Identification and quantification of degradome components in human synovial fluid reveals an increased proteolytic activity in knee osteoarthritis patients vs controls.

Synovial fluid (SF) may contain cleavage products of proteolytic activities. Our aim was to characterize the degradome through analysis of proteolytic activity and differential abundance of these components in a peptidomic analysis of SF in knee osteoarthritis (OA) patients versus controls (n = 23). SF samples from end-stage knee osteoarthritis patients undergoing total knee replacement surgery and controls, that is, deceased donors without known knee disease were previously run using liquid chromatography mass spectrometry (LC-MS). This data was used to perform new database searches generating results for non-tryptic and semi-tryptic peptides for studies of degradomics in OA. We used linear mixed models to estimate differences in peptide-level expression between the two groups. Known proteolytic events (from the MEROPS peptidase database) were mapped to the dataset, allowing the identification of potential proteases and which substrates they cleave. We also developed a peptide-centric R tool, proteasy, which facilitates analyses that involve retrieval and mapping of proteolytic events. We identified 429 differentially abundant peptides. We found that the increased abundance of cleaved APOA1 peptides is likely a consequence of enzymatic degradation by metalloproteinases and chymase. We identified metalloproteinase, chymase, and cathepsins as the main proteolytic actors. The analysis indicated increased activity of these proteases irrespective of their abundance.

Chryseobacterium aquifrigidense keratinase liberated essential and nonessential amino acids from chicken feather degradation.

Keratinous biomass valorization for value-added products presents a high prospect in ecological management and the advancement of the bio-economy. Consequently, soil samples from the poultry dumpsite were collected. The bacteria isolated on the basal salt medium were screened for keratinolytic activity. The potent chicken feathers degrading bacteria were identified through 16S rRNA gene sequencing and phylogenetic analysis. Fermentation process conditions were optimized, and the amino acid compositions of the feather hydrolysate were likewise quantified. Ten (10) proteolytic bacteria evaluated on skimmed milk agar showed intact chicken feather degradation ranging from 33% (WDS-03) to 88% (FPS-09). The extracellular keratinase activity ranged from 224.52 ± 42.46 U/mL (WDS-03) to 834.55 ± 66.86 U/mL (FPS-07). Based on 16S rRNA gene sequencing and phylogenetic analysis, the most potent keratinolytic isolates coded as FPS-07, FPS-09, FPS-01, and WDS-06 were identified as Chryseobacterium aquifrigidense FANN1, Chryseobacterium aquifrigidense FANN2, Stenotrophomonas maltophilia ANNb, and Bacillus sp. ANNa, respectively. C aquifrigidense FANN2 maximally produced keratinase (1460.90 ± 26.99 U/mL) at 72 h of incubation under optimal process conditions of pH (6), inoculum side (5%; v/v), temperature (30°C), and chicken feather (25 g/L). The feather hydrolysate showed a protein value of 67.54%, with a relative abundance of arginine (2.84%), serine (3.14%), aspartic acid (3.33%), glutamic acid (3.73%), and glycine (2.81%). C. aquifrigidense FANN2 yielded high keratinase titre and dismembered chicken feathers into amino acids-rich hydrolysate, highlighting its significance in the beneficiation of recalcitrant keratinous wastes into dietary proteins as potential livestock feed supplements.

Modifying a bacterial tyrosinase zymogen for use in protease activity assays.

Current clinical laboratory assays are not sufficient for determining the activity of many specific human proteases yet. In this study, we developed a general approach that enables the determination of activities of caspase-3 based on the proteolytic activation of the engineered zymogen of the recombinant tyrosinase from Verrucomicrobium spinosum (Vs-tyrosinase) by detecting the diphenolase activity in an increase in absorbance at 475 nm. Here, we designed three different zymogen constructs of Vs-tyrosinase, including RSL-pre-pro-TYR, Pre-pro-TYR, and Pro-TYR. The active domain was fused to the reactive site loop (RSL) of α1-proteinase inhibitor and/or its own signal peptide (pre) and/or its own C-terminal domain (pro) via a linker containing a specific caspase-3 cleavage site. Further studies revealed that both RSL peptide and TAT signal peptide were able to inhibit tyrosinase diphenolase activity, in which RSL-pre-pro-TYR had the lowest background signals. Therefore, a specific protease activity such as caspase-3 could be detected when a suitable zymogen was established. Our results could provide a new way to directly detect the activities of key human proteases, for instance, to monitor the efficacy and safety of tumor therapy by determining the activity of apoptosis-related caspase-3 in patients. KEY POINTS: • RSL inhibited the activity of Verrucomicrobium spinosum tyrosinase. • N-pre and C-terminal domain exerted stronger dual inhibition on the Vs-tyrosinase. • The activity of caspase-3 could be measured by the zymogen activation system.

