Active Protease Research Articles

Structural basis for the non-self RNA-activated protease activity of the type III-E CRISPR nuclease-protease Craspase

The RNA-targeting type III-E CRISPR-gRAMP effector interacts with a caspase-like protease TPR-CHAT to form the CRISPR-guided caspase complex (Craspase), but their functional mechanism is unknown. Here, we report cryo-EM structures of the type III-E gRAMPcrRNA and gRAMPcrRNA-TPR-CHAT complexes, before and after either self or non-self RNA target binding, and elucidate the mechanisms underlying RNA-targeting and non-self RNA-induced protease activation. The associated TPR-CHAT adopted a distinct conformation upon self versus non-self RNA target binding, with nucleotides at positions −1 and −2 of the CRISPR-derived RNA (crRNA) serving as a sensor. Only binding of the non-self RNA target activated the TPR-CHAT protease, leading to cleavage of Csx30 protein. Furthermore, TPR-CHAT structurally resembled eukaryotic separase, but with a distinct mechanism for protease regulation. Our findings should facilitate the development of gRAMP-based RNA manipulation tools, and advance our understanding of the virus-host discrimination process governed by a nuclease-protease Craspase during type III-E CRISPR-Cas immunity.

Proteolytic Activity against the Distal Polybasic Tract of the Gamma Subunit of the Epithelial Sodium Channel ENaC in Nephrotic Urine.

Experimental nephrotic syndrome in mice leads to proteolytic activation of the epithelial sodium channel ENaC, possibly involving the distal polybasic tract of its γ-subunit (183RKRK). We sought to determine if urine samples from both nephrotic mice and a cohort of patients with acute nephrotic syndrome contain a specific proteolytic activity against this region of γ-ENaC. A peptide substrate consisting of amino acids 180-194 of murine γ-ENaC was N-terminally coupled to a fluorophore, yielding AMCA-FTGRKRKISGKIIHK. The substrate was incubated with nephrotic urine samples from mice as well as patients with or without the serine protease inhibitor, aprotinin. The digested peptides were separated on a reverse phase HPLC and detected with a fluorescence detector (350/450 nm). Peptide masses of the peaks were determined with a MALDI-TOF mass spectrometer. In addition, urinary proteolytic activity was quantitated using AMC-coupled substrates reflecting different cleavage sites within the polybasic tract. No significant proteolytic activity against the substrate was found in the urine of healthy humans or mice. Incubation with urine samples of nephrotic patients (n = 8) or mice subjected to three different models of experimental nephrotic syndrome (n = 4 each) led to cleavage of the substrate within the polybasic tract prevented by the serine protease inhibitor aprotinin. The most dominant cleavage product was FTGRKR in both species, which was confirmed using quantitative measurements with FTGRKR- AMC. Nephrotic urine from both humans and mice contains aprotinin-sensitive proteolytic activity against the distal polybasic tract of γ-ENaC, reflecting excretion of active proteases in the urine or proteasuria.

Identification of Cysteine 270 as a Novel Site for Allosteric Modulators of SARS‐CoV‐2 Papain‐Like Protease**

AbstractThe coronavirus papain‐like protease (PLpro) plays an important role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. However, the development of inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) PLpro is challenging owing to the restricted S1/S2 sites in the substrate binding pocket. Here we report the discovery of two activators of SARS‐CoV‐2 PLpro and the identification of the unique residue, cysteine 270 (C270), as an allosteric and covalent regulatory site for the activators. This site is also specifically modified by glutathione, resulting in protease activation. Furthermore, a compound was found to allosterically inhibit the protease activity by covalent binding to C270. Together, these results elucidate an unrevealed molecular mechanism for allosteric modulation of SARS‐CoV‐2 PLpro and provid a novel site for allosteric inhibitors design.

