Showing papers on "Proteolytic enzymes published in 2018"

The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database

Abstract: The MEROPS database (http://www.ebi.ac.uk/merops/) is an integrated source of information about peptidases, their substrates and inhibitors. The hierarchical classification is: protein-species, family, clan, with an identifier at each level. The MEROPS website moved to the EMBL-EBI in 2017, requiring refactoring of the code-base and services provided. The interface to sequence searching has changed and the MEROPS protein sequence libraries can be searched at the EMBL-EBI with HMMER, FastA and BLASTP. Cross-references have been established between MEROPS and the PANTHER database at both the family and protein-species level, which will help to improve curation and coverage between the resources. Because of the increasing size of the MEROPS sequence collection, in future only sequences of characterized proteins, and from completely sequenced genomes of organisms of evolutionary, medical or commercial significance will be added. As an example, peptidase homologues in four proteomes from the Asgard superphylum of Archaea have been identified and compared to other archaean, bacterial and eukaryote proteomes. This has given insights into the origins and evolution of peptidase families, including an expansion in the number of proteasome components in Asgard archaeotes and as organisms increase in complexity. Novel structures for proteasome complexes in archaea are postulated.

Matrix Metalloproteinase Inhibitors in Cancer Therapy: Turning Past Failures Into Future Successes.

Abstract: The matrix metalloproteinases (MMP) are a family of proteolytic enzymes that degrade multiple components of the extracellular matrix. A large body of experimental and clinical evidence has implicated MMPs in tumor invasion, neoangiogenesis, and metastasis, and therefore they represent ideal pharmacologic targets for cancer therapy. From the 1990s to early 2000s, synthetic inhibitors of MMPs (MMPI) were studied in various cancer types. Unexpectedly, despite strongly promising preclinical data, all trials were unsuccessful in reducing tumor burden or improving overall survival; in addition, MMPIs had unforeseen, severe side effects. Two main reasons can explain the failure of MMPIs in clinical trials. It has now become apparent that some MMPs have antitumor effects; therefore, the broad-spectrum MMPIs used in the initial trials might block these MMPs and result in tumor progression. In addition, although MMPs are involved in the early stages of tumor progression, MMPIs were tested in patients with advanced disease, beyond the stage when these compounds could be effective. As more specific MMPIs are now available, MMP targeting could be reconsidered for cancer therapy; however, new trials should be designed to test their antimetastatic properties in early-stage tumors, and endpoints should focus on parameters other than decreasing metastatic tumor burden. Mol Cancer Ther; 17(6); 1147-55. ©2018 AACR.

Application of bacteriocins and protective cultures in dairy food preservation.

Abstract: In the last years, consumers are becoming increasingly aware of the human health risk posed by the use of chemical preservatives in foods. In contrast, the increasing demand by the dairy industry to extend shelf-life and prevent spoilage of dairy products has appeal for new preservatives and new methods of conservation. Bacteriocins are antimicrobial peptides, which can be considered as safe since they can be easily degraded by proteolytic enzymes of the mammalian gastrointestinal tract. Also, most bacteriocin producers belong to lactic acid bacteria (LAB), a group that occurs naturally in foods and have a long history of safe use in dairy industry. Since they pose no health risk concerns, bacteriocins, either purified or excreted by bacteriocin producing strains, are a great alternative to the use of chemical preservatives in dairy products. Bacteriocins can be applied to dairy foods on a purified/crude form or as a bacteriocin-producing LAB as a part of fermentation process or as adjuvant culture. A number of applications of bacteriocins and bacteriocin-producing LAB have been reported to successful control pathogens in milk, yogurt, and cheeses. One of the more recent trends consists in the incorporation of bacteriocins, directly as purified or semi-purified form or in incorporation of bacteriocin-producing LAB into bioactive films and coatings, applied directly onto the food surfaces and packaging. This review is focused on recent developments and applications of bacteriocins and bacteriocin-producing LAB for reducing the microbiological spoilage and improve safety of dairy products.

UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites

Abstract: Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.

Multifaceted Role of the Urokinase-Type Plasminogen Activator (uPA) and Its Receptor (uPAR): Diagnostic, Prognostic, and Therapeutic Applications.