Allergen Content and Protease Activity in Milk Feeds from Mothers of Preterm Infants.

Rationale: There is little information regarding the allergen content of milk feeds in the preterm population. Previous studies have not performed a broad analysis of the allergenic peptide content and protease activity of milk feeds in this population. Methods: To evaluate feasibility, we initially performed mass spectrometry on 4 human milk (HM) samples (2 term and 2 preterm) from the Mommy's Milk Human Milk Biorepository (HMB) and analyzed the results against the University of Nebraska FASTA database and UniProt for a total of 2,211 protein sequences. We then further analyzed five samples from the Microbiome, Atopy, and Prematurity (MAP) study including peptidomic and protease activity analysis. Results: Each HMB sample had between 806 and 1,007 proteins, with 37-44 nonhuman proteins/sample encompassing 26 plant and animal species. In the preterm MAP samples, 784 digested nonhuman proteins were identified, 30 were nonbovine in origin. Proteins from 23 different species including aeroallergens, food, and contact allergens were identified. Protease activity was highest in HM samples without human milk fortifier and lowest in preterm formula. Conclusions: These findings represent the first preterm milk feed mass spectrometry and protease analysis with identification of known allergenic proteins to food, contact, and aeroallergens. These results raise questions of whether the composition of milk feeds in the neonatal intensive care unit impact the development of atopic disease in the preterm population and whether the complex interaction between allergens, proteases, and other HM components can serve to induce sensitization or tolerance to allergens in infants. Clinical Trial Registration Number: NCT04835935.

Enhancing adhesive-dentin interface stability of primary teeth: From ethanol wet-bonding to plant-derived polyphenol application.

OBJECTIVES: To investigate whether the adhesive-dentin interface stability of primary teeth would be enhanced by epigallocatechin-3-gallate (EGCG) with ethanol wet-bonding. METHODS: Non-caries primary molars were sliced to achieve a flat dentin surface and etched then randomly distributed into five groups in accordance with different treatments: group 1, no treatment; group 2, applying absolute ethanol wet-bonding for 60 s; groups 3-5, applying 0.1%, 0.5%, and 1% (w/v) EGCG-incorporating ethanol wet-bonding (0.1%, 0.5%, and 1% EGCG) for 60 s. Singlebond universal adhesive was then applied followed by resin composite construction. Microtensile bond strength, fracture mode, and nanoleakage at adhesive-dentin interface were evaluated after 24 h of water storage or 10,000 times of thermocycling. Zymography of hybrid layer, biofilm formation of Streptococcus mutans by CLSM, FESEM, and MTT test, and cytotoxicity by CCK-8 assay were respectively assessed. RESULTS: Irrespective of thermocycling, the dentin bond strength was preserved with reduced nanoleakage in the 0.5% and 1% EGCG groups. Furthermore, the activity of endogenous proteases and the growth of Streptococcus mutans biofilm were inhibited after treatment with 0.5% and 1% EGCG/ethanol solutions (groups 4 and 5). CCK-8 results of the 0.1% and 0.5% EGCG groups showed acceptable biocompatibility. CONCLUSIONS: Treatment by EGCG/ethanol solutions effectively enhanced the bond stability of primary teeth at the adhesive-dentin interface. CLINICAL SIGNIFICANCE: Synergistic application of EGCG and ethanol wet-bonding suggesting a promising strategy to improve dentin bonding durability with bacterial biofilm inhibition, thus increasing resin-based restorations' service life in primary dentition.

Retention of Intrafibrillar Minerals Improves Resin-Dentin Bond Durability.

