NETosis occurs independently of neutrophil serine proteases.

Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity.

Noninvasive optical detection of granzyme B from natural killer cells with enzyme-activated fluorogenic probes.

Natural killer (NK) cells are key innate immunity effectors that combat viral infections and control several cancer types. For their immune function, human NK cells rely largely on five different cytotoxic proteases, called granzymes (A/B/H/K/M). Granzyme B (GrB) initiates at least three distinct cell death pathways, but key aspects of its function remain unexplored because selective probes that detect its activity are currently lacking. In this study, we used a set of unnatural amino acids to fully map the substrate preferences of GrB, demonstrating previously unknown GrB substrate preferences. We then used these preferences to design substrate-based inhibitors and a GrB-activatable activity-based fluorogenic probe. We show that our GrB probes do not significantly react with caspases, making them ideal for in-depth analyses of GrB localization and function in cells. Using our quenched fluorescence substrate, we observed GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with GrB attacked MDA-MB-231 target cells, and active GrB influenced its target cell-killing efficiency. In summary, we have developed a set of molecular tools for investigating GrB function in NK cells and demonstrate noninvasive visual detection of GrB with an enzyme-activated fluorescent substrate.

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils.

BACKGROUND: Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. RESULTS: Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. CONCLUSIONS: Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.

An alternative for proteinase K-heat-sensitive protease from fungus Onygena corvina for biotechnology: cloning, engineering, expression, characterization and special application for protein sequencing.

BACKGROUND: A neutral, heat-sensitive serine protease (NHSSP) originating from the feather-degrading fungus Onygena corvina (O. corvina) was described and defined as an alkaline serine protease of the subtilisin type S8 family, exhibiting an enzymatic activity at neutral pH. Generally, broad specificity proteases, such as proteinase K or trypsin, have found numerous applications in research and biotechnology. RESULTS: We report the cloning and expression in the yeast PichiaPink™ system, as well as purification, and characterization of the NHSSP. Recombinant, His6-tagged NHSSP was efficiently expressed from an optimized, synthetic gene and purified using a simple protocol based on ammonium sulfate fractionation and hydrophobic interaction chromatography. The enzyme shows atypical C-terminal processing, the coded preproprotein undergoes signal peptide removal and maturation through the clipping of a propeptide section and 10 amino acids (aa) from the C-terminus, including the His6-tag. The deletion variant has been constructed, devoid of the C-terminal ORF segment, thus eliminating the need for C-terminal processing. Both NHSSP variants exhibit very similar enzymatic characteristics. The purified enzymes were characterized to determine the optimal proteolytic conditions. We revealed that the mature NHSSP is reproducibly active over a wide pH range from neutral to mild acidic (pH of 5.0 to 8.5), with an optimum at pH 6.8, and at temperatures of 15 to 50 °C with an optimum at 38-42 °C. Interestingly, we demonstrated that the protease can be fully deactivated by a moderate increase in temperature of about 15 °C from the optimum to over 50 °C. The protease was partially sensitive to serine protease inhibitors, and not inhibited by chelating or reducing agents and detergents. SDS induced autolysis of NHSSP, which points to a high stimulation of its proteolytic activity. CONCLUSIONS: The NHSSP was produced as a recombinant protein with high efficiency. Compared to proteinase K, the most common serine protease used, NHSSP shows an approx. twofold higher specific activity. Protein sequencing can be a valuable technical application for the protease. The protein coverage is significantly higher in comparison to trypsin and reaches about 84-100% for ß-lactoglobulin (BLG), antibody (mAb) light and heavy chains. Furthermore, the option to perform digestions at neutral to slightly acidic pH-values down to pH 5.0 avoids modification of peptides, e.g. due to deamidation.

Toolbox of Fluorescent Probes for Parallel Imaging Reveals Uneven Location of Serine Proteases in Neutrophils.

Neutrophils, the front line defenders against infection, express four serine proteases (NSPs) that play roles in the control of cell-signaling pathways and defense against pathogens and whose imbalance leads to pathological conditions. Dissecting the roles of individual NSPs in humans is problematic because neutrophils are end-stage cells with a short half-life and minimal ongoing protein synthesis. To gain insight into the regulation of NSP activity we have generated a small-molecule chemical toolbox consisting of activity-based probes with different fluorophore-detecting groups with minimal wavelength overlap and highly selective natural and unnatural amino acid recognition sequences. The key feature of these activity-based probes is the ability to use them for simultaneous observation and detection of all four individual NSPs by fluorescence microscopy, a feature never achieved in previous studies. Using these probes we demonstrate uneven distribution of NSPs in neutrophil azurophil granules, such that they seem to be mutually excluded from each other, suggesting the existence of unknown granule-targeting mechanisms.