Abstract: The plasminogen activator (PA) system is an extracellular proteolytic enzyme system associated with various physiological and pathophysiological processes. A large body of evidence support that among the various components of the PA system, urokinase-type plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitor-1 and -2 (PAI-1 and PAI-2) play a major role in tumor progression and metastasis. The binding of uPA with uPAR is instrumental for the activation of plasminogen to plasmin, which in turn initiates a series of proteolytic cascade to degrade the components of the extracellular matrix, and thereby, cause tumor cell migration from the primary site of origin to a distant secondary organ. The components of the PA system show altered expression patterns in several common malignancies, which have identified them as ideal diagnostic, prognostic, and therapeutic targets to reduce cancer-associated morbidity and mortality. This review summarizes the various components of the PA system and focuses on the role of uPA-uPAR in different biological processes especially in the context of malignancy. We also discuss the current state of knowledge of uPA-uPAR-targeted diagnostic and therapeutic strategies for various malignancies.

Transcellular stomach absorption of a derivatized glucagon-like peptide-1 receptor agonist.

Abstract: Oral administration of therapeutic peptides is hindered by poor absorption across the gastrointestinal barrier and extensive degradation by proteolytic enzymes. Here, we investigated the absorption of orally delivered semaglutide, a glucagon-like peptide-1 analog, coformulated with the absorption enhancer sodium N-[8-(2-hydroxybenzoyl) aminocaprylate] (SNAC) in a tablet. In contrast to intestinal absorption usually seen with small molecules, clinical and preclinical dog studies revealed that absorption of semaglutide takes place in the stomach, is confined to an area in close proximity to the tablet surface, and requires coformulation with SNAC. SNAC protects against enzymatic degradation via local buffering actions and only transiently enhances absorption. The mechanism of absorption is shown to be compound specific, transcellular, and without any evidence of effect on tight junctions. These data have implications for understanding how highly efficacious and specific therapeutic peptides could be transformed from injectable to tablet-based oral therapies.

An overview of Bacillus proteases: from production to application.

Abstract: Proteases have a broad range of applications in industrial processes and products and are representative of most worldwide enzyme sales. The genus Bacillus is probably the most important bacterial source of proteases and is capable of producing high yields of neutral and alkaline proteolytic enzymes with remarkable properties, such as high stability towards extreme temperatures, pH, organic solvents, detergents and oxidizing compounds. Therefore, several strategies have been developed for the cost-effective production of Bacillus proteases, including optimization of the fermentation parameters. Moreover, there are many studies on the use of low-cost substrates for submerged and solid state fermentation. Other alternatives include genetic tools such as protein engineering in order to obtain more active and stable proteases and strain engineering to better secrete recombinant proteases from Bacillus through homologous and heterologous protein expression. There has been extensive research on proteases because of the broad number of applications for these enzymes, such as in detergent formulations for the removal of blood stains from fabrics, production of bioactive peptides, food processing, enantioselective reactions, and dehairing of skins. Moreover, many commercial proteases have been characterized and purified from different Bacillus species. Therefore, this review highlights the production, purification, characterization, and application of proteases from a number of Bacillus species.

The nutritional and health attributes of kiwifruit: a review

Abstract: To describe the nutritional and health attributes of kiwifruit and the benefits relating to improved nutritional status, digestive, immune and metabolic health. The review includes a brief history of green and gold varieties of kiwifruit from an ornamental curiosity from China in the 19th century to a crop of international economic importance in the 21st century; comparative data on their nutritional composition, particularly the high and distinctive amount of vitamin C; and an update on the latest available scientific evidence from well-designed and executed human studies on the multiple beneficial physiological effects. Of particular interest are the digestive benefits for healthy individuals as well as for those with constipation and other gastrointestinal disorders, including symptoms of irritable bowel syndrome. The mechanisms of action behind the gastrointestinal effects, such as changes in faecal (stool) consistency, decrease in transit time and reduction of abdominal discomfort, relate to the water retention capacity of kiwifruit fibre, favourable changes in the human colonic microbial community and primary metabolites, as well as the naturally present proteolytic enzyme actinidin, which aids protein digestion both in the stomach and the small intestine. The effects of kiwifruit on metabolic markers of cardiovascular disease and diabetes are also investigated, including studies on glucose and insulin balance, bodyweight maintenance and energy homeostasis. The increased research data and growing consumer awareness of the health benefits of kiwifruit provide logical motivation for their regular consumption as part of a balanced diet. Kiwifruit should be considered as part of a natural and effective dietary strategy to tackle some of the major health and wellness concerns around the world.

