An inconvenient association between granzyme A and Nicotinamide Nucleotide Transhydrogenase

Granzyme A (GzmA) is a serine protease secreted by cytotoxic lymphocytes, with GzmA-/- mouse studies informing our understanding of GzmAs physiological function. We show herein that GzmA-/- mice have a mixed C57BL/6J and C57BL/6N background and retain the full length Nicotinamide Nucleotide Transhydrogenase (Nnt) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in GzmA-/- mice; however, the presence of Nnt, rather than loss of GzmA, was responsible for this phenotype by constraining lymphocyte infiltration. A new CRISPR active site mutant C57BL/6J GzmAS211A mouse provided the first insights into GzmAs bioactivity free of background issues, with circulating proteolytically active GzmA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ~27% of Run Accessions and ~38% of Bioprojects listing C57BL/6J as the mouse strain, had Nnt sequencing reads inconsistent with a C57BL/6J background. The Nnt issue has clearly complicated our understanding of GzmA and may similarly have influenced studies across a broad range of fields.

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An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648387 ◽

1992 ◽

Vol 67 (01) ◽

pp. 095-100 ◽

Cited By ~ 18

Author(s):

Paul J Declerck ◽

Leen Van Keer ◽

Maria Verstreken ◽

Désiré Collen

Keyword(s):

Serine Protease ◽

Plasminogen Activator ◽

Active Site ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Protease Domain ◽

Serine Protease Domain ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

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Kinetic analysis of the interaction of alkyl glycosides with two human β-glucosidases

Biochemical Journal ◽

10.1042/bj2620541 ◽

1989 ◽

Vol 262 (2) ◽

pp. 541-548 ◽

Cited By ~ 11

Author(s):

V Gopalan ◽

L B Daniels ◽

R H Glew ◽

M Claeyssens

Keyword(s):

Active Site ◽

Alkyl Chain ◽

Physiological Function ◽

Alkyl Chain Length ◽

Alkyl Glycosides ◽

Structural Nature ◽

Similarities And Differences ◽

Beta Glucosidase ◽

Free Energy Of Binding ◽

Physiological Substrates

This paper addresses the similarities and differences in the topology of the catalytic centres of human liver cytosolic beta-glucosidase and placental lysosomal glucocerebrosidase, and utilizes well-documented reversible active-site-directed inhibitors. This comparative kinetic study was performed mainly to decipher the chemical and structural nature of the active site of the cytosolic beta-glucosidase, whose physiological function is unknown. Specifically, analysis of the effects of a family of alkyl beta-glucosides consistently displayed 100-250-fold lower inhibition constants with the cytosolic broad-specificity beta-glucosidase compared with the placental glucocerebrosidase; for example, with octyl beta-D-glucoside the Ki values were 10 microM and 1490 microM for the cytosolic and lysosomal beta-glucosidases respectively. Furthermore the higher affinity of the cytosolic beta-glucosidase than glucocerebrosidase for the amphipathic alkyl beta-D-glucosides was validated by the greater increase in the free energy of binding with increasing alkyl chain length [delta delta G0 (K,)/CH2: lysosomal enzyme, 2.01 kJ/mol (480 cal/mol); cytosolic enzyme, 3.05 kJ/mol (730 cal/mol)]. The implications of the presence of highly non-polar domains in the active site of the cytosolic beta-glucosidase are discussed with regard to its potential physiological substrates.

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Deficiency in T follicular regulatory cells promotes autoimmunity

Journal of Experimental Medicine ◽

10.1084/jem.20170901 ◽

2018 ◽

Vol 215 (3) ◽

pp. 815-825 ◽

Cited By ~ 74

Author(s):

Weiwei Fu ◽

Xindong Liu ◽

Xiang Lin ◽

Han Feng ◽

Lin Sun ◽

...

Keyword(s):

Influenza Virus ◽

Autoimmune Disease ◽

Autoimmune Diseases ◽

Germinal Center ◽

Physiological Function ◽

Late Onset ◽

Humoral Response ◽

Disease Development ◽

Lymphocyte Infiltration ◽

Regulatory Cells

T follicular regulatory (Tfr) cells are a new subset of regulatory T (T reg) cells localized in the germinal center to limit the humoral response. Until now, the physiological function of Tfr cells has been largely unknown. In this study, we developed a Bcl6fl/flFoxp3Cre mouse to analyze the function of Tfr cells in immune and autoimmune responses. These mice exhibited enhanced immunity to influenza virus; moreover, Bcl6fl/flFoxp3Cre/Cre mice developed late-onset spontaneous autoimmune diseases, affecting the salivary glands with lymphocyte infiltration and antibody deposition. In a mouse experimental Sjögren’s syndrome model, ablation of Bcl6 in T reg cells greatly enhanced disease development. Conversely, Bcl6fl/flCd4Cre mice were protected in the model. Thus, our study indicates that Tfr cells control autoimmune diseases and can be targeted in infectious and autoimmune disease.

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Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells

Science ◽

10.1126/science.aaz7548 ◽

2020 ◽

Vol 368 (6494) ◽

pp. eaaz7548 ◽

Cited By ~ 31

Author(s):

Zhiwei Zhou ◽

Huabin He ◽

Kun Wang ◽

Xuyan Shi ◽

Yupeng Wang ◽

...