Small Molecule Active Site Directed Tools for Studying Human Caspases.

Caspases are proteases of clan CD and were described for the first time more than two decades ago. They play critical roles in the control of regulated cell death pathways including apoptosis and inflammation. Due to their involvement in the development of various diseases like cancer, neurodegenerative diseases, or autoimmune disorders, caspases have been intensively investigated as potential drug targets, both in academic and industrial laboratories. This review presents a thorough, deep, and systematic assessment of all technologies developed over the years for the investigation of caspase activity and specificity using substrates and inhibitors, as well as activity based probes, which in recent years have attracted considerable interest due to their usefulness in the investigation of biological functions of this family of enzymes.

Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling.

The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1-S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones.

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme.

Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferon-stimulated gene 15 (ISG15) participate in the host defence of viral infections. Viruses, including the severe acute respiratory syndrome human coronavirus (SARS hCoV), have co-opted Ub-ISG15 conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub-ISG15-conjugated host proteins. In the present study, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle East respiratory syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that, similar to SARS PLpro, MERS PLpro is both a deubiquitinating (DUB) and a deISGylating enzyme. Further analysis of the intrinsic DUB activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, whereas SARS PLpro prefers to cleave Lys48-linked polyUb chains. Secondly, MERS PLpro cleaves polyUb chains in a 'mono-distributive' manner (one Ub at a time) and SARS PLpro prefers to cleave Lys48-linked polyUb chains by sensing a di-Ub moiety as a minimal recognition element using a 'di-distributive' cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP (Ub-specific protease)-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help to identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses.

Recent advances and concepts in substrate specificity determination of proteases using tailored libraries of fluorogenic substrates with unnatural amino acids.

Substrate specificity of proteases can be determined using several methods among which the most frequently used are positional scanning library, proteomics and phage display. Classic approaches can deliver information about preferences for natural amino acids in binding pockets of virtually all proteases. However, recent studies demonstrate the ability to obtain much more information by application of unnatural amino acids to positional scanning library approaches. This knowledge can be used for the design of more active and specific substrates, inhibitors and activity based probes. In this minireview we describe recent strategies and concepts for the design and application of fluorogenic substrates library tailored for exopeptidases and endopeptidases.

Substrate specificity profiling of heat-sensitive serine protease from the fungus Onygena corvina.

Proteases catalyze hydrolysis of amide bonds within peptides and proteins, therefore they play crucial functions for organism functioning, but also in industry to facilitate numerous processes. Feather-degrading fungus Onygena corvina (O. corvina) is loaded with numerous proteases that can be utilized for variety of applications. The most active species of these enzymes is heat-sensitive serine protease (NHSSP), from O. corvina fungi and due to its potential applications in industry is an alternative to proteinase K. The uniqueness of NHSSP relies on the ability of NHSSP to hydrolyze peptides at neutral to acidic pH values between 5.0 to 8.5, with an optimum of 6.8 and a temperature activity ranging from 15-50°C making NHSSP exceptionally universal enzyme. Thus, we have performed the in-depth characterization of NHSSP substrate specificity by using a positional scanning substrate combinatorial library (PS-SCL). Afterward, we obtained a set of fluorescent substrates hydrolyzed by NHSSP that served as a leading sequence for the first tailored covalent inhibitor of this enzyme, containing a diphenylphosphonate as a warhead and MeOSuc amine protecting group. Our first inhibitor for NHSSP binds potently with target protease and is a tool for future study of this enzyme functions.

Pulsed electric field induces exocytosis and overexpression of MAGE antigens in melanoma.

Nanosecond pulsed electric field (nsPEF) has emerged as a promising approach for inducing cell death in melanoma, either as a standalone treatment or in combination with chemotherapeutics. However, to date, there has been a shortage of studies exploring the impact of nsPEF on the expression of cancer-specific molecules. In this investigation, we sought to assess the effects of nsPEF on melanoma-specific MAGE (Melanoma Antigen Gene Protein Family) expression. To achieve this, melanoma cells were exposed to nsPEF with parameters set at 8 kV/cm, 200 ns duration, 100 pulses, and a frequency of 10 kHz. We also aimed to comprehensively describe the consequences of this electric field on melanoma cells' invasion and proliferation potential. Our findings reveal that following exposure to nsPEF, melanoma cells release microvesicles containing MAGE antigens, leading to a simultaneous increase in the expression and mRNA content of membrane-associated antigens such as MAGE-A1. Notably, we observed an unexpected increase in the expression of PD-1 as well. While we did not observe significant differences in the cells' proliferation or invasion potential, a remarkable alteration in the cells' metabolomic and lipidomic profiles towards a less aggressive phenotype was evident. Furthermore, we validated these results using ex vivo tissue cultures and 3D melanoma culture models. Our study demonstrates that nsPEF can elevate the expression of membrane-associated proteins, including melanoma-specific antigens. The mechanism underlying the overexpression of MAGE antigens involves the initial release of microvesicles containing MAGE antigens, followed by a gradual increase in mRNA levels, ultimately resulting in elevated expression of MAGE antigens post-experiment. These findings shed light on a novel method for modulating cancer cells to overexpress cancer-specific molecules, thereby potentially enhancing their sensitivity to targeted anticancer therapy.