Efficient and robust proteome-wide approaches for cross-linking mass spectrometry

Abstract: Cross-linking mass spectrometry (XL-MS) has received considerable interest, owing to its potential to investigate protein-protein interactions (PPIs) in an unbiased fashion in complex protein mixtures. Recent developments have enabled the detection of thousands of PPIs from a single experiment. A unique strength of XL-MS, in comparison with other methods for determining PPIs, is that it provides direct spatial information for the detected interactions. This is accomplished by the use of bifunctional cross-linking molecules that link two amino acids in close proximity with a covalent bond. Upon proteolytic digestion, this results in two newly linked peptides, which are identifiable by MS. XL-MS has received the required boost to tackle more-complex samples with recent advances in cross-linking chemistry with MS-cleavable or reporter-based cross-linkers and faster, more sensitive and more versatile MS platforms. This protocol provides a detailed description of our optimized conditions for a full-proteome native protein preparation followed by cross-linking using the gas-phase cleavable cross-linking reagent disuccinimidyl sulfoxide (DSSO). Following cross-linking, we demonstrate extensive sample fractionation and substantially simplified data analysis with XlinkX in Proteome Discoverer, as well as subsequent protein structure investigations with DisVis and HADDOCK. This protocol produces data of high confidence and can be performed within ~10 d, including structural investigations.

Ensemble cryoEM elucidates the mechanism of insulin capture and degradation by human insulin degrading enzyme.

Abstract: Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies.

Anti-inflammatory effects of polyphenols in arthritis.

Abstract: Polyphenols have been extensively investigated with regard to their antioxidant, anti-inflammatory, and immunomodulant properties in many inflammatory chronic conditions. The aim of this review is to summarise how these compounds can modulate the inflammatory pathways which characterise the most prevalent arthropathies including osteoarthritis, rheumatoid arthritis and crystal-induced arthritis. Among polyphenols, epigallocatechin gallate, carnosol, hydroxytyrosol, curcumin, resveratrol, kaempferol and genistein have been the most widely investigated in arthritis. The most important results of the studies outlined in this article show how polyphenolic compounds are able to inhibit the expression and the release of a number of pro-inflammatory mediators and proteolytic enzymes, the activity of different transcriptional factors and the production of reactive oxygen species in vitro. Studies on animal models of rheumatoid arthritis, osteoarthritis and gout show interesting results in terms of reduced tissue damage, restored cartilage homeostasis, and decreased levels of uric acid, respectively. Despite the multiple protective effects of polyphenols, there are no dietary recommendations for patients affected by rheumatic diseases. Future studies, including intervention trials, should be conducted to determine the relevance of polyphenols consumption or supplementation in arthritis. © 2017 Society of Chemical Industry.

Perineuronal nets decrease membrane capacitance of peritumoral fast spiking interneurons in a model of epilepsy.

Abstract: Brain tumor patients commonly present with epileptic seizures. We show that tumor-associated seizures are the consequence of impaired GABAergic inhibition due to an overall loss of peritumoral fast spiking interneurons (FSNs) concomitant with a significantly reduced firing rate of those that remain. The reduced firing is due to the degradation of perineuronal nets (PNNs) that surround FSNs. We show that PNNs decrease specific membrane capacitance of FSNs permitting them to fire action potentials at supra-physiological frequencies. Tumor-released proteolytic enzymes degrade PNNs, resulting in increased membrane capacitance, reduced firing, and hence decreased GABA release. These studies uncovered a hitherto unknown role of PNNs as an electrostatic insulator that reduces specific membrane capacitance, functionally akin to myelin sheaths around axons, thereby permitting FSNs to exceed physiological firing rates. Disruption of PNNs may similarly account for excitation-inhibition imbalances in other forms of epilepsy and PNN protection through proteolytic inhibition may provide therapeutic benefits.