The concept of extrafibrillar demineralization involves selective removal of apatite crystallites from the extrafibrillar spaces of mineralized dentin without disturbing the intrafibrillar minerals within collagen. This helps avoiding activation of endogenous proteases and enables air-drying of partially demineralized dentin without causing collapse of completely demineralized collagen matrix that adversely affects resin infiltration. The objective of the present study was to evaluate the potential of quaternized carboxymethyl chitosan (QCMC)-based extrafibrillar demineralization in improving resin-dentin bond durability. Isothermal titration calorimetry indicated that QCMC synthesized by quaternization of O-carboxymethyl chitosan had moderate affinity for Ca2+ (binding constant: 8.9 × 104 M-1). Wet and dry bonding with the QCMC-based demineralization produced tensile bond strengths equivalent to the phosphoric acid (H3PO4)-based etch-and-rinse technique. Those bond strengths were maintained after thermocycling. Amide I and PO43- mappings of QCMC-conditioned dentin were performed with atomic force microscope-infrared spectroscopy (AFM-IR). Whereas H3PO4-etched dentin exhibited an extensive reduction in PO43- signals corresponding to apatite depletion, QCMC-conditioned dentin showed scattered dark areas and bright PO43- streak signals. The latter were consistent with areas identified as collagen fibrils in the amide I mapping and were suggestive of the presence of intrafibrillar minerals in QCMC-conditioned dentin. Young's modulus mapping of QCMC-demineralized dentin obtained by AFM-based amplitude modulation-frequency modulation recorded moduli that were the same order of magnitude as those in mineralized dentin and at least 1 order higher than H3PO4-etched dentin. In situ zymography of the gelatinolytic activity within hybrid layers created with QCMC conditioning revealed extremely low signals before and after thermocycling, compared with H3PO4-etched dentin for both wet and dry bonding. Confocal laser scanning microscopy identified the antibacterial potential of QCMC against Streptococcus mutans and Enterococcus faecalis biofilms. Taken together, the QCMC-based demineralization retains intrafibrillar minerals, preserves the elastic modulus of collagen fibrils, reduces endogenous proteolytic activity, and inhibits bacteria biofilms to extend dentin bond durability.

Characterization of a Novel Antimicrobial Peptide Isolated from Moringa oleifera Seed Protein Hydrolysates and Its Membrane Damaging Effects on Staphylococcus aureus.

The present study sought to identify and characterize a novel antimicrobial peptide, named MOp2 from Moringa oleifera seed protein hydrolysates, and elucidate its potential antimicrobial effects on Staphylococcus aureus. MOp2, with the amino acid sequence of His-Val-Leu-Asp-Thr-Pro-Leu-Leu (HVLDTPLL), was characterized as a hydrophobic anionic AMP of the ß-sheet structure. MOp2 exhibited negligible hemolytic activity at 2.0× MIC, suggesting its inhibitory effect on the growth of S. aureus (MIC: 2.204 mM). It maintained more than 90% of antimicrobial activity under 5% salt and about 78% of antimicrobial activity at a high temperature of 115 °C for 30 min. Protease, especially acid protease, reduced its antimicrobial activity to different extents. Moreover, MOp2 caused irreversible membrane damage to S. aureus cells by increasing the membrane permeability, resulting in the release of intracellular nucleotide pools. Additionally, molecular docking revealed that MOp2 could inhibit S. aureus growth by interacting with dihydrofolate reductase and DNA gyrase through hydrogen bonding and hydrophobic interactions. Overall, MOp2 could be a potential novel antimicrobial agent against S. aureus in food processing.

A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection.

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.

Highly Efficient Electrogenerated Chemiluminescence Quenching on Lipid-Coated Multifunctional Magnetic Nanoparticles for the Determination of Proteases.

