intestines of different hosts (sea birds, salt-water fish, and freshwater fish) was investigated. Alcataenia larina, Arctotaenia tetrabothrioides, Tetrabothrius erostris, T. minor, Wardium cirrosa, Bothriocephalus scorpii, Eubothrium rugosum, and Triaenophorus nodulosus were able to inhibit the activity of the commercial trypsin and activity of proteinase homogenates of the intestinal mucosa of the hosts. It was suggested that the inhibitor produced by the cestodes is specific for trypsin and protects them from the digestive enzymes of the host.

Effect of pH on dentin protease inactivation by carbodiimide.

A water-soluble crosslinking agent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), has been used as a pretreatment of acid-etched dentin to inactivate matrix-bound endogenous dentin proteases. The aim of this study was to evaluate the effect of pH on the inactivation capacity of EDC. Demineralized dentin beams (1×2×6mm) were divided into six groups (n=8 per group). Then, EDC (0.3M) was solubilized in distilled water with pH of 2, 4, 6, 7, 9, or 11. Control EDC was solubilized in 0.1M 2-(N-morpholino) ethanesulfonic acid (MES) buffer and its pH was adjusted to 6. The dentin beams were pretreated for 1min with EDC at each pH or with EDC in MES buffer at pH6.0 and then incubated in 1ml of simulated body fluid (pH7.2) for 1, 3, 7, or 14d. Untreated beams served as controls. At each study time-point, the dry mass of dentin beams was assessed and the incubation media were analyzed for carboxyterminal telopeptide of type-I collagen (ICTP) and C-terminal telopeptide of type I collagen (CTX) using specific ELISAs. Data were subjected to repeat-measures anova. The results of the study indicated that specimens pretreated with EDC in MES buffer showed the lowest collagen degradation in terms of mass loss and release of telopeptides, while specimens pretreated in alkaline media showed the highest collagen degradation. This study indicates that the pH of the EDC solution plays an important role in the stability of dentin protease inactivation.

Inactivation of proteolytic enzymes by Eubothrium rugosum (Cestoda) from the gut of burbot Lota lota.

Parasitic organisms inhabiting the alimentary canal should permanently resist the destructive action of host digestive enzymes. The intestinal parasites were shown to produce specific protease inhibitors protecting them from proteolysis. However, little is known about this adaptive mechanism in cestodes so far, especially for the tapeworms dwelling inside the fish intestines. Here, we explored the ability to inactivate proteolytic enzymes in the fish tapeworm Eubothrium rugosum (Batsch, 1786) (Bothriocephalidea) parasitising the intestine of wild burbot, Lota lota (Linnaeus). The assays were conducted with different concentrations of commercial trypsin and homogenate of intestinal mucosa both being the sources of proteinases. The incubation of live E. rugosum in trypsin solutions of two different concentrations caused a significant decrease in the enzyme activity. The extent of activity reduction was dependent on trypsin concentration. At the same time, the inhibitory effect of the worm incubation medium turned out to be statistically insignificant. These findings suggest partial adsorption of the enzyme to the tegument surface, with its further inactivation. In contrast to the incubation medium, the worm extract suppressed over 80% of trypsin activity and nearly half of the proteolytic activity in the mucosa homogenate. Notably, the inhibitory activity of the tapeworms hardly depended on their size characteristics. Finally, the research has demonstrated secretion of proteinase inhibitor in E. rugosum, which appears to be essential for its survival in enzymatically hostile environment.

Catalytic properties and thermal stability of a crude protease from the keratinolytic Bacillus sp. CL33A

