Proteolytic Reactions Research Articles

Role of Salivary Protease Enzymatic Activity in Saliva of Children with and without Early Childhood Caries: A Randomized Clinical Trial.

Early childhood caries (ECC) is one of the most prevalent diseases in children worldwide. Early childhood caries is driven by a dysbiotic state of oral microorganisms, mainly caused by a sugar-rich diet. Additionally, poor oral hygiene or insufficient dental plaque removal leads to the rapid progression of ECC. Early childhood caries leads not only to dental destruction and pain in children but also affects the quality of life of the caregivers.Additionally, upon neutrophil activation at inflammatory locations, these proteases are externalized in an active state, aiding in the control of inflammatory and immunological responses. Any enzyme that catalyzes proteolysis reactions is known as a protease. Proteases are produced by human glands or derived from microbes in the oral cavity. Additionally, the oropharyngeal mucosae and crevicular fluids are sources of protease. This study is aimed at the estimation and correlation of salivary protease enzymatic activity in the saliva of children with and without ECC. A total of 50 children were included in the study, which was divided into two groups: group I (caries-active) and group II (caries-free)-each consisting of 25 children. Unstimulated saliva samples were collected and subjected to a spectrophotometer for analysis. Salivary protease levels were estimated and correlated between caries-active and caries-free children. The correlation between caries score and salivary protease activity was statistically significant with a moderate correlation. The comparison of mean salivary protease activity between caries-active and caries-free groups was statistically significant. However, the comparison of salivary protease activity based on different age-groups was not statistically significant, whereas gender and caries scores in group A were statistically significant. In conclusion, there is a substantial correlation between salivary protease enzyme levels and the severity of dental caries, and an increase in salivary protease enzyme levels is linked to a considerable rise in caries severity. As a result, prevention may be possible with early detection. Thimmegowda U, Kuri PN, Dhamnekar P. Role of Salivary Protease Enzymatic Activity in Saliva of Children with and without Early Childhood Caries: A Randomized Clinical Trial. Int J Clin Pediatr Dent 2024;17(8):877-880.

Biochemical Characterization of Recombinant Enterococcus faecalis EntV Peptide to Elucidate Its Antihyphal and Antifungal Mechanisms against Candida albicans.

Candida albicans is a common opportunistic fungus in humans, whose morphological switch between yeast and hyphae forms represents a key virulence trait. Developing strategies to inhibit C. albicans hyphal growth may provide insights into designs of novel antivirulent therapeutics. Importantly, the gut commensal bacterium, Enterococcus faecalis, secretes a bacteriocin EntV which has potent antivirulent and antifungal effects against C. albicans in infection models; however, hampered by the challenges to access large quantities of bioactive EntV, the detailed understanding of its mechanisms on C. albicans has remained elusive. In this work, we biochemically reconstituted the proteolytic cleavage reaction to obtain recombinant EntV88-His6 on a large preparative scale, providing facile access to the C-terminal EntV construct. Under in vitro C. albicans hyphal assay with specific inducers, we demonstrated that EntV88-His6 exhibits potent bioactivity against GlcNAc-triggered hyphal growth. Moreover, with fluorescent FITC-EntV88-His6, we revealed that EntV88-His6 enters C. albicans via endocytosis and perturbs the proper localization of the polarisome scaffolding Spa2 protein. Our findings provide important clues on EntV's mechanism of action. Surprisingly, we showed that EntV88-His6 does not affect C. albicans yeast cell growth but potently exerts cytotoxicity against C. albicans under hyphal-inducing conditions in vitro. The combination of EntV88-His6 and GlcNAc displays rapid killing of C. albicans, rendering it a promising antivirulent and antifungal agent.

Activation of the Calcium-Sensing Receptor by a Subfraction of Amino Acids Contained in Thyroid Drainage Fluid.

