Inflammasomes and Gasdermin D Signaling Pathways

Inflammasomes are multimeric protein complexes that comprise a sensor (e.g. NLRP3), an adaptor (ASC/Pycard) and a protease (pro-caspase-1) (1). An inflammasome assembles in response to a diverse range of pathogen-associated or danger-associated molecular patterns (PAMPs or DAMPs), or perturbations in cytoplasmic homeostasis (term ‘homeostasis-altering molecular processes’ (HAMPs)) (2). The inflammasome platform leads to activation of caspase-1, which further induces maturation of interleukin-1β and -18 (IL-1β and IL-18) through proteolytic cleavage of pro-IL-1β and pro-IL-18. Activated caspase-1, and also the recently characterized caspase-11 non-canonical inflammasome pathway, cleave the newly discovered intracellular protein Gasdermin D (3, 4). The Gasdermin family members contain N-terminal domains that are capable of forming membrane pores, whereas the C-terminal domains of Gasdermins function as inhibitors of such cytolysis through intramolecular domain association. Caspase-1 or -11 cleavage of Gasdermin D is required for regulation of Pyroptosis: upon caspase-1/11 cleavage of the Gasdermin N- and C-domain linker, the cleaved N-terminal fragment of Gasdermin D oligomerizes and forms pores on the host cell membrane (5), leading to a cell death called pyroptosis and further activation of inflammasomes by triggering K+ efflux (6). Gasdermin D forming pores regulate the non-conventional secretion of cytokines such as IL-1β, in response to cytosolic LPS and other activators of the inflammasome (7). Neutrophil extrusion of neutrophil extracellular traps (NETs) and concomitant cell death (NETosis), a particular neutrophil defense against pathogens, are dependent on Gasdermin D cleavage by caspase-11 (8). Gasdermin D-mediated pyroptosis is regulated at the level of lipid peroxidation (9) and seems to be a key effector in the LPS-induced lethal sepsis (10).

Caspase-8, an upstream activator of caspase-3, controls apoptotic cell death and prevents RIPK3–MLKL-dependent necroptosis. Caspase-8, activated by the Yersinia effector protein YopJ, also triggers Gasdermin D processing and cell death with different Yersinia species (11).

After formation of the pore at the cellular membrane by Gasdermin D N-terminal fragment, the role and fate of the C-terminus fragment of Gasdermin D is still unclear. Using the Gasdermin D (mouse) ELISA Kit (Prod. No. AG-45B-0011), that detects the C-terminal part of Gasdermin D (as well as the full-length protein), a signal is detected in the supernatant of cells dying by pyroptosis, suggesting that the C-terminal fragment is released from cells, either by chance due to the presence of a pore or for a specific task not yet clear.

Literature References:

  1. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta: F. Martinon, et al.; Mol. Cell 10, 417 (2002)
  2. Homeostasis-altering molecular processes as mechanisms of inflammasome activation: A. Liston & S.L. Masters; Nat. Rev. Immunol. 17, 208 (2017)
  3. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling: N. Kayagaki, et al.; Nature 526, 666 (2015)
  4. Mechanisms of Gasdermin Family Members in Inflammasome Signaling and Cell Death: S. Feng, et al.; J. Mol. Biol. 430, 3068 (2018)
  5. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores: X. Liu, et al.; Nature 535, 153 (2016)
  6. Gasdermin D Restrains Type I Interferon Response to Cytosolic DNA by Disrupting Ionic Homeostasis: I. Banerjee, et al.; Immunity 49, 413 (2018)
  7. The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages: C.L. Evavold, et al.; Immunity 48, 35 (2018)
  8. Noncanonical inflammasome signaling elicits gasdermin D-dependent neutrophil extracellular traps: K.W. Chen, et al.; Sci. Immunol. 3, 26 (2018)
  9. Lipid Peroxidation Drives Gasdermin D-mediated Pyroptosis in Lethal Polymicrobial Sepsis: R. Kang, et al.; Cell Host Microbe 24, 97 (2018)
  10. Chemical disruption of the pyroptotic pore-forming protein gasdermin D inhibits inflammatory cell death and sepsis: J.K. Rathkey, et al.; Sci. Immunol. 3, 26 (2018)
  11. Pathogen blockade of TAK1 triggers caspase-8-dependent cleavage of gasdermin D and cell death: P. Orning, et al.; Science (Epub ahead of print) 26 (2018)

 

New Reagents for Gasdermin Research

NEW Gasdermin D (mouse) ELISA Kit

The Gasdermin D (mouse) ELISA Kit (Prod. No. AG-45B-0011) is a sandwich ELISA for quantitative determination of mouse Gasdermin D in cell culture supernatants and in cell extracts. This ELISA is specific for the measurement of natural and recombinant mouse Gasdermin D (full-length and C-terminus cleaved fragment). It does not detect human Gasdermin D.

Specificity:

Gasdermin D is tested from supernatants of Bone Marrow-Derived Macrophages cells (BMDMs) transfected with LPS from different knockout mice strains (see Figure 1). Only the supernatants from WT and NLRP3-/- strains contain the protein Gasdermin D. Gasdermin D is also tested from cell extracts (lysed with a Triton X-100 buffer) of Bone Marrow-Derived Macrophages cells from WT and Gasdermin D knockout mice strains (see Figure 2).

