Nonspecific Protease Research Articles

TR-107, an Agonist of Caseinolytic Peptidase Proteolytic Subunit, Disrupts Mitochondrial Metabolism and Inhibits the Growth of Human Colorectal Cancer Cells.

Oxidative phosphorylation is an essential metabolic process for cancer proliferation and therapy resistance. The ClpXP complex maintains mitochondrial proteostasis by degrading misfolded proteins. Madera Therapeutics has developed a class of highly potent and selective small-molecule activators (TR compounds) of the ClpXP component caseinolytic peptidase proteolytic subunit (ClpP). This approach to cancer therapy eliminates substrate recognition and activates nonspecific protease function within mitochondria, which has shown encouraging preclinical efficacy in multiple malignancies. The class-leading compound TR-107 has demonstrated significantly improved potency in ClpP affinity and activation and enhanced pharmacokinetic properties over the multitargeting clinical agent ONC201. In this study, we investigate the in vitro efficacy of TR-107 against human colorectal cancer cells. TR-107 inhibited colorectal cancer cell proliferation in a dose- and time-dependent manner and induced cell cycle arrest at low nanomolar concentrations. Mechanistically, TR-107 downregulated the expression of proteins involved in the mitochondrial unfolded protein response and mitochondrial DNA transcription and translation. TR-107 attenuated oxygen consumption rate and glycolytic compensation, confirming inactivation of oxidative phosphorylation and a reduction in total cellular respiration. Multiomics analysis of treated cells indicated a downregulation of respiratory chain complex subunits and an upregulation of mitophagy and ferroptosis pathways. Further evaluation of ferroptosis revealed a depletion of antioxidant and iron toxicity defenses that could potentiate sensitivity to combinatory chemotherapeutics. Together, this study provides evidence and insight into the subcellular mechanisms employed by colorectal cancer cells in response to potent ClpP agonism. Our findings demonstrate a productive approach to disrupting mitochondrial metabolism, supporting the translational potential of TR-107.

Discovery of a Highly Promising Disulfide Derivative Scaffold as Inhibitor of SARS-CoV-2 Main Protease.

The main protease (Mpro) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) represents a promising target for antiviral drugs aimed at combating COVID-19. Consequently, the development of Mpro inhibitor is an ideal strategy for combating the virus. In this study, we identified twenty-two dithiocarbamates (1 a-h), dithiocarbamate-Cu(II) complexes (2 a-hCu) and disulfide derivatives (2 a-e, 2 i) as potent inhibitors of Mpro, with IC50 value range of 0.09-0.72, 0.9-24.7, and 15.1-111 μM, respectively, through FRET screening. The enzyme kinetics, inhibition mode, jump dilution, and DTT assay revealed that 1 g may be a partial reversible inhibitor, while 2 d and 2 f-Cu are the irreversible and dose- and time-dependent inhibitors, potentially covalently binding to the target. Binding of 2 d, 2 f-Cu, and 1 g to Mpro was found to decrease the stability of the protein. Additionally, DTT assays and thermal shift assays indicated that 2 f-Cu and 2 d are the nonspecific and promiscuous cysteine protease inhibitor. ICP-MS implied that the inhibitory activity of 2 f-Cu may stem from the uptake of Cu(II) by the enzyme. Cytotoxicity assays demonstrated that 2 d and 1 g exhibit low cytotoxicity, whereas 2 f-Cu show certain cytotoxicity in L929 cells. Overall, this work presents two promising scaffolds for the development of Mpro inhibitors to combat COVID-19.

Streamlining Protein Fractional Synthesis Rates Using SP3 Beads and Stable Isotope Mass Spectrometry: A Case Study on the Plant Ribosome.

Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context-dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context-dependent functionality of r-protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to high-resolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot-gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r-proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r-proteins and normalize their intensities by the sum of r-protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r-protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r-proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r-proteins determined by stable isotope-assisted mass spectrometry. Our method purifies the r-proteins in a reversibly denatured state, which offers the possibility for combined top-down and bottom-up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r-proteins. As such, our method is better suited to conserve post-translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r-proteins to select an appropriate protease for digestion. We avoid non-specific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r-proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find "false" enrichments in non-labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r-protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top-down and bottom-up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto high-resolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements. Key features • Efficient purification of the ribosomal proteome: streamlined procedure for the specific purification of the ribosomal proteome or complex Ome. • Accurate calculation of fractional synthesis rates: robust method for calculating fractional protein synthesis rates in macromolecular complexes under different physiological steady states. • Holistic ribosome methodology focused on plants: comprehensive approach that provides insights into the ribosomes and translational control of plants, demonstrated using cold acclimation [1]. • Tailored strategies for stable isotope labeling in plants: methodology focusing on materials and labeling considerations specific to free and proteinogenic amino acid analysis [2].

Abstract 5776: Target identification, selectivity profiling and binding site mapping of small molecule and peptide drugs by LiP-MS

Abstract Target identification in the cellular context is a critical step for both target-based and phenotypic drug discovery. Limited proteolysis coupled with mass spectrometry (LiP-MS) has emerged as a powerful technique for target deconvolution of small molecules or peptides in cell lysate without compound modification or genetic manipulation of cell lines [1,2,3,4]. Utilizing a non-specific protease under well-controlled conditions, LiP-MS exploits drug-induced structural alteration or steric hindrance on protein targets and utilizes quantitative mass spectrometry to probe over 250’000 peptides covering more than 9000 proteins in the proteome. To aid target ID, this workflow incorporates a 7-concentration dose response experiment and a machine-learning framework [1] to compute a LiP-score to rank identified target proteins, as well as predict potential binding site. In this study, we present target deconvolution results on four small molecule compounds and one peptide compound with very distinct pharmacological profile: 1) staurosporine, a broad-specific kinase inhibitor; 2) roniciclib, a failed clinical-stage pan-CDK inhibitor; 3) selumetinib, an allosteric, non-ATP-competitive MEK 1 and 2 inhibitor; 4) NST-628, a newly developed non-degradating molecular glue inhibiting the RAS/MAPK pathway; and 5) Acetyl-calpastatin, a 24 amino-acid polypeptide inhibitor of calpain 1 and 2. Target ID experiment with staurosporine and roniciclib yielded > 150 and 68 kinase targets were identified respectively. For the pan-kinase inhibitor roniciclib, both cell-cycle CDKs (CDK1/cyclin B, CDK2/cyclin E, CDK4 cyclin D) and transcriptional CDK9 were among the targets. For Selumetinib, LiP-score ranking identified MEK1 and MEK2 as the top two targets, and only kinases among a total of 23 targets. Furthermore, mapping the LiP-peptides from this experiment onto MEK1 showed that they are in close proximity to the selumetinib allosteric binding site on co-crystal structure. Last but not least, we assessed the target landscape of NST-628, a novel non-degrading molecular glue and identified MEK1 and MEK2 clearly as the primary targets. This finding is in line with orthogonal data obtained from X-ray crystallography [5]. Collectively, this data demonstrates that LiP-MS can be deployed to effectively identify protein drug targets and predict binding sites in complex cellular millieu independent of the compound’s mechanism of action and without compound modification or labeling. These capabilities make LiP-MS a powerful addition to the target deconvolution toolbox. [1] Piazza and Beaton et al. Nature Comm 2020 [2] Hendricks and Beaton et al. ACS Chem Bio 2021 [3] AACR 2022 [4] AACR 2023 [5] AACR-NCI-EORTC 2023 Citation Format: Fabio Sabino, Martin Soste, Dominique Kamber, Maria Stella Ritorto, Klaus Hoeflich, Michael Hale, Bradley Quade, Xin Huang, Nigel Beaton, Roland Bruderer, Yuehan Feng, Lukas Reiter. Target identification, selectivity profiling and binding site mapping of small molecule and peptide drugs by LiP-MS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5776.