Hypoparathyroidism is a common sequela of thyroid surgery; in this study, we aimed at exploring the pathogenesis behind it. The following premises suggest that wound fluid might be a causative agent. (i) Parathyroid hormone secretion is under feedback control by the calcium-sensing receptor, which responds to a diverse array of activating ligands. (ii) Postoperative hypoparathyroidism arises from a secretory deficiency of the parathyroid glands. Even in patients later unaffected by hypoparathyroidism, parathyroid hormone levels drop within hours after surgery. (iii) Wound fluid is bound to enter the tissue around the thyroid bed, where the parathyroid glands are located. Its composition is shaped by a series of proteolytic reactions triggered by wounding. Using thyroid drainage as a surrogate, we addressed the possibility that wound fluid contains compounds activating the calcium-sensing receptor. Drainage fluid ultrafiltrate was found to be rich in amino acids, and on separation by HPLC, compounds activating the calcium-sensing receptor partitioned with hydrophilic matter that rendered buffer acidic. The data show that glutamate and aspartate at millimolar concentrations supported activation of the calcium-sensing receptor, an effect contingent on low pH. In the presence of glutamate/aspartate, protons activated the calcium-sensing receptor with a pH50 of 6.1, and at pH 5, produced maximal activation. This synergistic mode of action was exclusive; glutamine/asparagine did not substitute for the acidic amino acids, nor did Ca2+ substitute for protons. NPS-2143, a negative allosteric receptor modulator completely blocked receptor activation by glutamate/aspartate and by fractionated drainage fluid. Thus, wound fluid may be involved in suppressing parathyroid hormone secretion.

Capturing acyl-enzyme intermediates with genetically encoded 2,3-diaminopropionic acid for hydrolase substrate identification.

Catalytic mechanism-based, light-activated traps have recently been developed to identify the substrates of cysteine or serine hydrolases. These traps are hydrolase mutants whose catalytic cysteine or serine are replaced with genetically encoded 2,3-diaminopropionic acid (DAP). DAP-containing hydrolases specifically capture the transient thioester- or ester-linked acyl-enzyme intermediates resulting from the first step of the proteolytic reaction as their stable amide analogs. The trapped substrate fragments allow the downstream identification of hydrolase substrates by mass spectrometry and immunoblotting. In this protocol, we provide a detailed step-by-step guide for substrate capture and identification of the peptidase domain of the large tegument protein deneddylase (UL36USP) from human herpesvirus 1, both in mammalian cell lysate and live mammalian cells. Four procedures are included: Procedure 1, DAP-mediated substrate trapping in mammalian cell lysate (~8 d); Procedure 2, DAP-mediated substrate trapping in adherent mammalian cells (~6 d); Procedure 3, DAP-mediated substrate trapping in suspension mammalian cells (~5 d); and Procedure 4, substrate identification and validation (~12-13 d). Basic skills to perform protein expression in bacteria or mammalian cells, affinity enrichment and proteomic analysis are required to implement the protocol. This protocol will be a practical guide for identifying substrates of serine or cysteine hydrolases either in a complex mixture, where genetic manipulation is challenging, or in live cells such as bacteria, yeasts and mammalian cells.

Proteolytic reaction-based versatile electrochemical biosensor chip for simple and quantitative point-of-care testing