 

NEW anti-Gasdermin D (mouse), pAb (IN110)

AdipoGen Life Sciences’ anti-Gasdermin D (mouse), pAb (IN110) (Prod. No. AG-25B-0036) is a polyclonal antibody immunised with the recombinant C-terminus domain of mouse Gasdermin D. The antibody recognizes full-length and the cleaved C-terminus of mouse Gasdermin D, does not cross-react with human Gasdermin D and works specifically in Western Blot application to detect the cleaved C-terminal Gasdermin D.

 

Specificity:

Figure: Mouse Gasdermin D (full-length and cleaved p22 fragments) are detected by immunoblotting using anti-Gasdermin D (mouse), pAb (IN110) (Prod. No. AG-25B-0036).

Method: Gasdermin D is analyzed by Western blot in cell extracts of bone marrow-derived macrophage cells (BMDMs) (WT, Gasdermin -/- or Asc -/-) treated with LPS (50ng/ml; Prod. No. AG-CU1-0001) for 3h and +/- Nigericin (5μM for 2.5h, Prod. No. AG-CN2-0020). Cell extracts are separated by SDS-PAGE under reducing conditions, transferred to nitrocellulose and incubated with anti-Gasdermin D (mouse), pAb (IN110) (0.5µg/ml). After addition of an anti-guinea pig secondary antibody coupled to HRP (1/5000), proteins are visualized by a chemiluminescence detection system.

Picture courtesy of Prof. Olaf Gross, University Medical Center Freiburg, Germany

 

The Standard Inflammasome Research Antibodies – Validated Reliable Antibodies used by the Experts

AdipoGen Life Sciences is the “No. 1” manufacturer and supplier of inflammasome signaling research reagents. The validated high quality inflammasome research tools are used and published by the experts in inflammasome research on a daily basis.

Product Name PIDProduct Description
anti-Caspase-1 (p20) (mouse), mAb (Casper-1)AG-20B-0042Recognizes endogenous full-length and activated (p20 fragment) mouse caspase-1.
anti-Caspase-1 (p10) (mouse), mAb (Casper-2)AG-20B-0044Recognizes endogenous full-length and activated (p10 fragment) mouse caspase-1.
anti-Caspase-1 (p20) (human), mAb (Bally-1)AG-20B-0048Recognizes endogenous full-length and activated (p20 fragment) human caspase-1.
anti-NLRP3/NALP3, mAb (Cryo-2)AG-20B-0014Recognizes mouse and human NLRP3/NALP3.

Over 1000 Citations!
anti-Asc, pAb (AL177)AG-25B-0006Recognizes human and mouse Asc.
anti-IL-1α (p18) (mouse), mAb (Teo-1)AG-20B-0064Recognizes full-length and cleaved p18 fragment of mouse IL-1α.
anti-Caspase-8 (human), mAb (C15)AG-20B-0057Recognizes the p18 subunit of human caspase-8.
anti-Caspase-8 (mouse), mAb (1G12)AG-20T-0137Recognizes full-length and the p18 cleaved fragment of mouse caspase-8.
anti-Caspase-8 (mouse), mAb (3B10)AG-20T-0138Recognizes full-length and the p18 cleaved fragment of mouse caspase-8.
anti-Caspase-4 /11 (p20), mAb (Flamy-1)AG-20B-0060Recognizes endogenous full-length protein and activated (p20) fragment of mouse and human caspase-4/11. Does not detect human Caspase-5.

The Key Inflammasome Activators and Inhibitors – Including New Gasdermin Inhibitor

Product Name PIDProduct Description
Monosodium urate (crystals)AG-CR1-3950Potent NLRP3 inflammasome activator.
Monosodium urate (ready-to-use)AG-CR1-3951Potent NLRP3 inflammasome activator.
Nigericin . NaAG-CN2-0020Potent NLRP3 inflammasome activator.
N-Acetyl-D-glucosamineAG-CN2-0489Acts as activator of NLRP3 inflammasome by dissociating the enzyme hexokinase from the mitochondria.
Necrosulfonamide NEWAG-CR1-3705Binds directly to Gasdermin D and inhibits the oligomerization of the N-terminus and therefore the pore formation and pyroptosis.
U-73122 NEWAG-CR1-3698Gasdermin D N-terminal fragment (GSDMD-N)-induced pyroptosis inhibitor. Protects against GSDMD-N cytotoxicity in macrophages or against lethal infection in mice.
MCC950 . Na (water soluble)AG-CR1-3615Potent and selective NLRP3 inflammasome inhibitor.
IsoliquiritigeninAG-CN2-0459Inhibits NLRP3-activated Asc oligomerization. Blocks priming and activation steps.
BAY 11-7082AG-CR1-0013Reduces ATPase activity of the NLRP3 inflammasome.
(R)-3-Hydroxybutyric acidAG-CR1-3616Prevent K+-efflux and consequently reduce Asc oligomerization and speck formation.
(S)-3-Hydroxybutyric acidAG-CR1-3617Prevent K+-efflux and consequently reduce Asc oligomerization and speck formation.
DL-3-Hydroxybutyric acid sodium saltCDX-H0080Prevent K+-efflux and consequently reduce Asc oligomerization and speck formation.
K777 [K11777] NEWAG-CR1-0158Broad-range cathepsin inhibitor useful for inflammasome inhibition.

 

 

 

Inflammasomes and Gasdermin D Signaling Pathways