This work reports a highly efficient electrogenerated chemiluminescence (ECL) quenching on lipid-coated multifunctional magnetic nanoparticles (MMNP) for the determination of proteases incorporating membrane-confined quenching with a specific cleavage reaction for the first time. A new ruthenium complex [Ru(bpy)2(ddcbpy)](PF6)2 (bpy = 2,2'-bipyridine, ddcbpy = 4,4'-didodecyl-carbonyl-2,2'-bipyridine with two hydrophobic long alkyl chains) was synthesized as a signal probe, while [cholesterol-(CH2)6-HSSKLQK(peptide)-ferrocene (quencher)] was designed as a specific peptide-quencher probe. The MMNP were prepared by inserting both the signal probe and the peptide-quencher probe into the cholesterol-phospholipid-coated Fe3O4 magnetic nanoparticles (Fe3O4 NP, ∼200 nm). When prostate specific antigen (PSA) taken as a model analyte was introduced into the suspension of MMNP, PSA cleaved the amide bond of SK in cholesterol-(CH2)6-HSSKLQK-Fc, and then the cleaved peptide-motif-Fc-quencher was deviated from the MMNP, resulting in the increase in the ECL intensity. It was found that the ECL quenching constant of [Ru(bpy)2(ddcbpy)]2+ on MMNP (KSV, NP/lipECL =2.68 × 107 M-1) is 137-folds higher than that on the lipid-coated electrode (KSV, lipECL=1.95 × 105 M-1) and 391-folds higher than that in the solution (KSV, aqECL =6.86 × 104 M-1). The ECL emission of Ru(bpy)32+ derivative-attached Fe3O4 NP was observed at ∼1.2 V, involving the tunnel-electron transfer pathway (TPA• + Ru(bpy)33+ = Ru(bpy)32+*). Based on the highly efficient ECL quenching of the ruthenium complex by ferrocene on the MMNP, a new ECL method was developed for PSA with a linear range from 0.01 to 1.0 ng/mL and a limit of detection of 3.0 pg/mL. This work demonstrates that the approach of ECL quenching by ferrocene on lipid-coated Fe3O4 NP is promising and could be easily extended to determine other proteases.

Mass Spectrometry-Cleavable Protein N-Terminal Tagging Strategy for System-Level Protease Activity Profiling.

Proteolysis is one of the most important protein post-translational modifications (PTMs) that influences the functions, activities, and structures of nearly all proteins during their lifetime. To facilitate the targeted identification of low-abundant proteolytic products, we devised a strategy incorporating a novel biotinylated reagent PFP (pentafluorophenyl)-Rink-biotin to specifically target, enrich and identify proteolytic N-termini. Within the PFP-Rink-biotin reagent, a mass spectrometry (MS)-cleavable feature was designed to assist in the unambiguous confirmation of the enriched proteolytic N-termini. The proof-of-concept study was performed with multiple standard proteins whose N-termini were successfully modified, enriched and identified by a signature ion (SI) in the MS/MS fragmentation, along with the determination of N-terminal peptide sequences by multistage tandem MS of the complementary fragment generated after the cleavage of MS-cleavable bond. For large-scale application, the enrichment and identification of protein N-termini from Escherichia coli cells were demonstrated, facilitated by an in-house developed NTermFinder bioinformatics workflow. We believe this approach will be beneficial in improving the confidence of identifying proteolytic substrates in a native cellular environment.

Newly Designed Decellularized Scaffolds for Scaffold-based Gene Therapy from Elastic Cartilages via Supercritical Carbon Dioxide Fluid and Alkaline/ Protease Treatments.

BACKGROUND: Scaffold-based gene therapy provides a promising approach for tissue engineering, which was important and popular as it combined medical applications and engineering materials' knowledge. OBJECTIVE: The decellularization techniques were employed to remove the cellular components from porcine elastic cartilages, leaving a native decellularized Extracellular Matrix (dECM) composition and architecture integrity of largely insoluble collagen, elastin, and tightly bound glycosaminoglycans. For newly designed collagen scaffold samples, elastic cartilages were hydrolyzed by protease with different concentrations to gain state completely and clearly. METHODS: An extraction process of Supercritical Carbon Dioxide (ScCO2) was used to remove cellular components from porcine elastic cartilage. The dECM scaffolds with collagen must be characterized by Fourier transform infrared spectroscopy(FTIR), Thermo-Gravimetric Analysis (TGA), and Scanning Electron Microscope (SEM). RESULTS: The study provided a new treatment combined with supercritical carbon dioxide and alkaline/ protease to prepare dECM scaffolds with hole-scaffold microstructures and introduce into a potential application on osteochondral tissue engineering using scaffold-based gene therapy. The new process is simple and efficient. The pore-scaffold microstructures were observed in dECM scaffolds derived from porcine elastic cartilages. The Tdmax values of the resulting dECM scaffolds were observed at over 330oC. CONCLUSION: A series of new scaffolds were successfully obtained from porcine tissue by using ScCO2 and alkaline/enzyme treatments such as a mixing aqueous solution of NH4OH and papain. The dECM scaffolds with high thermal stability were obtained. The resulting scaffold with clean pore-scaffold microstructure could be a potential application for scaffold-based gene therapy.