Enzymatic activity of extracts from higher fungi for manufacturing fermented dairy products

The present study investigates the enzymatic activity and chemical composition of extracts obtained from the substrate mycelium of higher fungi. The investigated object is the biomass of fungi Piptoporus betulinus (substrate mycelium) gathered after solid-phase cultivation on natural substrate. The extracts were obtained using distilled water (pH=7.0), acetate (pH=4.7) and phosphate (pH=7.4) buffers, and Mcllvaine buffer (pH=4.0). Milk-clotting, proteolytic, cellulosolytic and lipolytic activity, as well as protein content, were determined in both aqueous or buffer extracts. As a result, the values of cellulosolytic (3.75–3.90 units/g), lipolytic (40.00–44.24 units/g) and milk-clotting (65.80–66.60 units/mL) activity of the substrate mycelium was determined. These values differ slightly in the extracts prepared on distilled water and buffers. Moreover, the concentration of protein substances in the native aqueous extract from the substrate mycelium of P. betulinus was 14.50 mg/mL. The values of proteolytic activity varied from 0.22 to 0.78 units/mL. Distilled water was found to be the most effective solvent for achieving high values of milk-clotting activity. Extract purification by microfiltration or with bentonite leads to a significant decrease in protein concentration (up to 5.90 mg/mL), cellulose(up to 1.40 units/g), lipo(up to 5.30 units/g), and proteolytic (up to 0.11 units/mL) activity, and an increase in milk-clotting activity values (up to 285.80 units/mL). The bentonite sorbs cellulosolytic, lipolytic, and non-specific proteolytic enzymes, resulting in a noticeable increase in the value of milk-clotting activity. In the aqueous extract of P. betulinus fungi, a high ratio of milk-clotting to proteolytic activity was found with a value of 2598.20. This may lead to an increased yield and improvement of the organoleptic properties of cheese and its storage period.

Tutorial review for peptide assays: An ounce of pre-analytics is worth a pound of cure

The recent increase in peptidomimetic-based medications and the growing interest in peptide hormones has brought new attention to the quantification of peptides for diagnostic purposes. Indeed, the circulating concentrations of peptide hormones in the blood provide a snapshot of the state of the body and could eventually lead to detecting a particular health condition. Although extremely useful, the quantification of such molecules, preferably by liquid chromatography coupled to mass spectrometry, might be quite tricky. First, peptides are subjected to hydrolysis, oxidation, and other post-translational modifications, and, most importantly, they are substrates of specific and nonspecific proteases in biological matrixes. All these events might continue after sampling, changing the peptide hormone concentrations. Second, because they include positively and negatively charged groups and hydrophilic and hydrophobic residues, they interact with their environment; these interactions might lead to a local change in the measured concentrations. A phenomenon such as nonspecific adsorption to lab glassware or materials has often a tremendous effect on the concentration and needs to be controlled with particular care. Finally, the circulating levels of peptides might be low (pico- or femtomolar range), increasing the impact of the aforementioned effects and inducing the need for highly sensitive instruments and well-optimized methods. Thus, despite the extreme diversity of these peptides and their matrixes, there is a common challenge for all the assays: the need to keep concentrations unchanged from sampling to analysis. While significant efforts are often placed on optimizing the analysis, few studies consider in depth the impact of pre-analytical steps on the results. By working through practical examples, this solution-oriented tutorial review addresses typical pre-analytical challenges encountered during the development of a peptide assay from the standpoint of a clinical laboratory. We provide tips and tricks to avoid pitfalls as well as strategies to guide all new developments. Our ultimate goal is to increase pre-analytical awareness to ensure that newly developed peptide assays produce robust and accurate results.