Abstract Protease production by a feather-degrading mesophilic Bacillus sp. CL33A was performed through submerged cultivations using feather meal as low-cost organic substrate. Soluble protein concentration increased from 1.15 mg/mL at the beginning of cultivations (0 h) to 4.39 mg/mL after 72 h, indicating effective substrate solubilization. Crude protease, recovered as culture supernatants after 72 h of cultivation, was characterized. Optimal temperature and pH conditions for proteolytic activity were 48–62 °C and pH 7.2–9.2. Presence of MgCl2 (1–5 mM), MnCl2 (1 mM), Tween-20 and dimethyl sulfoxide (0.5–1.0%, v/v) increased protease activity at optimal conditions (55 °C; pH 8.0). Contrarily, CoCl2, CuSO4, FeSO4, ZnCl2, MnCl2 (5 mM), sodium dodecyl sulfate (0.05–1.00%, w/v), and β-mercaptoethanol (0.1–1.0%, v/v) decreased enzyme activity. Inhibition by phenylmethylsulfonyl-fluoride (25%), ethylenediaminotetraacetic acid (35%) and 1,10-phenanthroline (31%) indicates that crude protease contains serine and/or metalloproteases. Thermal inactivation of the crude protease at 50–60 °C followed apparent first-order kinetics. Inactivation rates (k) increased at higher temperatures, resulting in half-lives of 38.6 and 2.7 min, and D-values of 128.4 and 8.9 min, at 50 and 60 °C, respectively. Activity decay was modeled as a function of time and temperature by combining first-order and Arrhenius equations. Thermal inactivation was an endothermic process, resulting from conformational destabilization due to a highly positive apparent entropic contribution. Activity at mild conditions and low thermal stability might be beneficial for biocatalytic processes. Crude protease specificity might be explored for the hydrolysis and modification of food proteins, particularly casein and soy protein isolate.

Dysfunction of mitochondrial Lon protease and identification of oxidized protein in mouse brain following exposure to MPTP: Implications for Parkinson disease

Compelling evidence suggests that mitochondrial dysfunction leading to reactive oxygen species (ROS) production and protein oxidation could represent a critical event in the pathogenesis of Parkinson's disease (PD). Pioneering studies have shown that the mitochondrial matrix contains the Lon protease, which degrades oxidized, dysfunctional, and misfolded protein. Using the PD animal model of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) intoxication in mice, we showed that Lon protease expression increased in the ventral mesencephalon of intoxicated animals, concomitantly with the appearance of oxidized proteins and dopaminergic cell loss. In addition, we report that Lon is inactivated by ROS. Moreover, proteomic experiments provide evidence of carbonylation in α-ketoglutarate dehydrogenase (KGDH), aconitase or subunits of respiratory chain complexes. Lon protease inactivation upon MPTP treatment in mice raises the possibility that Lon protease dysfunction is an early event in the pathogenesis of PD.

Adsorption and inactivation of proteolytic enzymes by Triaenophorus nodulosus (Cestoda)

Summary The proteolytic activity in washings off the Triaenophorus nodulosus cestode tegument and the ability of the worms to inactivate proteolytic enzymes were studied. It was found that the major proteolytic activity in the washing samples is represented by the easily desorbed fraction most probably characterizing the activity of the host’s enzymes. Serine proteinases are an essential part of these enzymes. It was shown that the worms’ incubation medium and their homogenates can inhibit host proteinases and commercial trypsin samples. Suppressive activity of the incubation medium suggests that the inhibitors are rather spontaneously produced by the worms than induced by the presence of proteinases in the surrounding medium. The inhibitor produced by the cestode is hypothesized to be trypsin-specific.

Impaired inactivation of digestive proteases: The possible key factor for the high susceptibility of germ-free and antibiotic-treated animals to gut epithelial injury.

Recent study shows that germ-free and antibiotic-treated animals are highly susceptible to gut epithelial injury. This paper addresses that impaired inactivation of digestive proteases may be the key factor for the increased susceptibility.

Acute Phase Proteins and their Potential Role as an Indicator for Fish Health and in Diagnosis of Fish Diseases.

The acute phase proteins are biochemically and functionally unrelated protein predominantly synthesized in the liver. The local inflammatory cells i.e. macrophages and neutrophils secretes various cytokines like IL-1, IL-6, IL-8 (interleukins) and TNF-α into bloodstream in response to injury and tissue damage, which stimulate hepatocytes to produce protein and release them into the circulation; these proteins are called as acute phase protein (e.g. C-reactive protein (CRP), serum amyloid A (SAA), metal binding protein, lysozyme, lectin, etc.). The acute phase proteins are involved in variety of defence related activities e.g., inactivation of proteolytic enzymes, preventing the distribution of infectious agents (i.e. either by destruction of microorganism or making microbial cell suitable for cell response by modifying surface targets) and restoration of damage tissue and healthy condition. A number of well-known acute phase proteins have disease prognosis importance and change in the APPs level reflects the presence and intensity of inflammation during infection or injury. Further studies are still necessary to develop our knowledge in diagnostic importance of different acute phase proteins in fish and more efforts are needed to differentiate the APPs levels in case of viral, bacterial and parasitic diseases.