Assessment of gelatinolytic proteinases in chilled grass carp (Ctenopharyngodon idellus) fillets: characterization and contribution to texture softening.

BACKGROUND: Texture softening is always a problem during chilling of grass carp fillets. To solve this problem and provide for better quality of flesh, understanding the mechanism of softening is necessary. Gelatinolytic proteinases are suspected to play an essential role in the disintegration of collagen in softening of fish flesh. In the present study, the types and contribution of gelatinolytic proteinases in chilled fillets were investigated. RESULTS: Four active bands (G1, 250 kDa; G2, 68 kDa; G3, 66 kDa; G4, 29 kDa) of gelatinolytic proteinases were identified in grass carp fillets by gelatin zymography. The effect of inhibitors and metal ions revealed that G1 was possibly a serine proteinase, G2 and G3 were calcium-dependent metalloproteinases and G4 was a cysteine proteinase. The effect of the inhibitors phenylmethanesulfonyl fluoride (PMSF), l-3-carboxy-trans-2,3-epoxy-propionyl-l-leucine-4-guanidinobutylamide (E-64) and 1,10-phenanthroline (Phen) on chilled fillets revealed that gelatinolytic proteinase activities were significantly suppressed. Collagen solubility indicated that metalloproteinase and serine proteinase played critical roles in collagen breakdown during the first 3 days, and cysteine proteinase revealed its effect after 3 days. Meanwhile, during chilled storage for 11 days, the final values of shear force increased 19.68% and 24.33% in PMSF and E-64 treatments when compared to control fillets respectively, whereas the increase after Phen treatment was 49.89%. CONCLUSION: Our study concluded that the disintegration of collagen in post-mortem softening of grass carp fillets was mainly mediated by metalloproteinase and to a lesser extent by serine proteinase and cysteine proteinase. © 2021 Society of Chemical Industry.

Photoactivatable Reporter to Perform Multiplexed and Temporally Controlled Measurements of Kinase and Protease Activity in Single Cells.

Peptide bioreporters were developed to perform multiplexed measurements of the activation of epidermal growth factor receptor kinase (EGFR), Akt kinase (Akt/protein kinase B), and proteases/peptidases in single cells. The performance characteristics of the three reporters were assessed by measuring the reporter's proteolytic stability, kinetic constants for EGFR and Akt, and dephosphorylation rate. The reporter displaying optimal performance was composed of 6-carboxyfluorescein (6-FAM) on the peptide N-terminus, an Akt substrate sequence employing a threonine phosphorylation site for Akt, followed by a tri-D arginine linker, and finally an EGFR substrate sequence bearing a phosphatase-resistant 7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (L-htc) residue as the EGFR phosphorylation site. Importantly, use of a single electrophoretic condition separated the mono- and diphosphorylated products as well as proteolytic forms permitting the quantitation of multiple enzyme activities simultaneously using a single reporter. Because the Akt and EGFR substrates were linked, a known ratio (EGFR/Akt) of the reporter was loaded into cells. A photoactivatable version of the reporter was synthesized by adding two 4,5-dimethoxy-2-nitrobenzyl (DMNB) moieties to mask the EGFR and Akt phosphorylation sites. The DMNB moieties were readily photocleaved following exposure to 360 nm light, unmasking the phosphorylation sites on the reporter. The new photoactivatable reporter permitted multiplexed measurements of kinase signaling and proteolytic degradation in single cells in a temporally controlled manner. This work will facilitate the development of a new generation of multiplexed activity-based reporters capable of light-initiated measurement of enzymatic activity in single cells.

Surface Plasmon Resonance for Protease Detection by Integration of Homogeneous Reaction.