Engineering receptors in the secretory pathway for orthogonal signalling control

Synthetic receptors targeted to the secretory pathway often fail to exhibit the expected activity due to post-translational modifications (PTMs) and/or improper folding. Here, we engineered synthetic receptors that reside in the cytoplasm, inside the endoplasmic reticulum (ER), or on the plasma membrane through orientation adjustment of the receptor parts and by elimination of dysfunctional PTMs sites. The cytoplasmic receptors consist of split-TEVp domains that reconstitute an active protease through chemically-induced dimerization (CID) that is triggered by rapamycin, abscisic acid, or gibberellin. Inside the ER, however, some of these receptors were non-functional, but their activity was restored by mutagenesis of cysteine and asparagine, residues that are typically associated with PTMs. Finally, we engineered orthogonal chemically activated cell-surface receptors (OCARs) consisting of the Notch1 transmembrane domain fused to cytoplasmic tTA and extracellular CID domains. Mutagenesis of cysteine residues in CID domains afforded functional OCARs which enabled fine-tuning of orthogonal signalling in mammalian cells.

Identification of Cysteine 270 as a Novel Site for Allosteric Modulators of SARS-CoV-2 Papain-Like Protease.

The coronavirus papain-like protease (PLpro ) plays an important role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. However, the development of inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro is challenging owing to the restricted S1/S2 sites in the substrate binding pocket. Here we report the discovery of two activators of SARS-CoV-2 PLpro and the identification of the unique residue, cysteine 270 (C270), as an allosteric and covalent regulatory site for the activators. This site is also specifically modified by glutathione, resulting in protease activation. Furthermore, a compound was found to allosterically inhibit the protease activity by covalent binding to C270. Together, these results elucidate an unrevealed molecular mechanism for allosteric modulation of SARS-CoV-2 PLpro and provid a novel site for allosteric inhibitors design.

Genome Editing of Factor IX Paralogs in Zebrafish Demonstrates Subfunctionalization of Procoagulant Function

Hemophilia is an inherited monogenic bleeding disorder affecting ~400,000 individuals worldwide, with 20% of cases due to deficiency of factor IX (FIX, hemophilia B), affecting 1 in 30,000 male births. Although there are many exciting new treatments for this disorder, there are still unknown features of FIX. This includes potential distribution from plasma to tissue, which may affect some therapies. The zebrafish is a unique model to study coagulation disorders due to its high fecundity, external development, and optical transparency, as well as a high degree of genetic and functional conservation with the mammalian hemostatic system. Due to an ancestral gene duplication event in zebrafish, 30-40% of the zebrafish genome is duplicated, including the FIX gene (f9), resulting in the paralogs f9a and f9b. Such paralogs may arise due to subfunctionalization of the ancestral gene, raising the possibility that this could identify roles for mammalian FIX that have not previously been discovered. Here we utilize CRISPR/Cas9 genome editing to produce large deletions in f9a and f9b using guide RNAs that delete the intervening regions between exons 2 and 8, uncovering evidence for subfunctionalization. Assessment of hemostasis in 3 day post fertilization (dpf) mutant larvae using laser-mediated endothelial injury unexpectedly revealed that single and double f9a and f9b homozygous mutants exhibited normal thrombus formation. Upon crossing these mutants into an antithrombin (at3) deficient background (which exhibits spontaneous venous thrombosis), loss of F9b (f9b-/-;at3-/- mutants) prevented intravascular thrombosis development. f9a-/-;at3-/- double mutants were indistinguishable from f9a+/+;at3-/- siblings, both showing spontaneous thrombosis. These results demonstrated subfunctionalization of F9 activities, which could be due to differential functions or tissue-specific expression. The latter was investigated using whole mount in situ hybridization in wild-type larvae, revealing that f9a and f9b are both expressed in the liver. To evaluate function, microinjection of zebrafish f9a and f9b cDNAs regulated by a ubiquitous promoter was performed in one-cell stage f9b-/-;at3-/- zebrafish embryos. Analysis at 3 dpf found that only F9b could restore spontaneous thrombosis, confirming functional differences between F9a and F9b. Alignment of human FIX to F9a and F9b indicated conservation of functional domain sequences (signal sequence, propeptide, Gla, Egf1, Egf2, activation peptide, and protease). These domains were individually swapped between the f9a and f9b plasmids. Except for Egf1, all individual domains of F9a exchanged into F9b were able to restore thrombus formation in f9b-/-;at3-/- mutants. Replacement of F9b Egf1 with a F9aQ59G modified Egf1 domain (F9b has a glycine at this orthologous position), resulted in normal function. In summary, these data show clear evidence for subfunctionalization of F9 in zebrafish larvae, with F9b retaining procoagulant function. F9a possesses an active protease domain, but variation in Egf1 prevents it from contributing to canonical circulating FIX activity. We hypothesize that the alternative role for F9a might represent a previously unknown activity or tissue localization of human FIX. This putative role would also be deficient in hemophilia B patients and/or may be lacking in current therapies. An additional possibility is that this represents another means for regulation of FIX. In both cases, further study could lead to novel therapeutic ideas for patients with hemophilia B.

Mast Cells and Interleukins.

Mast cells play a critical role in inflammatory diseases and tumor growth. The versatility of mast cells is reflected in their ability to secrete a wide range of biologically active cytokines, including interleukins, chemokines, lipid mediators, proteases, and biogenic amines. The aim of this review article is to analyze the complex involvement of mast cells in the secretion of interleukins and the role of interleukins in the regulation of biological activities of mast cells.

ADAMTS6 cleaves the large latent TGFβ complex and increases the mechanotension of cells to activate TGFβ

The ADAMTS superfamily is composed of secreted metalloproteases and structurally related non-catalytic ADAMTS-like proteins. A subset of this superfamily, including ADAMTS6, ADAMTS10 and ADAMTSL2, are involved in elastic fiber assembly and bind to fibrillin and other matrix molecules that regulate the extracellular bioavailability of the potent growth factor TGFβ. Fibrillinopathies, that can also result from mutation of these ADAMTS/L proteins, have been linked to disrupted TGFβ homeostasis. ADAMTS6 and ADAMTS10 are homologous metalloproteases with poorly characterized substrates where ADAMTS10 is thought to process fibrillin-2 and ADAMTS6 latent TGFβ-binding protein (LTBP)-1. In order to understand the contribution of ADAMTS6, and these other members of the ADAMTS/L family, to TGFβ homeostasis, we have analyzed the effects of ADAMTS6, ADAMTS10 and ADAMTSL2 expression on TGFβ activation. We found that their expression increases TGFβ activation in a dose dependent manner, following stimulation with mature TGFβ1. For ADAMTS6, the catalytically active protease is required for effective TGFβ activation, where ADAMTS6 cleaves LTBP3 as well as LTBP1, and binds to the large latent TGFβ complexes of LTBP1 and LTBP3. Furthermore, ADAMTS6 expression increases the mechanotension of cells which results in inactivation of the Hippo Pathway, resulting in an increased translocation of YAP/TAZ complex to the nucleus. Together these findings suggest that when the balance of TGFβ is perturbed ADAMTS6 can influence TGFβ activation via two mechanisms. It directly cleaves the latent TGFβ complexes and also acts indirectly, along with ADAMTS10 and ADAMTSL2, by altering the mechanotension of cells. Together this increases activation of TGFβ from large latent complexes which may contribute to disease pathogenesis.

Dual role of an essential HtrA2/Omi protease in the human malaria parasite: Maintenance of mitochondrial homeostasis and induction of apoptosis-like cell death under cellular stress

Members of the HtrA family of serine proteases are known to play roles in mitochondrial homeostasis as well as in programmed cell death. Mitochondrial homeostasis and metabolism are crucial for the survival and propagation of the malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin-like protease activity that can be inhibited by its specific inhibitor, ucf-101. A transgenic parasite line was generated, using the HA-glmS C-terminal tagging approach, for localization as well as for inducible knock-down of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during the asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, the ucf-101 treatment did not affect the parasite growth, suggesting the non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase-like protease as well as parasite cell death, suggesting the involvement of protease activity of PfHtrA2 in apoptosis-like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show the specific roles of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating stress-induced cell death.

Design of programmable post-translational switch control platform for on-demand protein secretion in mammalian cells.

The development of novel strategies to program cellular behaviors is a central goal in synthetic biology, and post-translational control mediated by engineered protein circuits is a particularly attractive approach to achieve rapid protein secretion on demand. We have developed a programmable protease-mediated post-translational switch (POSH) control platform composed of a chimeric protein unit that consists of a protein of interest fused via a transmembrane domain to a cleavable ER-retention signal, together with two cytosolic inducer-sensitive split protease components. The protease components combine in the presence of the specific inducer to generate active protease, which cleaves the ER-retention signal, releasing the transmembrane-domain-linked protein for trafficking to the trans-Golgi region. A furin site placed downstream of the protein ensures cleavage and subsequent secretion of the desired protein. We show that stimuli ranging from plant-derived, clinically compatible chemicals to remotely controllable inducers such as light and electrostimulation can program protein secretion in various POSH-engineered designer mammalian cells. As proof-of-concept, an all-in-one POSH control plasmid encoding insulin and abscisic acid-activatable split protease units was hydrodynamically transfected into the liver of type-1 diabetic mice. Induction with abscisic acid attenuated glycemic excursions in glucose-tolerance tests. Increased blood levels of insulin were maintained for 12 days.

Multiomics Profiling of Toxins in the Venom of the Amazonian Spider Acanthoscurria juruenicola.

Acanthoscurria juruenicola is an Amazonian spider described for the first time almost a century ago. However, little is known about their venom composition. Here, we present a multiomics characterization of A. juruenicola venom by a combination of transcriptomics, proteomics, and peptidomics approaches. Transcriptomics of female venom glands resulted in 93,979 unique assembled mRNA transcript encoding proteins. A total of 92 proteins were identified in the venom by mass spectrometry, including 14 mature cysteine-rich peptides (CRPs). Quantitative analysis showed that CRPs, cysteine-rich secretory proteins, metalloproteases, carbonic anhydrases, and hyaluronidase comprise >90% of the venom proteome. Relative quantification of venom toxins was performed by DIA and DDA, revealing converging profiles of female and male specimens by both methods. Biochemical assays confirmed the presence of active hyaluronidases, phospholipases, and proteases in the venom. Moreover, the venom promoted in vivo paralytic activities in crickets, consistent with the high concentration of CRPs. Overall, we report a comprehensive analysis of the arsenal of toxins of A. juruenicola and highlight their potential biotechnological and pharmacological applications. Mass spectrometry data were deposited to the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD013149 and via the MassIVE repository with the dataset identifier MSV000087777.

Pharmacological inhibition of cathepsin C (CatC) as a potential approach for cancer therapeutics

Studies have established that proteolytic enzyme inhibition holds significant promise in cancer prevention and treatment. Cathepsin C (CatC) is conserved lysosomal cysteine dipeptidyl aminopeptidase, which is the key for pro-inflammatory neutrophil serine protease activation and biological functioning. This makes CatC as a promising therapeutic drug target for the management of different cancer types. Considering this, using a wide range of computer aided drug-designing applications, several inhibitors are shortlisted against CatC active pocket, which interact with the enzyme with high affinity and form strong intermolecular interaction network. Compared to control, three molecules ASN_06916232, ASN_06917112 and ASN_06916892 are filtered as best binders of the CatC active pocket with binding energy value of −10.9 kcal/mol, −10.9 kcal/mol and −10.7 kcal/mol, respectively. These compounds interact with several important active side residues of CatC such as Ser233, Cys234, Gly277, Asn380 and His38. Furthermore, the complexes of these compounds with CatC reveal very stable dynamics with average RMSD value less than 3 Å. The binding energy analysis further indicates the compounds to have very stable van der Waals and electrostatic energies. In conclusion, these molecules are promising and require experimental validation to prove them as anti-CatC molecules. Communicated by Ramaswamy H. Sarma

Characterization of a multipurpose NS3 surface patch coordinating HCV replicase assembly and virion morphogenesis.

The hepatitis C virus (HCV) life cycle is highly regulated and characterized by a step-wise succession of interactions between viral and host cell proteins resulting in the assembly of macromolecular complexes, which catalyse genome replication and/or virus production. Non-structural (NS) protein 3, comprising a protease and a helicase domain, is involved in orchestrating these processes by undergoing protein interactions in a temporal fashion. Recently, we identified a multifunctional NS3 protease surface patch promoting pivotal protein-protein interactions required for early steps of the HCV life cycle, including NS3-mediated NS2 protease activation and interactions required for replicase assembly. In this work, we extend this knowledge by identifying further NS3 surface determinants important for NS5A hyperphosphorylation, replicase assembly or virion morphogenesis, which map to protease and helicase domain and form a contiguous NS3 surface area. Functional interrogation led to the identification of phylogenetically conserved amino acid positions exerting a critical function in virion production without affecting RNA replication. These findings illustrate that NS3 uses a multipurpose protein surface to orchestrate the step-wise assembly of functionally distinct multiprotein complexes. Taken together, our data provide a basis to dissect the temporal formation of viral multiprotein complexes required for the individual steps of the HCV life cycle.

Acyldepsipeptide Analogues: A Future Generation Antibiotics for Tuberculosis Treatment.

Acyldepsipeptides (ADEPs) are a new class of emerging antimicrobial peptides (AMPs), which are currently explored for treatment of pathogenic infections, including tuberculosis (TB). These cyclic hydrophobic peptides have a unique bacterial target to the conventional anti-TB drugs, and present a therapeutic window to overcome Mycobacterium Tuberculosis (M. tb) drug resistance. ADEPs exerts their antibacterial activity on M. tb strains through activation of the protein homeostatic regulatory protease, the caseinolytic protease (ClpP1P2). ClpP1P2 is normally regulated and activated by the ClpP-ATPases to degrade misfolded and toxic peptides and/or short proteins. ADEPs bind and dysregulate all the homeostatic capabilities of ClpP1P2 while inducing non-selective proteolysis. The uncontrolled proteolysis leads to M. tb cell death within the host. ADEPs analogues that have been tested possess cytotoxicity and poor pharmacokinetic and pharmacodynamic properties. However, these can be improved by drug design techniques. Moreover, the use of nanomaterial in conjunction with ADEPs would yield effective synergistic effect. This new mode of action has potential to combat and eradicate the extensive multi-drug resistance (MDR) problem that is currently faced by the public health pertaining bacterial infections, especially TB.

The first Chinese oral anti-COVID-19 drug Azvudine launched

The first Chinese oral anti-COVID-19 drug Azvudine launched

Dipeptidyl peptidase-1 inhibition in patients hospitalised with COVID-19: a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial

BackgroundNeutrophil serine proteases are involved in the pathogenesis of COVID-19 and increased serine protease activity has been reported in severe and fatal infection. We investigated whether brensocatib, an inhibitor of dipeptidyl peptidase-1 (DPP-1; an enzyme responsible for the activation of neutrophil serine proteases), would improve outcomes in patients hospitalised with COVID-19.MethodsIn a multicentre, double-blind, randomised, parallel-group, placebo-controlled trial, across 14 hospitals in the UK, patients aged 16 years and older who were hospitalised with COVID-19 and had at least one risk factor for severe disease were randomly assigned 1:1, within 96 h of hospital admission, to once-daily brensocatib 25 mg or placebo orally for 28 days. Patients were randomly assigned via a central web-based randomisation system (TruST). Randomisation was stratified by site and age (65 years or ≥65 years), and within each stratum, blocks were of random sizes of two, four, or six patients. Participants in both groups continued to receive other therapies required to manage their condition. Participants, study staff, and investigators were masked to the study assignment. The primary outcome was the 7-point WHO ordinal scale for clinical status at day 29 after random assignment. The intention-to-treat population included all patients who were randomly assigned and met the enrolment criteria. The safety population included all participants who received at least one dose of study medication. This study was registered with the ISRCTN registry, ISRCTN30564012.FindingsBetween June 5, 2020, and Jan 25, 2021, 406 patients were randomly assigned to brensocatib or placebo; 192 (47·3%) to the brensocatib group and 214 (52·7%) to the placebo group. Two participants were excluded after being randomly assigned in the brensocatib group (214 patients included in the placebo group and 190 included in the brensocatib group in the intention-to-treat population). Primary outcome data was unavailable for six patients (three in the brensocatib group and three in the placebo group). Patients in the brensocatib group had worse clinical status at day 29 after being randomly assigned than those in the placebo group (adjusted odds ratio 0·72 [95% CI 0·57–0·92]). Prespecified subgroup analyses of the primary outcome supported the primary results. 185 participants reported at least one adverse event; 99 (46%) in the placebo group and 86 (45%) in the brensocatib group. The most common adverse events were gastrointestinal disorders and infections. One death in the placebo group was judged as possibly related to study drug.InterpretationBrensocatib treatment did not improve clinical status at day 29 in patients hospitalised with COVID-19.FundingSponsored by the University of Dundee and supported through an Investigator Initiated Research award from Insmed, Bridgewater, NJ; STOP-COVID19 trial.

Active antithrombin glycoforms are selectively physiosorbed on plasma extracellular vesicles

AbstractAntithrombin (AT) is a glycoprotein produced by the liver and a principal antagonist of active clotting proteases. A deficit in AT function leads to AT qualitative deficiency, challenging to diagnose. Here we report that active AT may travel physiosorbed on the surface of plasma extracellular vesicles (EVs), contributing to form the “EV‐protein corona.” The corona is enriched in specific AT glycoforms, thus suggesting glycosylation to play a key role in AT partitioning between EVs and plasma. Differences in AT glycoform composition of the corona of EVs separated from plasma of healthy and AT qualitative deficiency‐affected subjects were also noticed. This suggests deconstructing the plasma into its nanostructured components, as EVs, could suggest novel directions to unravel pathophysiological mechanisms.

A Phenotypic Screen Identifies Potent DPP9 Inhibitors Capable of Killing HIV-1 Infected Cells.

Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.

An optimized method of extracting and quantifying active Neutrophil serine proteases from human whole blood cells.

PurposeNeutrophil serine proteases (NSPs) are implicated in numerous inflammatory diseases. Thus, a robust methodology to monitor and quantify NSPs is important to study disease progression and evaluate the effect of pharmacological interventions. A comparison of the various methods used to extract NSPs from neutrophil granulocytes has not been published, providing the impetus to conduct this method optimization and comparison study.MethodsTwo NSP recovery methodologies were evaluated on samples from five human donors: zymosan stimulation and cell pellet extraction. For the zymosan stimulation method, 1 mL donor blood was added to zymosan and samples were incubated at 37°C for 30 min while shaking. Samples were then centrifuged, and the plasma was collected for quantitation of NSP activity. For the cell pellet extraction procedure, 2 mL whole blood samples were centrifuged into white blood cell pellets following red blood cell lysis. To each pellet, three sequential lysis steps were performed using either 0.05% Nonidet P-40 Substitute (NP40) or 0.02% Triton X-100 lysis buffers under agitation followed by centrifugation. NSP activities were quantified using an exogenous peptide substrate specific to each of the three NSPs being analyzed: neutrophil elastase, cathepsin G, and proteinase 3.Results and discussionThe zymosan stimulation method resulted in lower recovery of active NSPs and was unable to stimulate significant release of active cathepsin G. In contrast, the NP40 pellet extraction method showed consistent inter-donor NSP release with greater recoveries of active NSPs than the Triton method or the zymosan stimulation method. Overall, the pellet extraction procedure provided 13.3-fold greater recovery of active neutrophil elastase, 283-fold greater recovery of active cathepsin G, and 2.9-fold greater recovery of active proteinase 3 than the zymosan method.ConclusionThe NP40 cell pellet extraction method resulted in greater extraction of active NSPs compared to the other methods investigated here, which may allow for a more accurate and complete biomarker profile when evaluating human clinical samples.

Nontraditional translation is the key to UFMylation and beyond

The Ubiquitin-fold modifier 1 (Ufm1) is a ubiquitin-like protein that can also be conjugated to protein substrates and subsequently alter their fates. Both UFMylation and de-UFMylation are mediated by Ufm1-specific proteases (UFSPs). In humans, it is widely believed that UFSP2 is the only active Ufm1 protease involved in Ufm1 maturation and de-UFMylation, whereas UFSP1 is thought to be inactive. Here, Liang et al. provide strong evidence showing that human UFSP1 is also an active Ufm1 protease. These results solve an age-old mystery in the human Ufm1 conjugation system and could have a greater impact not only on Ufm1 biology but also on the translation of genes employing nontraditional start codons.