Electrochemical protease biosensors are well-suited for simple and quantitative point-of-care testing. However, there has been limited development of versatile chip formats similar to lateral-flow and glucose test strips for electrochemical proteases biosensors. This paper presents a versatile electrochemical biosensor chip for the wash-free and sensitive detection of proteases. The chip comprises (i) a reaction well, (ii) micropatterned electrodes on a glass substrate, and (iii) target protease-dependent filter paper. All (bio)chemicals required for the proteolytic reaction and signal amplification were predried on filter paper, which was then placed inside the reaction well. Upon injecting the sample solution into the reaction well, most of the dried (bio)chemicals dissolved rapidly. Wash-free and sensitive protease detection was achieved using electrochemical-enzymatic (EN) redox cycling of a redox-active species (4-amino-1-naphthol, AN) liberated from a peptide substrate through a rapid proteolytic reaction. The EN redox cycling involves a working electrode, AN, glucose dehydrogenase, and glucose. To enhance dissolvability and stability, (bio)chemicals were dried using a surfactant and stabilizer and stored at 4 °C with vacuum sealing. To minimize the effect of background variation among the different samples, a high concentration of glucose (200 mM) was used, and the difference between the two charge values obtained before and after the incubation period was measured. The biosensor chips successfully detected three proteases: thrombin, leucine aminopeptidase (LAP), and 3CL protease from coronavirus SARS-CoV-2 (Mpro). The detection limits of thrombin in artificial serum, Mpro in artificial saliva, and LAP in human serum were approximately 100, 100 pg/mL, and 8 U/L, respectively.

A versatile immunoassay based on functionalized nanoparticles for botulinum neurotoxin detection and sensor development

Botulinum neurotoxins (BoNTs) serotypes A and B are the two most common of the four BoNTs that cause the high mortality botulism disease in individuals consuming contaminated foods. The gold standard assay for BoNT detection is the live mouse bioassay, which has several major disadvantages, including tedious procedures and animal sacrifice requirements. In this study, we developed an immuno-based assay using magnetic streptavidin nanoparticles (mSNP) functionalized with specific synthetic biotinylated, 6xHis-tagged peptide substrates (Peptides A, PA and B, PB) designed for BoNT/A and BoNT/B proteolytic reactions, respectively. In the presence of active toxins that possess endopeptidase activity, upon cleavage, the released fragments with His-tag were dotted on a blotting membrane, ultimately producing color signals after incubation with anti-His antibody, alkaline phosphatase (AP)-conjugated antibody, and then AP substrates. The results showed that the efficiency of peptide-mSNP complex formation reached up to 81%, and the dot blot immunoassay allowed peptide detection from 10 ng of His-tagged peptides. Preliminary testing with the extracellular extracts from the isolated Clostridium botulinum strains indicated that the botulinum toxin in the 2020 botulism outbreak in Vietnam belonged to serotype A, the most potent BoNT. The established assay could be applied to construct a portable biosensor for BoNT detection and a high throughput device to screen potential BoNT inhibitors for drug development.

USE OF SUMO-EXPRESSION SYSTEM AND SUMO-PROTEASE FOR PRODUCTION OF ACTIVE INTERFERON α-2b

Recombinant proteins production in prokaryotic expression systems is often complicated by need of native, N-terminal formylmethionine free molecule extraction, refolding, and processing. To facilitate these stages, alternative expression systems and fusion of target proteins are being used, in particular the SUMO system. Aim. This research aimed to verify a feasibility of SUMO-protease and SUMO-expression system using to produce active interferon α-2b. The goal was to increase both expression and total yield of the target protein and enhance its quality characteristics, particularly its purity and medical safety, for potential application in acute respiratory diseases treating. Methods. The obtained SUMO-protease and SUMO-interferon hybrid protein, as well as the proteolytic reaction were analyzed by SDS-PAGE and HPLC techniques. The results confirmed that SUMO system appliance enhanced protein expression level, refolding efficiency; as well, it was useful for purification costs reduction. Conclusion. The proteolytic reaction efficiency was close to 100 %.

Integration of transcriptome and machine learning to identify the potential key genes and regulatory networks affecting drip loss in pork.