The role of nonspecific proteolysis in the development of alterative changes in lung tissue when using electronic cigarettes

In recent years, the number of electronic cigarettes (ES) users has been growing steadily around the world. ES heat and vaporize components of a mixture of propylene glycol, vegetable glycerin, nicotine, and flavors. Reports of severe forms of lung injury caused by the use of ES, some of which have resulted in death, urge studying the pathogenetic mechanisms of such complications. One of the mechanisms is the activation of nonspecific proteolysis accompanied by the destruction of connective tissue components of the blood-air barrier.Aim of the study. To study the dynamics of the proteinase-inhibitor system indicators and its role in the morphological changes of lung tissue in experimental modeling of the effects of ES.Methods. Experimental studies were performed in 24 white male Wistar rats weighing 160 – 180 g. ES use of was simulated by intermittent exposure to smoke mixture vapors using a POD system connected to a two-channel compressor.Results. Analysis of the state of the nonspecific proteinase inhibitory system in rat broncho-alveolar lavage and blood serum and the results of morphological studies indicate activation of trypsin-like proteinases and depletion of the local antitryptic potential, leading to biochemical changes with subsequent structural changes in lung tissue. The latter are characterized by congestion, leukocyte infiltration, and stromal proliferation. In addition, exposure to nicotine-containing mixtures has been shown to affect the proteinase inhibitory system and blood serum composition, which is associated with a compensatory increase in antitrypsin activity.Conclusion. The results of the study indicate the activation of trypsin-like proteinases in broncho-alveolar lavage, which have a damaging effect on lung tissue, leading to disturbances in tissue hemodynamics, extravasation of leukocytes, remodeling and thickening of the blood-air barrier.

N-Acetylation of Biodegradable Supramolecular Peptide Nanofilaments Selectively Enhances Their Proteolytic Stability for Targeted Delivery of Gold-Based Anticancer Agents.

Peptide materials are promising for various biomedical applications; however, a significant concern is their lack of stability and rapid degradation in vivo due to non-specific proteolysis. For materials specifically designed to respond to disease-specific proteases, it would be desirable to retain high susceptibility to target proteases while minimizing the impact of non-specific proteolysis. We describe N-terminal acetylation as a simple synthetic modification of amphiphilic self-assembling peptides that contain an MMP-9-cleavable segment and form soluble, nanoscale filaments. We found that the N-terminus capping of these peptides did not significantly impact their self-assembly behavior, critical aggregation concentration, or ability to encapsulate hydrophobic payloads. By contrast, their proteolytic stability in human plasma (especially for anionic peptide sequences) was considerably increased while susceptibility to hydrolysis by MMP-9 was retained when compared to non-acetylated peptides, especially during the first 12 h. We note, however, that due to the longer time scale required for in vitro studies (72 h), non-specific proteolysis of both anionic acetylated peptides leads to similar activity in vitro despite differing MMP-9 kinetics during the early stages. Overall, the enhanced stability against non-specific proteases, combined with the ability of these nanofilaments to enhance the effectiveness of gold-based drugs toward cancerous cells compared to healthy cells, brings these acetylated peptide filaments a step closer toward clinical translation.

Probing the mechanism of reduced in vivo potency of insulin detemir

ABSTRACT Background Insulin detemir (IDet) is an insulin analog used to treat diabetes. IDet shows full efficacy but reduced potency compared to human insulin (HI) in both man and rat. In contrast, in pigs and dogs, IDet appears to have full in vivo potency. Non-receptor mediated degradation (NRMD) has previously been suggested as an explanation for the low potency of IDet, but this hypothesis has not been investigated further until now. Bacitracin is a nonspecific protease inhibitor which we hypothesized could inhibit NRMD of IDet in rats. Research design and methods Healthy male rats instrumented with permanent catheters underwent euglycemic clamp during constant infusion of either HI or IDet at effect-matched doses with co-infusion of vehicle or bacitracin. Results Plasma concentrations of IDet increased significantly (p < 0.005) during bacitracin compared to vehicle co-infusion and the concomitant increase in glucose infusion rate (GIR, p < 0.001) required to maintain euglycemic clamp indicates that the IDet rescued from NRMD indeed was active. No significant differences were detected with co-infusions of HI with either bacitracin or vehicle. Conclusions A large proportion of NRMD of IDet which can be inhibited by bacitracin may partly explain the reduced potency of IDet observed in rats and likely also in man.