Mechanism and specificity of the human paracaspase MALT1.

The paracaspase domain of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of a gene translocation fused to the N-terminal domains of the cellular inhibitor of apoptosis protein 2. The paracaspase itself, commonly known as MALT1, participates in the NF-κB (nuclear factor κB) pathway, probably by driving survival signals downstream of the B-cell antigen receptor through MALT1 proteolytic activity. We have developed methods for the expression and purification of recombinant full-length MALT1 and its constituent catalytic domain alone. Both are activated by dimerization without cleavage, with a similar dimerization barrier to the distantly related cousins, the apical caspases. By using positional-scanning peptidyl substrate libraries we demonstrate that the activity and specificity of full-length MALT1 is recapitulated by the catalytic domain alone, showing a stringent requirement for cleaving after arginine, and with striking peptide length constraints for efficient hydrolysis. Rates of cleavage (kcat/Km values) of optimal peptidyl substrates are in the same order (10(3)-10(4) M(-1)·s(-1)) as for a putative target protein CYLD. Thus MALT1 has many similarities to caspase 8, even cleaving the putative target protein CYLD with comparable efficiencies, but with diametrically opposite primary substrate specificity.

A protocol for visualizing active cathepsin K in osteoclasts with a quenched-fluorescence-activity-based probe.

Herein, we provide a protocol for visualizing active osteoclast cathepsin K (CatK) with the quenched-fluorescent-activity-based probe qTJK17. We describe steps for isolating peripheral blood mononuclear cells, their differentiation into osteoclasts, and TRAP staining using an acid phosphatase leukocyte kit. We then detail visualization of active CatK. The probe qTJK17 includes a reactive group, acyloxymethylketone, that binds to the CatK active site, recognition sequence, and fluorescence donor-acceptor pair. This protocol can determine the exact localization of active CatK in osteoclasts. For complete details on the use and execution of this protocol, please refer to Janiszewski et al. (2023).1.

Fluorescent Activity-Based Probe To Image and Inhibit Factor XIa Activity in Human Plasma.

Anticoagulation therapy is a mainstay of the treatment of thrombotic disorders; however, conventional anticoagulants trade antithrombotic benefits for bleeding risk. Factor (f) XI deficiency, known as hemophilia C, rarely causes spontaneous bleeding, suggesting that fXI plays a limited role in hemostasis. In contrast, individuals with congenital fXI deficiency display a reduced incidence of ischemic stroke and venous thromboembolism, indicating that fXI plays a role in thrombosis. For these reasons, there is intense interest in pursuing fXI/factor XIa (fXIa) as targets for achieving antithrombotic benefit with reduced bleeding risk. To obtain selective inhibitors of fXIa, we employed libraries of natural and unnatural amino acids to profile fXIa substrate preferences. We developed chemical tools for investigating fXIa activity, such as substrates, inhibitors, and activity-based probes (ABPs). Finally, we demonstrated that our ABP selectively labels fXIa in the human plasma, making this tool suitable for further studies on the role of fXIa in biological samples.

Nanosecond pulsed electric field suppresses growth and reduces multi-drug resistance effect in pancreatic cancer.