Low level of drip loss is an important quality characteristic of meat with high economic value. However, the key genes and regulatory networks contributing to drip loss in pork remain largely unknown. To accurately identify the key genes affecting drip loss in muscles postmortem, 12 Duroc × (Landrace × Yorkshire) pigs with extremely high (n ＝ 6, H group) and low (n ＝ 6, L group) drip loss at both 24h and 48h postmortem were selected for transcriptome sequencing. The analysis of differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA) were performed to find the overlapping genes using the transcriptome data, and functional enrichment and protein-protein interaction (PPI) network analysis were conducted using the overlapping genes. Moreover, we used machine learning to identify the key genes and regulatory networks related to drip loss based on the interactive genes of the PPI network. Finally, nine potential key genes (IRS1, ESR1, HSPA6, INSR, SPOP, MSTN, LGALS4, MYLK2, and FRMD4B) mainly associated with the MAPK signaling pathway, the insulin signaling pathway, and the calcium signaling pathway were identified, and a single-gene set enrichment analysis (GSEA) was performed to further annotate the functions of these potential key genes. The GSEA results showed that these genes are mainly related to ubiquitin mediated proteolysis and oxidative reactions. Taken together, our results indicate that the potential key genes influencing drip loss are mainly related to insulin signaling mediated differences in glycolysis and ubiquitin mediated changes in muscle structure and improves the understanding of gene expression and regulation related to drip loss and contributes to future molecular breeding for improving pork quality.

PROPOSAL OF THE METHOD FOR QUANTITATIVE DETERMINATION OF PROTEOLYTIC ACTIVITY OF A PANCREATIN REFERENCE STANDARD

Relevance. Pancreatin is a polyenzyme drug with lipolytic, amylolytic and proteolytic activity, and is prescribed as a replacement therapy for exocrine pancreatic insufficiency. Modern requirements for quality control of drugs involve the use of highly specific and highly sensitive methods of analysis using reference standards (RS).For RS certification it is necessary to establish the values of the certifiable characteristic. For enzyme preparations the certifiable characteristic is catalytic activity. In this connection for certification of pancreatin RS it is necessary to establish the value of its catalytic ac-tivity.The analysis of the proteolytic activity of pancreatin consists in performing a proteolysis reaction and quantifying the reaction products. The most common methods used for this purpose are those in which the quantitative content of proteolysis products is determined by measuring the optical density of the solution using tyrosine as a standard. It should be noted that the value of proteolytic activity established in this way is relative, since the measured optical density actually corresponds to a mixture of tyrosine and tryptophan. The absolute method for determining proteolytic activity is the Kjeldahl method for analyzing fragmented peptide nitrogen. Aim. Propose the method for quantitative determination of the proteolytic activity of a pancreatin RS. Material and methods. The object of the study was a pharmaceutical substance pancreatin. Quantitative determination of proteolytic activity in pancreatin substance samples was performed according to the proposed method by determining protein nitrogen by the Kjeldahl method after the en-zymatic reaction. Results. As a result of testing the samples of pancreatin substance using the proposed method of quantitative analysis of proteolytic activity, we ob-tained reliable results, indicating the high sensitivity, accuracy and reliability of this method. Conclusions. The present study proposed the method for quantitative determination of the proteolytic activity of pancreatin substance and evaluated the acceptability of its use in the quality control of a pancreatin RS. Conflict of interest. The authors declare that they have no obvious and potential conflicts of interest related to the publication of this article.

Magnetically-assisted electrochemical immunoplatform for simultaneous detection of active and total prostate-specific antigen based on proteolytic reaction and sandwich affinity analysis

Simultaneous detection of active and inactive proteases is clinically meaningful for improving diagnostic specificity. In this work, we reported an electrochemical method for simultaneous immunoassays of active and total proteases. Magnetic beads (MBs) were used as the solid supports for immobilization of capture antibodies and enrichment of targets. For the detection of active protease, the proteolytic-reaction-based analysis was carried out by the generation of Cu2+-binding peptide, in which a label-free peptide was used as the proteolytic substrate. The redox potential of the resulting peptide-Cu2+ complex was intrinsically distinguished from that of free Cu2+, thus allowing the “signal-on” detection of active protease. For the immunoassay of total protease in a sandwich-like format, electroactive metal-organic frameworks (Cu-MOFs) were used as the signal tags. The captured Cu-MOFs could directly produce a well-defined electrochemical signal from the reduction of Cu2+ ions. The analytical performances of the immunoplatform were evaluated by determining the model analytes of free and total prostate-specific antigen (fPSA and tPSA) in buffer and serum. The detection limits were found to be 0.3 pM for fPSA and 2 pM for tPSA. This work proposed a new strategy for simultaneous detection of active and total proteases, which should be evaluable for clinical diagnosis and treatment of protease-relative diseases.