Functional profiling of circulating tumor cells with an integrated vortex capture and single-cell protease activity assay

Abstract: Tumor cells are hypothesized to use proteolytic enzymes to facilitate invasion. Whether circulating tumor cells (CTCs) secrete these enzymes to aid metastasis is unknown. A quantitative and high-throughput approach to assay CTC secretion is needed to address this question. We developed an integrated microfluidic system that concentrates rare cancer cells >100,000-fold from 1 mL of whole blood into ∼50,000 2-nL drops composed of assay reagents within 15 min. The system isolates CTCs by size, exchanges fluid around CTCs to remove contaminants, introduces a matrix metalloprotease (MMP) substrate, and encapsulates CTCs into microdroplets. We found CTCs from prostate cancer patients possessed above baseline levels of MMP activity (1.7- to 200-fold). Activity of CTCs was generally higher than leukocytes from the same patient (average CTC/leukocyte MMP activity ratio, 2.6 ± 1.5). Higher MMP activity of CTCs suggests active proteolytic processes that may facilitate invasion or immune evasion and be relevant phenotypic biomarkers enabling companion diagnostics for anti-MMP therapies.

Identification of Bioactive Peptides with α-Amylase Inhibitory Potential from Enzymatic Protein Hydrolysates of Red Seaweed (Porphyra spp).

Abstract: Inhibition of α-amylase enzyme is one therapeutic approach in lowering glucose level in the blood to manage diabetes mellitus. In this study α-amylase inhibitory peptides were identified from proteolytic enzymes hydrolysates of red seaweed laver (Porphyra species) using consecutive chromatographic techniques. In the resultant fractions from RP-HPLC (D1-10), D2 inhibited α-amylase activity (88.67 ± 1.05%) significantly (p ≤ 0.5) at 1 mg/mL protein concentration. A mass spectrometry (ESI-Q-TOF- MS) analysis was used to identify peptides from this fraction. Two novel peptides were identified as Gly-Gly-Ser-Lys and Glu-Leu-Ser. To validate their α-amylase inhibitory activity, these peptides were synthesized chemically. The peptides were demonstrated inhibitory activity at IC50 value: 2.58 ± 0.08 mM (Gly-Gly-Ser-Lys) and 2.62 ± 0.05 mM (Glu-Leu-Ser). The inhibitory kinetics revealed that these peptides exhibited noncompetitive binding mode. Thus, laver can be a potential source of novel ingredients in food and...

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis.

Abstract: The preponderance and diversity of charge variants in therapeutic monoclonal antibodies has implications for antibody efficacy and degradation. Understanding the extent and impact of minor antibody variants is of great interest, and it is also a critical regulatory requirement. Traditionally, a combination of approaches is used to characterize antibody charge heterogeneity, including ion exchange chromatography and independent mass spectrometric variant site mapping after proteolytic digestion. Here, we describe charge variant native mass spectrometry (CVMS), an integrated native ion exchange mass spectrometry-based charge variant analytical approach that delivers detailed molecular information in a single, semi-automated analysis. We utilized pure volatile salt mobile phases over a pH gradient that effectively separated variants based on minimal differences in isoelectric point. Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. We demonstrate that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Our CVMS method allows confident assignment of intact monoclonal antibody isoforms of similar mass and relative abundance measurements across three orders of magnitude dynamic range.

Candida albicans-Induced NETosis Is Independent of Peptidylarginine Deiminase 4.

Abstract: Neutrophils are the most abundant innate immune cells and the first line of defense against many pathogenic microbes, including the human fungal pathogen Candida albicans Among the neutrophils' arsenal of effector functions, neutrophil extracellular traps (NETs) are thought to be of particular importance for trapping and killing the large fungal filaments by means of their web-like structures that consist of chromatin fibers decorated with proteolytic enzymes and host defense proteins Peptidylarginine deiminase 4 (PAD4)-mediated citrullination of histones in activated neutrophils correlates with chromatin decondensation and extrusion and is widely accepted to act as an integral process of NET induction (NETosis) However, the requirement of PAD4-mediated histone citrullination for NET release during C albicans infection remains unclear In this study, we show that although PAD4-dependent neutrophil histone citrullination is readily induced by C albicans, PAD4 is dispensable for NETosis in response to the fungus and other common NET-inducing stimuli Moreover, PAD4 is not required for antifungal immunity during mucosal and systemic C albicans infection Our results demonstrate that PAD4 is dispensable for C albicans-induced NETosis, and they highlight the limitations of using histone citrullination as a marker for NETs and PAD4-/- mice as a model of NET-deficiency

Lysosomal membrane permeabilization as a cell death mechanism in cancer cells.