Nanosecond pulsed electric fields (nsPEF) have been shown to exert anticancer effects; however, little is known about the mechanisms triggered in cancer cells by nanosecond-length pulses, especially when low, sub-permeabilization voltage is used. In this study, three human pancreatic cancer cell lines were treated with nsPEF and molecular changes at the cellular level were analyzed. Further, we assessed the efficacy of paclitaxel chemotherapy following nsPEF treatment and correlated that with the changes in the expression of multi-drug resistance (MDR) proteins. Finally, we examined the influence of nsPEF on the adhesive properties of cancer cells as well as the formation and growth of pancreatic cancer spheroids. Cell line response differed with the application of a 200 ns, 100 pulses, 8 kV/cm, 10 kHz PEF treatment. PEF treatment led to (1) the release of microvesicles (MV) in EPP85-181RDB cells, (2) electropermeabilization in EPP85-181RNOV cells and (3) cell shrinkage in EPP85-181P cells. The release of MV's in EPP85-181RDB cells reduced the membrane content of P-gp and LRP, leading to a transient increase in vulnerability of the cells towards paclitaxel. In all cell lines we observed an initial reduction in size of the cancer spheroids after the nsPEF treatment. Cell line EPP85-181RNOV exhibited a permanent reduction in the spheroid size after nsPEF. We propose a mechanism in which the surface tension of the membrane, regulated by the organization of actin fibers, modulates the response of cancer cells towards nsPEF. When a membrane's surface tension remains low, we observed some cells form protrusions and release MVs containing MDR proteins. In contrast, when cell surface tension remains high, the cell membrane is being electroporated. The latter effect may be responsible for the reduced tumor growth following nsPEF treatment.

Investigation of osteoclast cathepsin K activity in osteoclastogenesis and bone loss using a set of chemical reagents.

Cathepsin K (CatK) is a lysosomal cysteine protease whose highest expression is found in osteoclasts, which are the cells responsible for bone resorption. Investigations of the functions and physiological relevance of CatK have often relied on antibody-related techniques, which makes studying its activity patterns a challenging task. Hence, we developed a set of chemical tools for the investigation of CatK activity. We show that our probe is a valuable tool for monitoring the proteolytic activation of CatK during osteoclast formation. Moreover, we demonstrate that our inhibitor of CatK impedes osteoclastogenesis and bone resorption and that CatK is stored in its active form in osteoclasts within their lysosomal compartment and mainly in the ruffled borders of osteoclasts. Given that our probe recognizes active CatK within living cells without exhibiting any observed cytotoxicity in the several models tested, we expect that it would be well suited to theranostic applications in CatK-related diseases.

Current and prospective applications of non-proteinogenic amino acids in profiling of proteases substrate specificity.

Proteases recognize their endogenous substrates based largely on a sequence of proteinogenic amino acids that surrounds the cleavage site. Currently, several methods are available to determine protease substrate specificity based on approaches employing proteinogenic amino acids. The knowledge about the specificity of proteases can be significantly extended by application of structurally diverse families of non-proteinogenic amino acids. From a chemical point of view, this information may be used to design specific substrates, inhibitors, or activity-based probes, while biological functions of proteases, such as posttranslational modifications can also be investigated. In this review, we discuss current and prospective technologies for application of non-proteinogenic amino acids in protease substrate specificity profiling.

Parallel imaging of coagulation pathway proteases activated protein C, thrombin, and factor Xa in human plasma.

Activated protein C (APC), thrombin, and factor (f) Xa are vitamin K-dependent serine proteases that are key factors in blood coagulation. Moreover, they play important roles in inflammation, apoptosis, fibrosis, angiogenesis, and viral infections. Abnormal activity of these coagulation factors has been related to multiple conditions, such as bleeding and thrombosis, Alzheimer's disease, sepsis, multiple sclerosis, and COVID-19. The individual activities of APC, thrombin, and fXa in coagulation and in various diseases are difficult to establish since these proteases are related and have similar substrate preferences. Therefore, the development of selective chemical tools that enable imaging and discrimination between coagulation factors in biological samples may provide better insight into their roles in various conditions and potentially aid in the establishment of novel diagnostic tests. In our study, we used a large collection of unnatural amino acids, and this enabled us to extensively explore the binding pockets of the enzymes' active sites. Based on the specificity profiles obtained, we designed highly selective substrates, inhibitors, and fluorescent activity-based probes (ABPs) that were used for fast, direct, and simultaneous detection of APC, thrombin, and fXa in human plasma.

Simple phosphonic inhibitors of human neutrophil elastase.

Herein, we describe the synthesis and resulting activity of a complex series of α-aminophosphonate diaryl esters as irreversible human neutrophil elastase inhibitors and their selectivity preference for human neutrophil elastase over several other serine proteases such as porcine pancreatic elastase, trypsin, and chymotrypsin. We synthesized and examined the inhibitory potency of several new simple Cbz-protected α-aminoalkylphosphonate diaryl esters that yielded several new HNE inhibitors, where one of the obtained compounds Cbz-Val(P)(OC(6)H(4)-4-COOMe)(2) displayed an apparent second-order inhibition value at 33,015 M(-1) s(-1).