Comparison of Pronase versus Manual Dechorionation of Zebrafish Embryos for Small Molecule Treatments.

Zebrafish are a powerful animal model for small molecule screening. Small molecule treatments of zebrafish embryos usually require that the chorion, an acellular envelope enclosing the embryo, is removed in order for chemical compounds to access the embryo from the bath medium. For large-scale studies requiring hundreds of embryos, manual dechorionation, using forceps, can be a time-consuming and limiting process. Pronase is a non-specific protease that is widely used as an enzymatic alternative for dechorionating zebrafish embryos. However, whether pronase treatments alter the effects of subsequent small molecule treatments has not been addressed. Here, we provide a detailed protocol for large-scale pronase dechorionation of zebrafish embryos. We tested whether pronase treatment can influence the efficacy of drug treatments in zebrafish embryos. We used a zebrafish model for Duchenne muscular dystrophy (DMD) to investigate whether the efficacies of trichostatin-A (TSA) or salermide + oxamflatin, small molecule inhibitors known to ameliorate the zebrafish dmd muscle degeneration phenotype, are significantly altered when embryos are treated with pronase versus manual dechorionation. We also tested the effects of pronase on the ability of the anthracycline cancer drug doxorubicin to induce cardiotoxicity in zebrafish embryos. When comparing pronase- versus forceps-dechorionated embryos used in these small molecule treatments, we found no appreciable effects of pronase on animal survival or on the effects of the small molecules. The significant difference that was detected was a small improvement in the ability of salermide + oxamflatin to ameliorate the dmd phenotype in pronase-treated embryos when compared with manual dechorionation. Our study supports the use of pronase treatment as a dechorionation method for zebrafish drug screening experiments.

Optimization of nonspecific protease activity fabrication by Bacillus subtilis N30 isolated from Taiwan using different models of response surface methodology

This study aimed to identify the best growth conditions for Bacillus subtilis N30 to produce viable cell counts, pH values, and protease activity through fermentation. The research employed response surface criteria and desirability function analysis. Box-Behnken and central composite designs (BBD and CCD) were used to determine the number of processing variables and experimental runs. The results of screening single factor experimental design as maltose content (0.5–1.5% w/v), malt extract concentration (0.25%–0.75% w/v), and incubation time between 30 and 42 h were applied for coded levels (−1, 0, +1) of each independent variable correlated to steps of 0.5, 0.25, and 6, respectively. Viable cell count, pH level, and protease activity were response variables. Analysis of variance (ANOVA) revealed no significant difference when using CCD but significant linear, quadratic, and interaction influences of fermentation parameters on the response of protease activity when using BBD. Both the BBD and CCD models did not fit the viable cell count and pH. According to numerical optimization, the ideal fermentation parameters were a maltose concentration of 1.19%, a malt extract of 0.56%, and a culture time of 33 h, with a predicted protease activity yield of 5.88 U/ml. This optimum condition for growing Bacillus subtilis N30 with high protease activity will be applied in the fermentation of soy milk since it contains many short-chain peptides (2–20 amino acids), which may help prevent and manage high blood pressure for human beings.