The heterogeneous assays of proteases usually require the immobilization of peptide substrates on the solid surface for enzymatic hydrolysis reactions. However, immobilization of peptides on the solid surface may cause a steric hindrance to prevent the interaction between the substrate and the active center of protease, thus limiting the enzymatic cleavage of the peptide. In this work, we reported a heterogeneous surface plasmon resonance (SPR) method for protease detection by integration of homogeneous reaction. The sensitivity was enhanced by the signal amplification of streptavidin (SA)-conjugated immunoglobulin G (SA-IgG). Caspase-3 (Cas-3) was determined as the model. A peptide labeled with two biotin tags at the N- and C-terminals (bio-GDEVDGK-bio) was used as the substrate. In the absence of Cas-3, the substrate peptide was captured by neutravidin (NA)-covered SPR chip to facilitate the attachment of SA-IgG by the avidin-biotin interaction. However, once the peptide substrate was digested by Cas-3 in the aqueous phase, the products of bio-GDEVD and GK-bio would compete with the substrate to bond NA on the chip surface, thus limiting the attachment of SA-IgG. The method integrated the advantages of both heterogeneous and homogeneous assays and has been used to determine Cas-3 inhibitor and evaluate cell apoptosis with satisfactory results.

Mapping Salivary Proteases in Sjögren's Syndrome Patients Reveals Overexpression of Dipeptidyl Peptidase-4/CD26.

Sjögren's Syndrome (SS) is an autoimmune exocrinopathy characterized by the progressive damage of salivary and lacrimal glands associated with lymphocytic infiltration. Identifying new non-invasive biomarkers for SS diagnosis remains a challenge, and alterations in saliva composition reported in patients turn this fluid into a source of potential biomarkers. Among these, proteases are promising candidates since they are involved in several key physio-pathological processes. This study evaluated differentially expressed proteases in SS individuals' saliva using synthetic fluorogenic substrates, zymography, ELISA, and proteomic approaches. Here we reported, for the first time, increased activity of the serine protease dipeptidyl peptidase-4/CD26 (DPP4/CD26) in pSS saliva, the expression level of which was corroborated by ELISA assay. Gelatin zymograms showed that metalloproteinase proteolytic band profiles differed significantly in intensity between control and SS groups. Focusing on matrix metalloproteinase-9 (MMP9) expression, an increased tendency in pSS saliva (p = 0.0527) was observed compared to the control group. Samples of control, pSS, and sSS were analyzed by mass spectrometry to reveal a general panorama of proteases in saliva. Forty-eight protein groups of proteases were identified, among which were the serine proteases cathepsin G (CTSG), neutrophil elastase (ELANE), myeloblastin (PRTN3), MMP9 and several protease inhibitors. This work paves the way for proteases to be explored in the future as biomarkers, emphasizing DPP4 by its association in several autoimmune and inflammatory diseases. Besides its proteolytic role, DPP4/CD26 acts as a cell surface receptor, signal transduction mediator, adhesion and costimulatory protein involved in T lymphocytes activation.

Analysis of Murein Hydrolases and Proteases Through Zymography.

Zymography has been used to analyze enzymatic activity and processing of enzymes for many years. We have used bacterial cells copolymerized into the acrylamide gel to analyze specific activity of murein hydrolases of interest. In addition, this method has been widely used to examine and distinguish protease activities using different substrates. This chapter provides instruction for zymography of both extracellular murein hydrolases and proteases produced by Staphylococcus aureus.

Near-Infrared Fluorescent Probes for the Detection of Cancer-Associated Proteases.

Proteases are enzymes capable of catalyzing protein breakdown, which is critical across many biological processes. There are several families of proteases, each of which perform key functions through the degradation of specific proteins. As our understanding of cancer improves, it has been demonstrated that several proteases can be overactivated during the progression of cancer and contribute to malignancy. Optical imaging systems that employ near-infrared (NIR) fluorescent probes to detect protease activity offer clinical promise, both for early detection of cancer as well as for the assessment of personalized therapy. In this Review, we review the design of NIR probes and their successful application for the detection of different cancer-associated proteases.

An Integrative Bioinformatic Analysis for Keratinase Detection in Marine-Derived Streptomyces.