Abstract: Lysosomes are acidic organelles that contain hydrolytic enzymes that mediate the intracellular degradation of macromolecules. Damage of these organelles often results in lysosomal membrane permeabilization (LMP) and the release into the cytoplasm of the soluble lysosomal contents, which include proteolytic enzymes of the cathepsin family. This, in turn, activates several intracellular cascades that promote a type of regulated cell death, called lysosome-dependent cell death (LDCD). LDCD can be inhibited by pharmacological or genetic blockade of cathepsin activity, or by protecting the lysosomal membrane, thereby stabilizing the organelle. Lysosomal alterations are common in cancer cells and may increase the sensitivity of these cells to agents that promote LMP. In this review, we summarize recent findings supporting the use of LDCD as a means of killing cancer cells.

Degradation Behavior of Silk Nanoparticles-Enzyme Responsiveness.

Abstract: Silk nanoparticles are viewed as promising vectors for intracellular drug delivery as they can be taken up into cells by endocytosis and trafficked to lysosomes, where lysosomal enzymes and the low pH trigger payload release. However, the subsequent degradation of the silk nanoparticles themselves still requires study. Here, we report the responsiveness of native and PEGylated silk nanoparticles to degradation following exposure to proteolytic enzymes (protease XIV and α-chymotrypsin) and papain, a cysteine protease. Both native and PEGylated silk nanoparticles showed similar degradation behavior over a 20 day exposure period (degradation rate: protease XIV > papain ≫ α-chymotrypsin). Within 1 day, the silk nanoparticles were rapidly degraded by protease XIV, resulting in a ∼50% mass loss, an increase in particle size, and a reduction in the amorphous content of the silk secondary structure. By contrast, 10 days of papain treatment was necessary to observe any significant change in nanoparticle properties...

The Role of Trypsin:Chymotrypsin in Tissue Repair

Abstract: Tissue damage of all types, such as surgical or accidental injuries, fractures, and burns, stimulates a well-orchestrated, physiological process of healing, which ultimately leads to structural and functional restoration of the damaged tissues. The tissue repair process can be broadly divided into four continuous and overlapping phases—hemostasis and coagulation, inflammation, proliferation, and remodeling. If the process is interrupted or halted during any stage, it leads to impaired healing and formation of a chronic wound. Chronic wounds are associated with significant morbidity, mortality, and poor quality of life. Therefore, prompt and effective management of acute tissue injury is necessary to prevent it from progressing to a chronic wound. Proteolytic enzymes have been used to facilitate tissue repair since ancient times. Trypsin:chymotrypsin is an oral proteolytic enzyme preparation which has been in clinical use since the 1960s. It provides better resolution of inflammatory symptoms and promotes speedier recovery of acute tissue injury than several of the other existing enzyme preparations. This review article revisits the role and clinical utility of trypsin:chymotrypsin combination in tissue repair. Funding: Torrent Pharmaceuticals Limited.

Influence of ultrasound and proteolytic enzyme inhibitors on muscle degradation, tenderness, and cooking loss of hens during aging

Abstract: Xiong G.-Y., Zhang L.-L., Zhang W., Wu J. (2012): Influence of ultrasound and proteolytic enzyme inhibitors on muscle degradation, tenderness, and cooking loss of hens during aging. Czech J. Food Sci., 30: 195–205. The potential contribution of mechanical disruption by ultrasonics and endogenous proteolytic enzymes on the tenderisation of hen muscle were investigated. The importance of endogenous enzymes was evaluated using various specific inhibitors. Freshly obtained breast muscles of culled hens, from the 6 groups investigated were treated with different proteolytic enzyme inhibitors and/or ultrasonics, group was treated with different methods, and then stored at 4°C for 0, 1, 3, and 7 days. Shear force decreased by 1.19 kg, and shear force and cooking loss were reduced by 0.69 kg and 4.27%, respectively, in the incorporated group treatment. The calpastatin activity was affected by all treatments except in the Z-DEVD-fmk-treated group, caspase-3 activity decreased in Z-DEVD-fmk-treated group. Therefore, the results suggest that ultrasonics and endogenous proteases contributed to muscle degradation, thereby improving hen meat tenderness and decreasing the cooking loss. Thus muscle degradation, tenderness, and water-retaining properties of hens were improved by a combination of ultrasound and exogenous proteolytic enzyme inhibitors.