4 results on '"Christopher J. Farady"'
Search Results
2. Evaluation of protein kinase D auto-phosphorylation as biomarker for NLRP3 inflammasome activation
- Author
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Diane Heiser, Joëlle Rubert, Adeline Unterreiner, Claudine Maurer, Marion Kamke, Ursula Bodendorf, Christopher J. Farady, Ben Roediger, and Frédéric Bornancin
- Subjects
Medicine ,Science - Abstract
Background The NLRP3 inflammasome is a critical component of sterile inflammation, which is involved in many diseases. However, there is currently no known proximal biomarker for measuring NLRP3 activation in pathological conditions. Protein kinase D (PKD) has emerged as an important NLRP3 kinase that catalyzes the release of a phosphorylated NLRP3 species that is competent for inflammasome complex assembly. Methods To explore the potential for PKD activation to serve as a selective biomarker of the NLRP3 pathway, we tested various stimulatory conditions in THP-1 and U937 cell lines, probing the inflammasome space beyond NLRP3. We analyzed the correlation between PKD activation (monitored by its auto-phosphorylation) and functional inflammasome readouts. Results PKD activation/auto-phosphorylation always preceded cleavage of caspase-1 and gasdermin D, and treatment with the PKD inhibitor CRT0066101 could block NLRP3 inflammasome assembly and interleukin-1β production. Conversely, blocking NLRP3 either genetically or using the MCC950 inhibitor prevented PKD auto-phosphorylation, indicating a bidirectional functional crosstalk between NLRP3 and PKD. Further assessments of the pyrin and NLRC4 pathways, however, revealed that PKD auto-phosphorylation can be triggered by a broad range of stimuli unrelated to NLRP3 inflammasome assembly. Conclusion Although PKD and NLRP3 become functionally interconnected during NLRP3 activation, the promiscuous reactivity of PKD challenges its potential use for tracing the NLRP3 inflammasome pathway.
- Published
- 2021
3. Danger-associated extracellular ATP counters MDSC therapeutic efficacy in acute GVHD
- Author
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Jonathan S. Serody, Jenny P.-Y. Ting, Christopher J. Farady, Vincenzo Bronte, Michael Loschi, Brent H. Koehn, Takao Iwawaki, Asim Saha, Govindarajan Thangavelu, Mark E. Cooper, Cameron McDonald-Hyman, Jamie Panthera, Lie Ma, Keli L. Hippen, Walker Krepps, Bruce R. Blazar, Josh Dysthe, Jeffrey S. Miller, Stephen C. Jameson, Robert Zeiser, Peter J. Murray, William J. Murphy, David H. Munn, Michael Zaiken, and Geoffrey R. Hill
- Subjects
Inflammasomes ,Receptor expression ,Immunology ,Graft vs Host Disease ,Inflammation ,Biochemistry ,Mice ,Adenosine Triphosphate ,In vivo ,medicine ,Extracellular ,Animals ,Receptor ,Mice, Knockout ,Apyrase ,Chemistry ,Myeloid-Derived Suppressor Cells ,Inflammasome ,Cell Biology ,Hematology ,medicine.disease ,Graft-versus-host disease ,Cancer research ,Female ,medicine.symptom ,medicine.drug - Abstract
Myeloid-derived suppressor cells (MDSCs) can subdue inflammation. In mice with acute graft-versus-host disease (GVHD), donor MDSC infusion enhances survival that is only partial and transient because of MDSC inflammasome activation early posttransfer, resulting in differentiation and loss of suppressor function. Here we demonstrate that conditioning regimen-induced adenosine triphosphate (ATP) release is a primary driver of MDSC dysfunction through ATP receptor (P2x7R) engagement and NLR pyrin family domain 3 (NLRP3) inflammasome activation. P2x7R or NLRP3 knockout (KO) donor MDSCs provided significantly higher survival than wild-type (WT) MDSCs. Although in vivo pharmacologic targeting of NLRP3 or P2x7R promoted recipient survival, indicating in vivo biologic effects, no synergistic survival advantage was seen when combined with MDSCs. Because activated inflammasomes release mature interleukin-1�� (IL-1��), we expected that IL-1�� KO donor MDSCs would be superior in subverting GVHD, but such MDSCs proved inferior relative to WT. IL-1�� release and IL-1 receptor expression was required for optimal MDSC function, and exogenous IL-1�� added to suppression assays that included MDSCs increased suppressor potency. These data indicate that prolonged systemic NLRP3 inflammasome inhibition and decreased IL-1�� could diminish survival in GVHD. However, loss of inflammasome activation and IL-1�� release restricted to MDSCs rather than systemic inhibition allowed non-MDSC IL-1�� signaling, improving survival. Extracellular ATP catalysis with peritransplant apyrase administered into the peritoneum, the ATP release site, synergized with WT MDSCs, as did regulatory T-cell infusion, which we showed reduced but did not eliminate MDSC inflammasome activation, as assessed with a novel inflammasome reporter strain. These findings will inform future clinical using MDSCs to decrease alloresponses in inflammatory environments.
- Published
- 2019
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4. The Mechanism of Inhibition of Antibody-Based Inhibitors of Membrane-Type Serine Protease 1 (MT-SP1)
- Author
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Christopher J. Farady, Susan M. Miller, Molly R. Darragh, Jeonghoon Sun, and Charles S. Craik
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Models, Molecular ,Serine Proteinase Inhibitors ,medicine.medical_treatment ,Immunoglobulin Variable Region ,Epitope ,Article ,Epitopes ,Structural Biology ,medicine ,Animals ,Humans ,Point Mutation ,Matriptase ,Molecular Biology ,chemistry.chemical_classification ,Serine protease ,Protease ,biology ,Serine Endopeptidases ,Active site ,Alanine scanning ,Protein Structure, Tertiary ,Enzyme ,chemistry ,Biochemistry ,biology.protein - Abstract
The mechanisms of inhibition of two novel scFv antibody inhibitors of the serine protease MT-SP1/matriptase reveal the basis of their potency and specificity. Kinetic experiments characterize the inhibitors as extremely potent inhibitors with K(I) values in the low picomolar range that compete with substrate binding in the S1 site. Alanine scanning of the loops surrounding the protease active site provides a rationale for inhibitor specificity. Each antibody binds to a number of residues flanking the active site, forming a unique three-dimensional binding epitope. Interestingly, one inhibitor binds in the active site cleft in a substrate-like manner, can be processed by MT-SP1 at low pH, and is a standard mechanism inhibitor of the protease. The mechanisms of inhibition provide a rationale for the effectiveness of these inhibitors, and suggest that the development of specific antibody-based inhibitors against individual members of closely related enzyme families is feasible, and an effective way to develop tools to tease apart complex biological processes.
- Published
- 2007
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