Generation of kokumi γ-glutamyl short peptides in Spanish dry-cured ham during its processing

The typical dry-cured ham flavor is rich in umami and brothy perceptions, for which short peptides may contribute. Particularly, γ-glutamyl peptides could be the responsible of these previously reported attributes, as they exert a synergistic interaction with other basic tastes and modify the intensity of salty, sweet, and umami tastes. The content of peptides has been reported to evolve along the processing, but no kokumi γ-glutamyl peptides have been identified in Spanish dry-cured hams yet. In this research, nine γ-glutamyl dipeptides (γ-EA, γ-EC, γ-EE, γ-EF, γ-EL, γ-EM, γ-EV, γ-EW, and γ-EY) and two γ-glutamyl tripeptides (GSH and γ-EVG) have been quantitated at 6, 12, 18 and 24 months of traditional processing of Spanish dry-cured ham by performing a Q Exactive Orbitrap-based tandem mass spectrometry. The results show an increase of γ-EA, γ-EE, γ-EF, γ-EL, γ-EM and γ-EVG, obtaining maximums at 24 months of curing ranging from 0.14 (γ-EVG) to 18.86 (γ-EL) μg/g dry-cured ham. Otherwise, γ-EV, γ-EW and γ-EY accumulated until the 18th month of storage to 15.10, 0.54 and 3.17 μg/g dry-cured ham, respectively; whereas γ-EC and GSH amounts decreased starting from 0.0676 and 4.41 μg/g dry-cured ham, respectively at earlier stages. The concentration dynamics of these compounds may be linked with proteolytic and oxidative reactions during processing. In addition, due to their synergistic effect on kokumi activity, this could constitute insights of the brothy perceptions of dry-cured ham, and these peptides probably contribute to the sensory differences existing in long processed Spanish dry-cured hams.

Flaxseed- and chia seed-derived protein hydrolysates exhibiting enhanced in vitro antidiabetic, anti-obesity, and antioxidant properties

This study investigated in vitro antidiabetic, anti-obesity, and antioxidant activities of chia seed protein hydrolysates (CSPHs) and flaxseed protein hydrolysates (FSPHs) generated using three food-grade proteases at the hydrolysis at intervals of 120 min for 6 h. The inhibitory potentials of CSPHs and FSPHs on the enzymatic biomarkers related to diabetes (α-glucosidase and DPP-4) and obesity (pancreatic lipase and cholesteryl esterase) were determined. The antioxidant capacity of CSPHs and FSPHs was also assessed using ABTS, DPPH, and FRAP antioxidant assays. Increasing the proteolytic reaction time was shown to significantly increase the degree of hydrolysis values of CSPHs and FSPHs. Protein hydrolysates resulted in higher inhibitory potential against enzymatic biomarkers related to metabolic diseases (diabetes and obesity) as well as higher antioxidant activities compared with undigested proteins. Overall, the lowest IC50 inhibition values demonstrated in DPP-IV were observed by alcalase-catalyzed CSPH at 6 h (IC50 = 272.17 μg/mL) and bromelain-catalyzed FSPH at 6 h (IC50 = 338.70 μg/mL). The strongest inhibition of pancreatic lipase was observed in bromelain-catalyzed hydrolysates (IC50 = 292.43 μg/mL and IC50 = 307.62 μg/mL for CSPH and FSPH, respectively) after 6 h. These findings indicate that CSPHs and FSPHs exhibited enhanced antidiabetic, anti-obesity, and antioxidant properties. Therefore, these hydrolysates can be successfully used for their applications in the development of functional foods.

Proteolytic rewiring of mitochondria by LONP1 directs cell identity switching of adipocytes.

Mitochondrial proteases are emerging as key regulators of mitochondrial plasticity and acting as both protein quality surveillance and regulatory enzymes by performing highly regulated proteolytic reactions. However, it remains unclear whether the regulated mitochondrial proteolysis is mechanistically linked to cell identity switching. Here we report that cold-responsive mitochondrial proteolysis is a prerequisite for white-to-beige adipocyte cell fate programming during adipocyte thermogenic remodelling. Thermogenic stimulation selectively promotes mitochondrial proteostasis in mature white adipocytes via the mitochondrial protease LONP1. Disruption of LONP1-dependent proteolysis substantially impairs cold- or β3 adrenergic agonist-induced white-to-beige identity switching of mature adipocytes. Mechanistically, LONP1 selectively degrades succinate dehydrogenase complex iron sulfur subunit B and ensures adequate intracellular succinate levels. This alters the histone methylation status on thermogenic genes and thereby enables adipocyte cell fate programming. Finally, augmented LONP1 expression raises succinate levels and corrects ageing-related impairments in white-to-beige adipocyte conversion and adipocyte thermogenic capacity. Together, these findings reveal that LONP1 links proteolytic surveillance to mitochondrial metabolic rewiring and directs cell identity conversion during adipocyte thermogenic remodelling.

Effect of Vacuum Packaging on the Biochemical, Viscoelastic, and Sensory Properties of a Spanish Cheese during Chilled Storage

The unique qualities of Spanish cheeses, such as the San Simón da Costa (SSC) cheese, are protected by the Protected Designation of Origin (PDO) status. The technological importance of chilled storage at 4 °C of vacuum-packaged (V) and natural (N) (unpackaged) cheeses was examined. For this purpose, the physico-chemical, biochemical, mechanical (puncture tests), viscoelastic (oscillatory and transient tests) and sensory properties of V and N cheeses were compared and analysed. During chilled storage, the caseins in V cheeses did not undergo proteolytic reactions. Low temperature maintained a low intensity of proteolytic phenomena for up to 6 months. Lipolysis was more intense in the N than in the V samples. The moisture content decreased in the N cheeses during chilled storage, and thus, the casein matrix concentration and ionic strength increased, resulting in an increase in the gel strength (S) parameter and complex modulus (G*), and the conformational stability-high stress amplitude (σmax). The low and similar values of the n' and n'' exponents (mechanical spectra) and the n parameter (transient tests) indicated the high degree of the temporal stability of the cheese network in both the N and V samples, irrespective of storage time. Likewise, the similar values of the phase angle (δ) for the N and V cheeses during storage indicate energy-stable bonds in the SSC cheese matrix. The attributes of the oral tactile phase (firmness, friability, gumminess, and microstructure perception), mechanical parameters and viscoelastic moduli enabled the discrimination of the packaged and unpackaged cheeses. Cheeses chilled and stored without packaging were awarded the highest scores for sensory attributes (preference) by trained panellists.

Different modeling approaches in the simulation of extrinsic coagulation factor X activation: Limitations and areas of applicability.

Proteolytic reactions on the phospholipid membrane surface, so-called "membrane-dependent" reactions, play central role in the process of blood clotting. One particularly important example is FX activation by the extrinsic tenase (VIIa/TF). Here we constructed three mathematical models of FX activation by VIIa/TF: (A) a homogeneous "well-mixed" model, (B) a two-compartment "well-mixed" model, (C) a heterogeneous model with diffusion, to investigate the impact and importance of inclusion of each complexity level. All models provided good description of the reported experimental data and were equivalently applicable for <40 μM of phospholipids. Model C provided better predictions than A, B in the presence of TF-negative phospholipid microparticles. Models predicted that for high TF surface density (STF ) and FX deficiency the FX activation rate was limited by the rate of FX binding to the membrane. For low STF and excess of FX the reaction rate was limited by the tenase formation rate. The analysis of the substrate delivery pathways revealed that FX bound to VIIa/TF predominantly from solution for STF >2.8 × 10-3 nmol/cm2 and from the membrane for lower STF . We proposed the experimental setting to distinguish between the collision-limited and non-collision-limited binding. The analysis of models in flow and non-flow conditions revealed that the model of a vesicle in flow might be substituted by model C in the absence of the substrate depletion. Together, this study was the first which provided the direct comparison of more simple and more complex models. The reaction mechanisms were studied in a wide range of conditions.

Proteolysis of Micellar β-Casein by Trypsin: Secondary Structure Characterization and Kinetic Modeling at Different Enzyme Concentrations.

Tryptic proteolysis of protein micelles was studied using β-casein (β-CN) as an example. Hydrolysis of specific peptide bonds in β-CN leads to the degradation and rearrangement of the original micelles and the formation of new nanoparticles from their fragments. Samples of these nanoparticles dried on a mica surface were characterized by atomic force microscopy (AFM) when the proteolytic reaction had been stopped by tryptic inhibitor or by heating. The changes in the content of β-sheets, α-helices, and hydrolysis products during proteolysis were estimated by using Fourier-transform infrared (FTIR) spectroscopy. In the current study, a simple kinetic model with three successive stages is proposed to predict the rearrangement of nanoparticles and the formation of proteolysis products, as well as changes in the secondary structure during proteolysis at various enzyme concentrations. The model determines for which steps the rate constants are proportional to the enzyme concentration, and in which intermediate nano-components the protein secondary structure is retained and in which it is reduced. The model predictions were in agreement with the FTIR results for tryptic hydrolysis of β-CN at different concentrations of the enzyme.

Characterization of Low-Molecular-Weight Collagen from Korean Native Chicken Feet Hydrolyzed Using Alcalase.

The aims of this study were to optimize the preparation of low-molecular-weight collagen using a proteolytic enzyme (alcalase) derived from the feet of Korean native chickens, and to characterize the process of collagen hydrolysis. Foreign bodies from chicken feet were removed using ultrasonication at 28 kHz with 1.36 kW for more than 25 min. The hydrolytic pattern and molecular weight distribution of enzyme-treated collagen from chicken feet were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography, respectively. Ideally, chicken feet should be treated at 100°C for 8 h to obtain a high collagen content using hot water extraction. The collagen content of the chicken foot extract was 13.9 g/100 g, and the proportion of low-molecular-weight collagen increased with increasing proteolytic enzyme concentration and reaction time. When treated with 1% alcalase, the average molecular weight of collagen decreased rapidly to 4,929 Da within 5 h and thereafter decreased at a slower rate, reaching 4,916 Da after 7 h. Size exclusion chromatography revealed that low-molecular-weight collagen peptides of approximately 1,000-5,000 Da were obtained after hydrolysis with 1% alcalase for 1 h.

Deforestation alters protease regulation by amino acids

Protease activity is regulated by numerous complex mechanisms in soil. In this study, we investigated the regulatory effect of the end product of the proteolytic reaction, amino acids, on protease activity in two forest soils and their deforested counterparts, which are located in two mountainous regions (Delvara and Minoodasht) of Iran. The protease activity was measured during the incubation of the soils treated with l-arginine and l-histidine. Our result indicated that, in general, amending the soils with the amino acids caused significant increases in the protease activity after a lag time of 2–5 days. However, the stimulatory effects of the amino acids on the protease activity were clearly affected by the land use so that the activity ratios of the amino acid-amended to the non-amended soils were between 3.1 and 3.5 in forest and 4.7–4.9 in the deforested counterparts of Delvara soil. Similarly, in the Minoodasht region, the ratio ranged 1.6–2.0 for the forest and 4.4–6.5 for the deforested counterpart. In addition, deforestation in the Minoodasht region significantly decreased indices of protease production and persistence. In l-arginine-amended soils, deforestation decreased protease production by 25 % while the protease persistence was decreased by 88 %. In l-histidine-amended soils, protease production was decreased by 70 % and protease persistence was decreased by 59 %, due to deforestation. Overall, we conclude that amending the soils with the amino acids increased the activity of protease and that the rate of increase is clearly affected by the management history.