Keyword(s):

Antitumor Immunity ◽

Interferon Γ ◽

Target Cells ◽

Cytotoxic Lymphocytes ◽

Cytotoxic Lymphocyte ◽

Granzyme A ◽

Tumor Clearance ◽

Ifn Γ ◽

Underlying Mechanisms ◽

Pore Forming Proteins

Cytotoxic lymphocyte–mediated immunity relies on granzymes. Granzymes are thought to kill target cells by inducing apoptosis, although the underlying mechanisms are not fully understood. Here, we report that natural killer cells and cytotoxic T lymphocytes kill gasdermin B (GSDMB)–positive cells through pyroptosis, a form of proinflammatory cell death executed by the gasdermin family of pore-forming proteins. Killing results from the cleavage of GSDMB by lymphocyte-derived granzyme A (GZMA), which unleashes its pore-forming activity. Interferon-γ (IFN-γ) up-regulates GSDMB expression and promotes pyroptosis. GSDMB is highly expressed in certain tissues, particularly digestive tract epithelia, including derived tumors. Introducing GZMA-cleavable GSDMB into mouse cancer cells promotes tumor clearance in mice. This study establishes gasdermin-mediated pyroptosis as a cytotoxic lymphocyte–killing mechanism, which may enhance antitumor immunity.

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Influenza virus RNA polymerase PA subunit is a novel serine protease with Ser624 at the active site

Genes to Cells ◽

10.1046/j.1365-2443.2001.00399.x ◽

2001 ◽

Vol 6 (2) ◽

pp. 87-97 ◽

Cited By ~ 53

Author(s):

Koyu Hara ◽

Mayumi Shiota ◽

Hiroshi Kido ◽

Yasushi Ohtsu ◽

Takahito Kashiwagi ◽

...

Keyword(s):

Influenza Virus ◽

Rna Polymerase ◽

Serine Protease ◽

Active Site ◽

Virus Rna

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The Herpesvirus Proteases as Targets for Antiviral Chemotherapy

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020001100101 ◽

2000 ◽

Vol 11 (1) ◽

pp. 1-22 ◽

Cited By ~ 85

Author(s):

Lloyd Waxman ◽

Paul L Darke

Keyword(s):

Serine Protease ◽

Active Site ◽

Serine Proteases ◽

Catalytic Triad ◽

Active Site Residues ◽

Virus Family ◽

Single Domain Protein ◽

Inhibitor Discovery ◽

Antiviral Chemotherapy ◽

Simplex Virus

Viruses of the family Herpesviridae are responsible for a diverse set of human diseases. The available treatments are largely ineffective, with the exception of a few drugs for treatment of herpes simplex virus (HSV) infections. For several members of this DNA virus family, advances have been made recently in the biochemistry and structural biology of the essential viral protease, revealing common features that may be possible to exploit in the development of a new class of anti-herpesvirus agents. The herpesvirus proteases have been identified as belonging to a unique class of serine protease, with a Ser-His-His catalytic triad. A new, single domain protein fold has been determined by X-ray crystallography for the proteases of at least three different herpesviruses. Also unique for serine proteases, dimerization has been shown to be required for activity of the cytomegalovirus and HSV proteases. The dimerization requirement seriously impacts methods needed for productive, functional analysis and inhibitor discovery. The conserved functional and catalytic properties of the herpesvirus proteases lead to common considerations for this group of proteases in the early phases of inhibitor discovery. In general, classical serine protease inhibitors that react with active site residues do not readily inactivate the herpesvirus proteases. There has been progress however, with activated carbonyls that exploit the selective nucleophilicity of the active site serine. In addition, screening of chemical libraries has yielded novel structures as starting points for drug development. Recent crystal structures of the herpesvirus proteases now allow more direct interpretation of ligand structure—activity relationships. This review first describes basic functional aspects of herpesvirus protease biology and enzymology. Then we discuss inhibitors identified to date and the prospects for their future development.

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Characterization of the enzymatic activity of the serine protease domain of Factor VII activating protease (FSAP)

Scientific Reports ◽

10.1038/s41598-019-55531-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Nis V. Nielsen ◽

Elfie Roedel ◽

Dipankar Manna ◽

Michael Etscheid ◽

Jens Preben Morth ◽

...

Keyword(s):

Enzymatic Activity ◽

Serine Protease ◽

Active Site ◽

Factor Vii ◽

Protease Domain ◽

Functional Changes ◽

E Coli ◽

Catalytic Activation ◽

Serine Protease Domain ◽

Tissue Factor Pathway

AbstractFactor VII (FVII) activating protease (FSAP) is a circulating serine protease. Human genetic studies, based on the Marburg I (MI) (Gly221Glu, chymotrypsin numbering system) polymorphism, implicate FSAP in the pathogenesis of many diseases. Here, we describe the molecular and functional changes caused by the Gly221Glu substitution in the 220 loop using recombinant proteins expressed in E. coli. The serine protease domain (SPD) of wild type (WT) FSAP displayed auto-catalytic activation whereas the MI isoform displayed very low autocatalytic activation and low proteolytic activity against the chromogenic substrate S-2288, Factor VII, tissue factor pathway inhibitor as well as pro-urokinase. Introduction of a thermolysin cleavage site in the activation position (Arg15Gln) led to cleavage of both WT- and MI-SPD and the resulting WT-SPD, but not the MI-SPD, was active. Mutating the Gly221 position to Asp, Gln and Leu led to a loss of activity whereas the Ala substitution was partially active. These results suggest a disturbance of the active site, or non-accessibility of the substrate to the active site in MI-SPD. With respect to regulation with metal ions, calcium, more than sodium, increased the enzymatic activity of WT-SPD. Thus, we describe a novel method for the production of recombinant FSAP-SPD to understand the role of the MI-single nucleotide polymorphism (SNP) in the regulation of its activity.

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