Comparative molecular dynamics study of dimeric and monomeric forms of HIV-1 protease in ligand bound and unbound state.

Human immunodeficiency virus type 1 protease is a viral-encoded enzyme and it is essential for replication and assembly of the virus. Inactivation of HIV-1 protease causes production of immature, noninfectious viral particles and thus HIV-1 protease is an attractive target in anti-AIDS drug design. In our current work, we performed molecular dynamics (MD) calculations (500 ns) for two different ligands (COM5 - designed in our previous study, and Darunavir) and made effort to understand dynamics behaviour of our designed compound COM5. An apo form of HIV-1 protease as monomer and dimer form was also studied in order to analyze response of protein to the ligand. MD results suggest that presence of ligand in hinders the stability of HIV-1 protease and one monomer from dimer systems is dominant on other monomer in terms of interaction made with ligands. We were able to trace functional residues as well as continuous motion of opening and closing (clapping) of flap region in HIV-1 protease (apo form) during entire 1000 ns of MD simulation. COM5 showed almost similar behaviour towards HIV-1 protease enzyme as Darunavir and propose as promising lead compound for the development of new inhibitor for HIV-1 protease.

Is the inactivation of dentin proteases by crosslinkers reversible?

ObjectiveInactivation of dentin proteases by crosslinkers has been suggested as a way to prevent the degradation of dentin collagen in the hybrid layer. However, it is not known if the inhibition is reversible. The aim of this study was to evaluate the inactivation effect of various crosslinkers on dentin protease activity over a period of 6 months. MethodsDemineralized dentin beams (1×2×6mm, n=10/group) were treated with (1) 1% glutaraldehyde (GA1), (2) 5% glutaraldehyde (GA5), (3) 1% grape seed extract (GS1), (4) 5% grape seed extract (GS5), (5) 10% sumac berry extract (S), (6) 20μM curcumin (CR20), and (7) 200μM curcumin (CR200) for 5min. Untreated beams served as control. The beams were incubated up to 6 months and incubation media were used to analyze solubilized telopeptide (ICTP and CTX) fragments as indicators of MMP- and cathepsin K-mediated degradation after 1, 3 and 6 months of incubation. The relative MMP activity of dentin beams was tested using a generic MMP assay. Data were analyzed using repeated-measures ANOVA, α=0.05. ResultsAll treated groups showed significant decrease in CTX release (32.2–469.5pg/mg dentin) and ICTP (1.8–47.6ng/mg dentin) fragments during the first month of incubation compared to control (1159pg/mg and 72.9ng/mg dentin, respectively). GA5, GS5 and CR200 maintained their inhibitory effect during 6-month incubation. The results were confirmed by dry mass loss and relative MMP activity following 6 months. SignificanceThe results of this study indicate that the long-term effect is both crosslinker and dose dependent.

Understanding Interactions of Surfactants and Enzymes: Impact of Individual Surfactants on Stability and Wash Performance of Protease Enzyme in Detergents

Abstract Enzymes and surfactants are both essential ingredients that determine the performance of modern laundry detergents. We have conducted an investigation of the interaction of surfactants and enzymes under laundry detergent application conditions in order to understand the influence of individual ingredients and to optimize detergent performance. We can show that for a given protease enzyme, individual surfactants in a constant detergent matrix have a significant impact on relevant stability and performance parameter. While certain anionic surfactants like e.g. linear alkylbenzene sulfonate show strong protease inactivation, nonionic surfactants did only show slight inactivation over time. On the other hand, proteolytic performance of protease on test stains was most driven by fatty alcohol ether sulfate. Knowledge about the impact of individual surfactants on proteases will enable the best choice of ingredients for mixed surfactant systems with optimized enzyme performance and stability.