Keratinases present promising biotechnological applications, due to their ability to degrade keratin. Streptomyces appears as one of the main sources of these enzymes, but complete genome sequences of keratinolytic bacteria are still limited. This article reports the complete genomes of three marine-derived streptomycetes that show different levels of feather keratin degradation, with high (strain G11C), low (strain CHD11), and no (strain Vc74B-19) keratinolytic activity. A multi-step bioinformatics approach is described to explore genes encoding putative keratinases in these genomes. Despite their differential keratinolytic activity, multiplatform annotation reveals similar quantities of ORFs encoding putative proteases in strains G11C, CHD11, and Vc74B-19. Comparative genomics classified these putative proteases into 140 orthologous groups and 17 unassigned orthogroup peptidases belonging to strain G11C. Similarity network analysis revealed three network communities of putative peptidases related to known keratinases of the peptidase families S01, S08, and M04. When combined with the prediction of cellular localization and phylogenetic reconstruction, seven putative keratinases from the highly keratinolytic strain Streptomyces sp. G11C are identified. To our knowledge, this is the first multi-step bioinformatics analysis that complements comparative genomics with phylogeny and cellular localization prediction, for the prediction of genes encoding putative keratinases in streptomycetes.

Substrate-biased activity-based probes identify proteases that cleave receptor CDCP1.

CUB domain-containing protein 1 (CDCP1) is an oncogenic orphan transmembrane receptor and a promising target for the detection and treatment of cancer. Extracellular proteolysis of CDCP1 by poorly defined mechanisms induces pro-metastatic signaling. We describe a new approach for the rapid identification of proteases responsible for key proteolytic events using a substrate-biased activity-based probe (sbABP) that incorporates a substrate cleavage motif grafted onto a peptidyl diphenyl phosphonate warhead for specific target protease capture, isolation and identification. Using a CDCP1-biased probe, we identify urokinase (uPA) as the master regulator of CDCP1 proteolysis, which acts both by directly cleaving CDCP1 and by activating CDCP1-cleaving plasmin. We show that coexpression of uPA and CDCP1 is strongly predictive of poor disease outcome across multiple cancers and demonstrate that uPA-mediated CDCP1 proteolysis promotes metastasis in disease-relevant preclinical in vivo models. These results highlight CDCP1 cleavage as a potential target to disrupt cancer and establish sbABP technology as a new approach to identify disease-relevant proteases.

Validation of an admission coagulation panel for risk stratification of COVID-19 patients.

BACKGROUND: There is limited data on the markers of coagulation and hemostatic activation (MOCHA) profile in Coronavirus disease 2019 (COVID-19) and its ability to identify COVID-19 patients at risk for thrombotic events and other complications. METHODS: Hospitalized patients with confirmed SARS-COV-2 from four Atlanta hospitals were included in this observational cohort study and underwent admission testing of MOCHA parameters (plasma d-dimer, prothrombin fragment 1.2, thrombin-antithrombin complex, fibrin monomer). Clinical outcomes included deep vein thrombosis, pulmonary embolism, myocardial infarction, ischemic stroke, access line thrombosis, ICU admission, intubation and mortality. MAIN RESULTS: Of 276 patients (mean age 59 ± 6.4 years, 47% female, 62% African American), 45 (16%) had a thrombotic endpoint. Each MOCHA parameter was independently associated with a thrombotic event (p<0.05) and ≥ 2 abnormalities was associated with thrombotic endpoints (OR 3.3, 95% CI 1.2-8.8) as were admission D-dimer ≥ 2000 ng/mL (OR 3.1, 95% CI 1.5-6.6) and ≥ 3000 ng/mL (OR 3.6, 95% CI 1.6-7.9). However, only ≥ 2 MOCHA abnormalities were associated with ICU admission (OR 3.0, 95% CI 1.7-5.2) and intubation (OR 3.2, 95% CI 1.6-6.4). MOCHA and D-dimer cutoffs were not associated with mortality. MOCHA with <2 abnormalities (26% of the cohort) had 89% sensitivity and 93% negative predictive value for a thrombotic endpoint. CONCLUSIONS: An admission MOCHA profile is useful to risk-stratify COVID-19 patients for thrombotic complications and more effective than isolated d-dimer for predicting risk of ICU admission and intubation.