Impact of Fusarium Head Blight on Wheat Flour Quality: Examination of Protease Activity, Technological Quality and Rheological Properties

Wheat infections caused by Fusarium represent a global agricultural problem that reduces grain yield and negatively impacts wheat’s technological and rheological quality. Although fungal proteases or an increase in endogenous proteases due to Fusarium infection could negatively influence wheat storage proteins and dough performance, little research has been performed on either of these topics. The primary objective of this study was to identify the effect of Fusarium infection on protease activity in 25 wheat cultivars grown in two distinct locations in eastern Croatia. Apart from proteolytic activity, this paper describes the impact of Fusarium head blight (FHB) infection on the technological quality parameters of wheat flour and the dough’s rheological properties. The first treatment consisted of naturally grown, healthy wheat without fungicides, while the second treatment utilized wheat varieties subjected to intense FHB infection. Protein and wet gluten content in wheat grain and flour of uninfected cultivars were heavily influenced by testing location, soil type, and quality. Fusarium infection increased the activity of nonspecific proteases by 43% in flour samples from Osijek and 125% in flour samples from Tovarnik. Estimates of effect size showed that FHB infection had twice as big an effect on protease activity in Tovarnik as in Osijek, and a similar trend was found for dough softening. Moreover, the infection significantly impacted wheat cultivars’ extensograph values, indicating a lower resistance to stretching, extensibility, and total stretching energy in infected flour samples, indicating that dough functionality and volume loss can be attributed to exogenous fungal proteases. Still, the magnitude of the effect varied depending on the growth location and the cultivar’s traits. Multivariate data analysis identified three clusters of wheat cultivars, each with varying degrees of the Fusarium infection’s effects. Some cultivars displayed consistent protease activity and flour quality across sites. In contrast, others showed variability in their responses due to environmental conditions. To conclude, genetic resistance could provide adequate control of FHB, guaranteeing the successful protection of wheat quality. However, the possibility of confounding factors influencing genetic and cultivation conditions must be considered, and further research is needed to understand their interaction.

Protease inhibitor ASP enhances freezing tolerance by inhibiting protein degradation in kumquat.

Cold acclimation is a complex biological process leading to the development of freezing tolerance in plants. In this study, we demonstrated that cold-induced expression of protease inhibitor FmASP in a Citrus-relative species kumquat [Fortunella margarita (Lour.) Swingle] contributes to its freezing tolerance by minimizing protein degradation. Firstly, we found that only cold-acclimated kumquat plants, despite extensive leaf cellular damage during freezing, were able to resume their normal growth upon stress relief. To dissect the impact of cold acclimation on this anti-freezing performance, we conducted protein abundance assays and quantitative proteomic analysis of kumquat leaves subjected to cold acclimation (4°C), freezing treatment (-10°C) and post-freezing recovery (25°C). FmASP (Against Serine Protease) and several non-specific proteases were identified as differentially expressed proteins induced by cold acclimation and associated with stable protein abundance throughout the course of low-temperature treatment. FmASP was further characterized as a robust inhibitor of multiple proteases. In addition, heterogeneous expression of FmASP in Arabidopsis confirmed its positive role in freezing tolerance. Finally, we proposed a working model of FmASP and illustrated how this extracellular-localized protease inhibitor protects proteins from degradation, thereby maintaining essential cellular function for post-freezing recovery. These findings revealed the important role of protease inhibition in freezing response and provide insights on how this role may help develop new strategies to enhance plant freezing tolerance.

Target Deconvolution by Limited Proteolysis Coupled to Mass Spectrometry.

Limited proteolysis coupled to mass spectrometry (LiP-MS) is a recent proteomics technique that allows structure-based target engagement profiling on a proteome-wide level. To achieve this, native lysates are first incubated with a compound, followed by a short incubation with a nonspecific protease. Binding of a compound can change accessibility at the binding site or induce other structural changes in the target. This leads to treatment-specific proteolytic fingerprints upon limited proteolysis, which can be analyzed by standard bottom-up MS-based proteomics. Here, we describe a basic LiP-MS protocol using the natural product rapamycin as an example compound. Along with the provided LiP-MS reference data available via ProteomeXchange with identifier PXD035183, this enables the straightforward implementation of the method by scientists with a basic biochemistry and mass spectrometry background. We describe how the procedure can easily be adapted to other protein samples and small molecules.

Mango extract in tambaqui (Colossoma macropomum) diet: an in vitro and in vivo study

ABSTRACT Two experiments were carried out in order to evaluate the functional effect of mango extract in vitro and in vivo. The in vitro assay evaluated the growth of Lactobacillus spp; different treatments with mango extract (1% and 3%), cellulose (1% and 3%) and a control group were used. In the in vivo test, 100 juvenile tambaquis were used, with an initial average weight of 57.03 ± 1.05 g, distributed in twenty 1000 L experimental units, in a water recirculation system with a biofilter. Five diets were tested, with different concentrations of mango extract (0, 20, 40, 60 and 80 g kg−1 of diet). The in vitro assay showed that the addition of 3% mango extract presented functional-prebiotic potential against the Lactobacillus spp. strain. The recommended concentration of 40 g. kg−1 of mango extract in the diet for tambaqui improves performance. Some hematological variables showed significant variations but did not harm tambaqui. Mango extract improved muscle glycogen stores. Plasma albumin and nonspecific alkaline protease activity were responsive in fish fed mango extract. Mango extract increases the lactate concentration in the tambaqui intestine.

Dynamics of indicators of the proteinase-inhibitory system of the body in destructive forms of chronic apical periodontitis during treatment

BACKGROUND: Destructive forms of chronic apical periodontitis (CAP) remain one of the most difficult problems not only in dentistry and endodontics but also in medicine. In addition to the progressive local process in the alveolar process of the jaw, they are a source of autoinfection and auto-intoxication, causing or exacerbating pathologies in other organs.&#x0D; AIM: This study aimed to analyze the dynamics of the activity of nonspecific proteinases and their inhibitors in patients with destructive forms of CAP when performing a standard endodontic treatment.&#x0D; MATERIALS AND METHODS: Some indicators of the proteinase-inhibitory system of the oral fluid and blood plasma were studied in 28 somatically healthy patients aged 2555 years with destructive forms of CAP in multi-root teeth. The canals were treated according to the protocol of the European Society of Endodontics. After instrumental and medical treatments of the root canal, it was dried and temporarily sealed with calcium hydroxide-based preparation. The tooth was closed with temporary fillers for 1014 days.&#x0D; RESULTS: The comparative analysis of the indicators of the proteinase-inhibitory system showed that destructive forms of CAP and rehabilitation of the pathological focus, to a greater or lesser extent, affect its condition.&#x0D; CONCLUSIONS: Changes in the parameters of the proteinase-inhibitory system in the development of CAP during endodontic treatment were observed both locally (oral fluid) and systemically (blood plasma).

Immunochemical studies of Toxocara canis proteases.

Proteases of nematodes play a crucial role in larval molting and, in addition to their active role in egg hatching, proteases are also considered a crucial factor in tissue invasion and connective tissue remodeling. In Toxocara canis, proteases play important roles throughout the complex life cycle. They can degrade components of a model of extracellular matrix, basement membranes and different physiological substrates. In the present study, measurements of the proteolytic activity of the perivitelline fluid (PF) surrounding Toxocara canis embryos at different stages of development, the hatching fluid (HF) surrounding the infective larvae, as well as the excretory secretory (ES) products of the larvae in the culture media were performed. Measurements were made using casein as substrate following the Sigma non-specific protease activity assay. The results showed that enzyme activity increased as the embryo matured. The infective larvae were found to continuously produce proteases in the surrounding HF and ES products after in vitro cultivation indicating that Toxocara canis proteases might be important for the worm in the egg and the host. Optimal enzymatic activity was found at pH 8. Incubation of the antiserum from infected mice with the HF and ES products decreased their proteolytic activities, suggesting that there may be a link between the proteases present in these fluids and the immune response.

Investigating the protein conformational landscape associated with cognitive decline in aging with mass spectrometry

Cognitive decline in aging is becoming a major issue for families and healthcare providers as the population ages. Protein misfolding and aggregation are frequently implicated as driving many neurodegenerative diseases; however, this view is informed by a relatively small number of proteins, such as Aβ, α-synuclein, and tau. Limited proteolysis mass spectrometry (LiP-MS) is an emerging technique which can provide structural information about protein conformational states proteome-wide. In this approach, pulse proteolysis with a non-specific protease enables accessible regions within proteins to be recorded as cleavage sites, which can be subsequently read out with mass spectrometry.

Blocking serine protease activity prevents semenogelin degradation leading to hyperviscous semen in humans.

Prostate-specific antigen (PSA) is a prostate-specific serine protease enzyme that hydrolyzes gel-forming proteins (semenogelins) and changes the semen from gel-like to watery viscosity, a process called semen liquefaction. Highly viscous semen and abnormal liquefaction reduce sperm motility and contribute to infertility. Previously, we showed that nonspecific serine protease inhibitor (AEBSF) prevented proteolytic degradation of semenogelin in mice. However, it is unclear whether similar effect could be recapitulated in fresh human ejaculates. Therefore, in this study we evaluated the effect of AEBSF on the degradation of semenogelin (SEMG1) and its subsequent impact on semen liquefaction and sperm motility in fresh semen ejaculates collected from healthy men. We found that AEBSF showed a dual contraceptive action where it effectively 1) prevented degradation of SEMG1 resulting in viscous semen and 2) decreased sperm motility in human semen samples. However, the impact of AEBSF on sperm motility and viability could be due to its inhibitory activity toward other serine proteases or simply due to its toxicity. Therefore, to determine whether inhibition of PSA activity alone could disrupt SEMG1 degradation and contribute to hyperviscous semen, a neutralizing PSA antibody was used. We found that PSA antibody effectively prevented SEMG1 degradation with a subtle impact on sperm motility. These findings suggest that the target inhibition of PSA activity can prevent proteolytic degradation of SEMG1 and block liquefaction process, resulting in hyperviscous semen. As it is currently unknown if blocking semen liquefaction alone could prevent pregnancy, it needs further extensive studies before drawing any translational conclusions.

Control of Staphylococcal-mediated endogenous protease activity alters wound closure time in a complex wound model

BackgroundElevated protease activity is a characteristic feature of chronic wounds, where the inflammatory phase of wound healing is prolonged. The choice of dressings in treatment of chronic wounds can change the nature of the wound base and have a significant impact on healing. ObjectiveTo evaluate the impact of oxidised regenerated cellulose/collagen dressings on Staphylococcal-mediated protease activity in an inflamed wound model. MethodsWe developed an in vitro 3D inflamed wound model, and simulated inflammation by exposing the models to Staphylococcal spent culture supernatant. Protease activity and wound healing were assessed in the presence/absence of the dressings. ResultsHistological analysis of the wound model revealed two distinct layers, an epidermal and dermal layer, similar to the organisation of human skin. Inflammation with Staphylococcal spent culture supernatant elevated protease levels by 1.7x and consequently prevented the wound from progressing to the proliferative phase of healing, without having a negative effect on cell viability. Adding a collagen dressing, known to have non-specific protease modulating properties, reduced Staphylococcal-mediated protease activity back to baseline, with a concomitant reduction in wound closure time. Inflamed wounds thus resembled unwounded skin after 10 days of treatment with the dressings. ConclusionOur findings support the further evaluation and use of oxidised regenerated cellulose/collagen dressings for inflamed, non-healing wounds in the clinical setting. The model used in this study has the potential to be applied in preclinical research; to test wound dressing performance, such as healing and cell viability, and to also assess key markers of inflammation.