The role of Pseudomonas aeruginosa alkaline protease in activation of the antimicrobial activity in Galleria mellonella larvae

The role of Pseudomonas aeruginosa metalloprotease - alkaline protease in activation of theantimicrobial activity in Galleria mellonella larvae was investigated. The results of our in vivo study showed that injection of alkaline protease at a sublethal dose enhanced the antimicrobial activity in the hemolymph of G. mellonella larvae as a result of induction of defense peptides synthesis. We observed that the antibacterial activity against E. coli appeared in the hemolymph 4 h after the injection of both metalloprotease or heat-killed P. aeruginosa, reached the maximum level 24 h post injection, and next decreased slightly. Antifungal activity against A. niger was detected in the hemolymph 15 h and 24 h after the challenge in the case of the alkaline protease and P. aeruginosa cell treatment, respectively. We also noted that the antimicrobial activity level induced by the presence of the metalloprotease in the hemolymph was higher than the activity measured after the injection of the insects with P. aeruginosa. The results of our in vitro studies indicated that inducible antimicrobial peptides present in the hemolymph of protease- or P. aeruginosa-challenged larvae were digested by alkaline protease.

Can Crohn's Disease Really be Caused by Mycobacterium avium Subspecies Paratuberculosis?-With My Alternative Theory that Reduction in Commensal Gut Bacteria and Resultant Impaired Inactivation of Digestive Proteases as the Primary Cause

The role of Mycobacterium avium subspecies paratuberculosis (MAP) in Crohn's disease (CD) has been debated for more than a century. Up to date, it remains a highly controversy issue as there are a large amounts of “solid” scientific evidence on both sides. However, I feel many of these conflicts are superficial and the core issue is just the extreme scarcity of MAP in CD and thus the still unresolved conflict between sensitivity and specificity. Along with in-depth analyses of the likely intimate nature of CD, MAP, and the so-called cell-wall deficient spheroplasts, as well as weighted assessment of findings from treatment and epidemiology, here I suggested that the evidences against a critical role of MAP in CD greatly overweigh those support it. Here I also shared a unified hypothesis I developed during the last 15 years regarding the etiology of inflammatory bowel disease (IBD), including the cause and mechanism of IBD as well as the relationship between CD and ulcerative colitis (UC). I proposed that reduction in commensal microbiota in modern society and the resultant impairment in inactivation of pancreatic digestive protease in the lower gut rather than any specific pathogens may have played the primary causative role in both CD and UC.

Inactivation of pancreatic digestive proteases by deconjugated bilirubin from the liver: A critical mechanism for gut protection

Each day, a large amount of different kinds of digestive enzymes are produced by the pancreas and discharged into the gut to digest the food. Among these enzymes, those such as amylase are made in the pancreas as the active form.

Targeted expression of cystatin restores fertility in cysteine protease induced male sterile tobacco plants

Fertility restoration in male sterile plants is an essential requirement for their utilization in hybrid seed production. In an earlier investigation, we have demonstrated that the targeted expression of a cysteine protease in tapetal cell layer resulted in complete male sterility in tobacco transgenic plants. In the present investigation, we have used a cystatin gene, which encodes for a cysteine protease inhibitor, from a wild peanut, Arachis diogoi and developed a plant gene based restoration system for cysteine protease induced male sterile transgenic tobacco plants. We confirmed the interaction between the cysteine protease and a cystatin of the wild peanut, A. diogoi through in silico modeling and yeast two-hybrid assay. Pollen from primary transgenic tobacco plants expressing cystatin gene under the tapetum specific promoter- TA29 restored fertility on cysteine protease induced male sterile tobacco plants developed earlier. This has confirmed the in vivo interaction of cysteine protease and cystatin in the tapetal cells, and the inactivation of cysteine protease and modulation of its negative effects on pollen fertility. Both the cysteine protease and cystatin genes are of plant origin in contrast to the analogous barnase–barstar system that deploys genes of prokaryotic origin. Because of the deployment of genes of plant origin, this system might not face biosafety problems in developing hybrids in food crops.

Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells

Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells.