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ACS Chemical Biology Sep 2023Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and...
Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, , comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. shows outstanding target engagement and proteome-wide selectivity in living cells. Importantly, as well as other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 on lysine 31. We present a route to future OTUB2-related therapeutics and have shown that the OTUB2 inhibitor developed in this study can help to uncover new aspects of the related biology and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.
Topics: Ubiquitination; Protein Processing, Post-Translational; Ubiquitin; Cysteine; Cysteine Proteases
PubMed: 37642399
DOI: 10.1021/acschembio.3c00227 -
Frontiers in Cellular and Infection... 2018The intestinal protozoan parasite () causes amebiasis associated with severe diarrhea and/or liver abscess. pathogenesis is multifactorial requiring both parasite...
The intestinal protozoan parasite () causes amebiasis associated with severe diarrhea and/or liver abscess. pathogenesis is multifactorial requiring both parasite virulent molecules and host-induced innate immune responses. -induced host pro-inflammatory responses plays a critical role in disease pathogenesis by causing damage to tissues allowing parasites access to systemic sites. cyclooxygenase (Cox) derived prostaglandin E stimulates the chemokine IL-8 from mucosal epithelial cells that recruits neutrophils to the site of infection to exacerbate disease. At present, it is not known how Cox is regulated or whether it affects the expression of other proteins in . In this study, we found that gene silencing of Cox () markedly increased endogenous cysteine protease (CP) protein expression and virulence without altering CP gene transcripts. Live virulent pretreated with arachidonic acid substrate to enhance PGE production or aspirin to inhibit Cox enzyme activity or addition of exogenous PGE to had no effect on CP activity. Increased CP enzyme activity in was stable and significantly enhanced erythrophagocytosis, cytopathic effects on colonic epithelial cells and elicited pro-inflammatory cytokines in mice colonic loops. Acute infection with in colonic loops increased inflammation associated with high levels of myeloperoxidase activity. This study has identified Cox protein as one of the important endogenous regulators of cysteine protease activity. Alterations of CP activity in response to Cox gene silencing may be a negative feedback mechanism in to limit proteolytic activity during colonization that can inadvertently trigger inflammation in the gut.
Topics: Animals; Cysteine Proteases; Entamoeba histolytica; Epithelial Cells; Gene Expression Regulation; Gene Knockdown Techniques; Mice; Prostaglandin-Endoperoxide Synthases; Virulence; Virulence Factors
PubMed: 30687644
DOI: 10.3389/fcimb.2018.00447 -
Viruses Sep 2022Enteroviruses (EVs) are medically important RNA viruses that cause a broad spectrum of human illnesses for which limited therapy exists. Although EVs have been shown to...
Enteroviruses (EVs) are medically important RNA viruses that cause a broad spectrum of human illnesses for which limited therapy exists. Although EVs have been shown to usurp the cellular recycling process of autophagy for pro-viral functions, the precise manner by which this is accomplished remains to be elucidated. In the current manuscript, we sought to address the mechanism by which EVs subvert the autophagy pathway using Coxsackievirus B3 (CVB3) as a model. We showed that CVB3 infection selectively degrades the autophagy cysteine protease ATG4A but not other isoforms. Exogenous expression of an N-terminally Flag-labeled ATG4A demonstrated the emergence of a 43-kDa cleavage fragment following CVB3 infection. Furthermore, bioinformatics analysis coupled with site-directed mutagenesis and in vitro cleavage assays revealed that CVB3 protease 2A cleaves ATG4A before glycine 374. Using a combination of genetic silencing and overexpression studies, we demonstrated a novel pro-viral function for the autophagy protease ATG4A. Additionally, cleavage of ATG4A was associated with a loss of autophagy function of the truncated cleavage fragment. Collectively, our study identified ATG4A as a novel substrate of CVB3 protease, leading to disrupted host cellular function and sheds further light on viral mechanisms of autophagy dysregulation.
Topics: Autophagy; Autophagy-Related Proteins; Coxsackievirus Infections; Cysteine Endopeptidases; Cysteine Proteases; Endopeptidases; Enterovirus B, Human; Enterovirus Infections; Glycine; HeLa Cells; Humans; Peptide Hydrolases
PubMed: 36146840
DOI: 10.3390/v14092026 -
Veterinary Research Aug 2021The aim of this study was to investigate the biological properties of a novel gut-specific cysteine protease in Trichinella spiralis (TsGSCP) and its role in larval...
The aim of this study was to investigate the biological properties of a novel gut-specific cysteine protease in Trichinella spiralis (TsGSCP) and its role in larval intrusion, development and fecundity. TsGSCP has a functional C1 peptidase domain; C1 peptidase belongs to cathepsin B family. The TsGSCP gene cloned and expressed in Escherichia coli BL21 showed intensive immunogenicity. qPCR and Western blotting revealed that TsGSCP mRNA and protein were expressed at various T. spiralis stages, but their expression levels in intestinal infectious larvae (IIL) were clearly higher than those in muscle larvae (ML), adult worms (AWs) and new-born larvae (NBL). Indirect immunofluorescence (IIF) analysis showed that TsGSCP was primarily located at the outer cuticle and the intrauterine embryos of this parasite. rTsGSCP showed the ability to specifically bind with IECs, and the binding site is within the IEC cytoplasm. rTsGSCP accelerated larval intrusion into host intestinal epithelial cells (IECs), whereas anti-rTsGSCP antibodies suppressed larval intrusion; the acceleration and suppression was induced by rTsGSCP and anti-rTsGSCP antibodies, respectively, in a dose-dependent manner. When ML were transfected with TsGSCP-specific dsRNA, TsGSCP expression and enzymatic activity were reduced by 46.82 and 37.39%, respectively, and the capacity of the larvae to intrude into IECs was also obviously impeded. Intestinal AW burden and adult female length and fecundity were significantly decreased in the group of mice infected with dsRNA-transfected ML compared to the control dsRNA and PBS groups. The results showed that TsGSCP plays a principal role in gut intrusion, worm development and fecundity in the T. spiralis lifecycle and might be a candidate target for vaccine development against Trichinella intrusion and infection.
Topics: Amino Acid Sequence; Animals; Cysteine Proteases; Female; Fertility; Helminth Proteins; Larva; Mice; Phylogeny; Sequence Alignment; Trichinella spiralis; Trichinellosis
PubMed: 34446106
DOI: 10.1186/s13567-021-00983-1 -
The European Respiratory Journal Apr 2022Biomarkers of disease progression and treatment response are urgently needed for patients with lymphangioleiomyomatosis (LAM). Activity-based nanosensors, an emerging...
BACKGROUND
Biomarkers of disease progression and treatment response are urgently needed for patients with lymphangioleiomyomatosis (LAM). Activity-based nanosensors, an emerging biosensor class, detect dysregulated proteases and release a reporter to provide a urinary readout of disease. Because proteases are dysregulated in LAM and may directly contribute to lung function decline, activity-based nanosensors may enable quantitative, real-time monitoring of LAM progression and treatment response. We aimed to assess the diagnostic utility of activity-based nanosensors in a pre-clinical model of pulmonary LAM.
METHODS
-null cells were injected intravenously into female nude mice to establish a mouse model of pulmonary LAM. A library of 14 activity-based nanosensors, designed to detect proteases across multiple catalytic classes, was administered into the lungs of LAM mice and healthy controls, urine was collected, and mass spectrometry was performed to measure nanosensor cleavage products. Mice were then treated with rapamycin and monitored with activity-based nanosensors. Machine learning was performed to distinguish diseased from healthy and treated from untreated mice.
RESULTS
Multiple activity-based nanosensors (PP03 (cleaved by metallo, aspartic and cysteine proteases), p<0.0001; PP10 (cleaved by serine, aspartic and cysteine proteases), p=0.017)) were differentially cleaved in diseased and healthy lungs, enabling strong classification with a machine learning model (area under the curve (AUC) 0.95 from healthy). Within 2 days after rapamycin initiation, we observed normalisation of PP03 and PP10 cleavage, and machine learning enabled accurate classification of treatment response (AUC 0.94 from untreated).
CONCLUSIONS
Activity-based nanosensors enable noninvasive, real-time monitoring of disease burden and treatment response in a pre-clinical model of LAM.
Topics: Animals; Cysteine Proteases; Female; Humans; Lymphangioleiomyomatosis; Mice; Mice, Nude; Peptide Hydrolases; Sirolimus
PubMed: 34561286
DOI: 10.1183/13993003.00664-2021 -
Nature Communications Aug 2023Proteolytic activation of cytokines regulates immunity in diverse organisms. In animals, cysteine-dependent aspartate-specific proteases (caspases) play central roles in...
Proteolytic activation of cytokines regulates immunity in diverse organisms. In animals, cysteine-dependent aspartate-specific proteases (caspases) play central roles in cytokine maturation. Although the proteolytic production of peptide cytokines is also essential for plant immunity, evidence for cysteine-dependent aspartate-specific proteases in regulating plant immunity is still limited. In this study, we found that the C-terminal proteolytic processing of a caspase-like substrate motif "CNYD" within Pathogenesis-related protein 1 (PR1) generates an immunomodulatory cytokine (CAPE9) in Arabidopsis. Salicylic acid enhances CNYD-targeted protease activity and the proteolytic release of CAPE9 from PR1 in Arabidopsis. This process involves a protease exhibiting caspase-like enzyme activity, identified as Xylem cysteine peptidase 1 (XCP1). XCP1 exhibits a calcium-modulated pH-activity profile and a comparable activity to human caspases. XCP1 is required to induce systemic immunity triggered by pathogen-associated molecular patterns. This work reveals XCP1 as a key protease for plant immunity, which produces the cytokine CAPE9 from the canonical salicylic acid signaling marker PR1 to activate systemic immunity.
Topics: Animals; Humans; Arabidopsis; Arabidopsis Proteins; Aspartic Acid; Caspases; Cysteine; Cysteine Proteases; Peptide Hydrolases; Plant Immunity; Salicylic Acid; Xylem
PubMed: 37542077
DOI: 10.1038/s41467-023-40406-7 -
Nutrients Aug 2022Although fibrinolytic enzymes and thrombolytic agents help in cardiovascular disease treatment, those currently available have several side effects. This warrants the...
Exploring the Fibrin(ogen)olytic, Anticoagulant, and Antithrombotic Activities of Natural Cysteine Protease (Ficin) with the κ-Carrageenan-Induced Rat Tail Thrombosis Model.
Although fibrinolytic enzymes and thrombolytic agents help in cardiovascular disease treatment, those currently available have several side effects. This warrants the search for safer alternatives. Several natural cysteine protease preparations are used in traditional medicine to improve platelet aggregation and thrombosis-related diseases. Hence, this study aimed to investigate the effect of ficin, a natural cysteine protease, on fibrin(ogen) and blood coagulation. The optimal pH (pH 7) and temperature (37 °C) for proteolytic activity were determined using the azocasein method. Fibrinogen action and fibrinolytic activity were measured both electrophoretically and by the fibrin plate assay. The effect of ficin on blood coagulation was studied by conventional coagulation tests: prothrombin time (PT), activated partial thromboplastin time (aPTT), blood clot lysis assay, and the κ-carrageenan thrombosis model. The Aα, Bβ, and γ bands of fibrinogen are readily cleaved by ficin, and we also observed a significant increase in PT and aPTT. Further, the mean length of the infarcted regions in the tails of Sprague-Dawley rats was shorter in rats administered 10 U/mL of ficin than in control rats. These findings suggest that natural cysteine protease, ficin contains novel fibrin and fibrinogenolytic enzymes and can be used for preventing and/or treating thrombosis-associated cardiovascular disorders.
Topics: Animals; Anticoagulants; Carrageenan; Cysteine Proteases; Estrone; Fibrin; Fibrinogen; Fibrinolytic Agents; Ficain; Rats; Rats, Sprague-Dawley; Thrombosis
PubMed: 36079810
DOI: 10.3390/nu14173552 -
Virulence Dec 2023Extracellular vesicles (EVs) can be released from gram-positive bacteria and would participate in the delivery of bacterial toxins. (group A , GAS) is one of the most...
