Publications by authors named "Aaron Gupta"

4 Publications

  • Page 1 of 1

Discovery of a caspase cleavage motif antibody reveals insights into noncanonical inflammasome function.

Proc Natl Acad Sci U S A 2021 Mar;118(12)

Department of Antibody Engineering, Genentech, Inc., South San Francisco, CA 94080;

Inflammasomes sense a number of pathogen and host damage signals to initiate a signaling cascade that triggers inflammatory cell death, termed pyroptosis. The inflammatory caspases (1/4/5/11) are the key effectors of this process through cleavage and activation of the pore-forming protein gasdermin D. Caspase-1 also activates proinflammatory interleukins, IL-1β and IL-18, via proteolysis. However, compared to the well-studied apoptotic caspases, the identity of substrates and therefore biological functions of the inflammatory caspases remain limited. Here, we construct, validate, and apply an antibody toolset for direct detection of neo-C termini generated by inflammatory caspase proteolysis. By combining rabbit immune phage display with a set of degenerate and defined target peptides, we discovered two monoclonal antibodies that bind peptides with a similar degenerate recognition motif as the inflammatory caspases without recognizing the canonical apoptotic caspase recognition motif. Crystal structure analyses revealed the molecular basis of this strong yet paradoxical degenerate mode of peptide recognition. One antibody selectively immunoprecipitated cleaved forms of known and unknown inflammatory caspase substrates, allowing the identification of over 300 putative substrates of the caspase-4 noncanonical inflammasome, including caspase-7. This dataset will provide a path toward developing blood-based biomarkers of inflammasome activation. Overall, our study establishes tools to discover and detect inflammatory caspase substrates and functions, provides a workflow for designing antibody reagents to study cell signaling, and extends the growing evidence of biological cross talk between the apoptotic and inflammatory caspases.
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http://dx.doi.org/10.1073/pnas.2018024118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000503PMC
March 2021

The role of IgG Fc receptors in antibody-dependent enhancement.

Nat Rev Immunol 2020 10 11;20(10):633-643. Epub 2020 Aug 11.

Laboratory of Molecular Genetics and Immunology, The Rockefeller University, New York, NY, USA.

Antibody-dependent enhancement (ADE) is a mechanism by which the pathogenesis of certain viral infections is enhanced in the presence of sub-neutralizing or cross-reactive non-neutralizing antiviral antibodies. In vitro modelling of ADE has attributed enhanced pathogenesis to Fcγ receptor (FcγR)-mediated viral entry, rather than canonical viral receptor-mediated entry. However, the putative FcγR-dependent mechanisms of ADE overlap with the role of these receptors in mediating antiviral protection in various viral infections, necessitating a detailed understanding of how this diverse family of receptors functions in protection and pathogenesis. Here, we discuss the diversity of immune responses mediated upon FcγR engagement and review the available experimental evidence supporting the role of FcγRs in antiviral protection and pathogenesis through ADE. We explore FcγR engagement in the context of a range of different viral infections, including dengue virus and SARS-CoV, and consider ADE in the context of the ongoing SARS-CoV-2 pandemic.
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http://dx.doi.org/10.1038/s41577-020-00410-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418887PMC
October 2020

Caspase-11 auto-proteolysis is crucial for noncanonical inflammasome activation.

J Exp Med 2018 09 22;215(9):2279-2288. Epub 2018 Aug 22.

Department of Physiological Chemistry, Genentech Inc., South San Francisco, CA

Intracellular LPS sensing by caspase-4/5/11 triggers proteolytic activation of pore-forming gasdermin D (GSDMD), leading to pyroptotic cell death in Gram-negative bacteria-infected cells. Involvement of caspase-4/5/11 and GSDMD in inflammatory responses, such as lethal sepsis, makes them highly desirable drug targets. Using knock-in (KI) mouse strains, we herein provide genetic evidence to show that caspase-11 auto-cleavage at the inter-subunit linker is essential for optimal catalytic activity and subsequent proteolytic cleavage of GSDMD. Macrophages from caspase-11-processing dead KI mice ( ) exhibit defective caspase-11 auto-processing and phenocopy and caspase-11 enzymatically dead KI ( ) macrophages in attenuating responses to cytoplasmic LPS or Gram-negative bacteria infection. KI macrophages also fail to cleave GSDMD and are hypo-responsive to inflammasome stimuli, confirming that the GSDMD Asp residue is a nonredundant and indispensable site for proteolytic activation of GSDMD. Our data highlight the role of caspase-11 self-cleavage as a critical regulatory step for GSDMD processing and response against Gram-negative bacteria.
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http://dx.doi.org/10.1084/jem.20180589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122968PMC
September 2018

GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes.

Proc Natl Acad Sci U S A 2016 07 23;113(28):7858-63. Epub 2016 Jun 23.

Department of Early Discovery Biochemistry, Genentech, Inc., South San Francisco, CA 94080;

Gasdermin-D (GsdmD) is a critical mediator of innate immune defense because its cleavage by the inflammatory caspases 1, 4, 5, and 11 yields an N-terminal p30 fragment that induces pyroptosis, a death program important for the elimination of intracellular bacteria. Precisely how GsdmD p30 triggers pyroptosis has not been established. Here we show that human GsdmD p30 forms functional pores within membranes. When liberated from the corresponding C-terminal GsdmD p20 fragment in the presence of liposomes, GsdmD p30 localized to the lipid bilayer, whereas p20 remained in the aqueous environment. Within liposomes, p30 existed as higher-order oligomers and formed ring-like structures that were visualized by negative stain electron microscopy. These structures appeared within minutes of GsdmD cleavage and released Ca(2+) from preloaded liposomes. Consistent with GsdmD p30 favoring association with membranes, p30 was only detected in the membrane-containing fraction of immortalized macrophages after caspase-11 activation by lipopolysaccharide. We found that the mouse I105N/human I104N mutation, which has been shown to prevent macrophage pyroptosis, attenuated both cell killing by p30 in a 293T transient overexpression system and membrane permeabilization in vitro, suggesting that the mutants are actually hypomorphs, but must be above certain concentration to exhibit activity. Collectively, our data suggest that GsdmD p30 kills cells by forming pores that compromise the integrity of the cell membrane.
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http://dx.doi.org/10.1073/pnas.1607769113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4948338PMC
July 2016