Sensitive Affinity-Based Biosensor Using the Autocatalytic Activation of Trypsinogen Mutant by Trypsin with Low Self-activation.

Self-propagating autocatalytic reactions of proteases that can provide high signal amplification have not been applied to affinity-based biosensors owing to the limited number of fast autocatalytic proteolytic reactions available and the self-activation of protease proenzymes. Here, we report that a self-propagating autocatalytic reaction based on the autocatalytic activation of the trypsinogen mutant by trypsin facilitates high signal amplification and a low background level, resulting in a low detection limit for prostate-specific antigen (PSA). A commercially available trypsinogen mutant minimizes the self-activation of trypsinogen by trypsinogen. Trypsin, which is used as a catalytic label in a sandwich-type immunosensor, converts the trypsinogen mutant into trypsin; the generated trypsin then further converts the trypsinogen mutant into trypsin. The autocatalytically produced trypsin proteolytically cleaves the peptide bond of a trypsin substrate, resulting in the liberation of electrochemically active 4-aminophenol (AP). The electrochemical oxidation of AP at a modified indium tin oxide (ITO) electrode induces electrochemical-chemical redox cycling involving the ITO electrode, AP, and a reductant. The triple combination of autocatalytic activation, proteolytic cleavage, and redox cycling results in a high electrochemical signal level. The detection limit for PSA obtained using a trypsin label and trypsinogen (∼7 pg/mL) is lower than that obtained using a trypsin label alone (∼100 pg/mL). This study demonstrated that autocatalytically activating a proenzyme is a very useful method for highly amplifying signals.

Using a peptide-based mass spectrometry approach to quantitate proteolysis of an intact heterogeneous procollagen substrate by BMP1 for antagonistic antibody screening.

Proteases are critical proteins involved in cleaving substrates that may impact biological pathways, cellular processes, or disease progression. In the biopharmaceutical industry, modulating the levels of protease activity is an important strategy for mitigating many types of diseases. While a variety of analytical tools exist for characterizing substrate cleavages, in vitro functional screening for antibody inhibitors of protease activity using physiologically relevant intact protein substrates remains challenging. In addition, detecting such large protein substrates with high heterogeneity using high-throughput mass spectrometry screening has rarely been reported in the literature with concerns for assay robustness and sensitivity. In this study, we established a peptide-based in vitro functional screening assay for antibody inhibitors of mouse bone morphogenic protein 1 (mBMP1) metalloprotease using a heterogeneous recombinant 66-kDa mouse Procollagen I alpha 1 chain (mProcollagen) substrate. We compared several analytical tools including capillary gel electrophoresis Western blot (CE-Western blot), as well as both intact protein and peptide-based mass spectrometry (MS) to quantitate the mBMP1 proteolytic activity and its inhibition by antibodies using this heterogeneous mProcollagen substrate. We concluded that the peptide-based mass spectrometry screening assay was the most suitable approach in terms of throughput, sensitivity, and assay robustness. We then optimized our mBMP1 proteolysis reaction after characterizing the enzyme kinetics using the peptide-based MS assay. This assay resulted in Z' values ranging from 0.6 to 0.8 from the screening campaign. Among over 1200 antibodies screened, IC50 characterization was performed on the top candidate hits, which showed partial or complete inhibitory activities against mBMP1.