Extracellular vesicles (EVs) can be released from gram-positive bacteria and would participate in the delivery of bacterial toxins. (group A , GAS) is one of the most common pathogens of monomicrobial necrotizing fasciitis. Spontaneous inactivating mutation in the CovR/CovS two-component regulatory system is related to the increase of EVs production via an unknown mechanism. This study aimed to investigate whether the CovR/CovS-regulated RopB, the transcriptional regulator of GAS exoproteins, would participate in regulating EVs production. Results showed that the size, morphology, and number of EVs released from the wild-type strain and the mutant were similar, suggesting RopB is not involved in controlling EVs production. Nonetheless, RopB-regulated SpeB protease degrades streptolysin O and bacterial proteins in EVs. Although SpeB has crucial roles in modulating protein composition in EVs, the SpeB-positive EVs failed to trigger HaCaT keratinocytes pyroptosis, suggesting that EVs did not deliver SpeB into keratinocytes or the amount of SpeB in EVs was not sufficient to trigger cell pyroptosis. Finally, we identified that EV-associated enolase was resistant to SpeB degradation, and therefore could be utilized as the internal control protein for verifying SLO degradation. This study revealed that RopB would participate in modulating protein composition in EVs via SpeB-dependent protein degradation and suggested that enolase is a potential internal marker for studying GAS EVs.
Topics: Streptococcus pyogenes; Cysteine Proteases; Bacterial Proteins; Extracellular Vesicles; Phosphopyruvate Hydratase
PubMed: 37621107
DOI: 10.1080/21505594.2023.2249784 -
Chemistry (Weinheim An Der Bergstrasse,... Nov 2022Rhodesain is the major cysteine protease of the protozoan parasite Trypanosoma brucei and a therapeutic target for sleeping sickness, a fatal neglected tropical disease....
Rhodesain is the major cysteine protease of the protozoan parasite Trypanosoma brucei and a therapeutic target for sleeping sickness, a fatal neglected tropical disease. We designed, synthesized and characterized a bimodal activity-based probe that binds to and inactivates rhodesain. This probe exhibited an irreversible mode of action and extraordinary potency for the target protease with a k /K value of 37,000 M s . Two reporter tags, a fluorescent coumarin moiety and a biotin affinity label, were incorporated into the probe and enabled highly sensitive detection of rhodesain in a complex proteome by in-gel fluorescence and on-blot chemiluminescence. Furthermore, the probe was employed for microseparation and quantification of rhodesain and for inhibitor screening using a competition assay. The developed bimodal rhodesain probe represents a new proteomic tool for studying Trypanosoma pathobiochemistry and antitrypanosomal drug discovery.
Topics: Biotin; Cysteine Proteases; Fluorescence; Proteomics; Trypanosoma brucei brucei; Trypanosoma; Structure-Activity Relationship
PubMed: 35852812
DOI: 10.1002/chem.202201636 -
International Journal of Molecular... Nov 2022Cathepsin K (CatK) is a part of the family of cysteine proteases involved in many important processes, including the degradation activity of collagen 1 and elastin in... (Review)
Review
Cathepsin K (CatK) is a part of the family of cysteine proteases involved in many important processes, including the degradation activity of collagen 1 and elastin in bone resorption. Changes in levels of CatK are associated with various pathological conditions, primarily related to bone and cartilage degradation, such as pycnodysostosis (associated with CatK deficiency), osteoporosis, and osteoarthritis (associated with CatK overexpression). Recently, the increased secretion of CatK is being highly correlated to vascular inflammation, hypersensitivity pneumonitis, Wegener granulomatosis, berylliosis, tuberculosis, as well as with tumor progression. Due to the wide spectrum of diseases in which CatK is involved, the design and validation of active site-specific inhibitors has been a subject of keen interest in pharmaceutical companies in recent decades. In this review, we summarized the molecular background of CatK and its involvement in various diseases, as well as its clinical significance for diagnosis and therapy.
Topics: Cathepsin K; Collagen Type I; Bone and Bones; Cysteine Proteases
PubMed: 36430239
DOI: 10.3390/ijms232213762