Inflammasomes and Gasdermin D Signaling Pathways

更新日期:2019年8月15日

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:

The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta: F. Martinon, et al.; Mol. Cell 10, 417 (2002)

Homeostasis-altering molecular processes as mechanisms of inflammasome activation: A. Liston & S.L. Masters; Nat. Rev. Immunol. 17, 208 (2017)

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling: N. Kayagaki, et al.; Nature 526, 666 (2015)Mechanisms of Gasdermin Family Members in Inflammasome Signaling and Cell Death: S. Feng, et al.; J. Mol. Biol. 430, 3068 (2018)

Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores: X. Liu, et al.; Nature 535, 153 (2016)

The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages: C.L. Evavold, et al.; Immunity 48, 35 (2018)

Noncanonical inflammasome signaling elicits gasdermin D-dependent neutrophil extracellular traps: K.W. Chen, et al.; Sci. Immunol. 3, 26 (2018)

Lipid Peroxidation Drives Gasdermin D-mediated Pyroptosis in Lethal Polymicrobial Sepsis: R. Kang, et al.; Cell Host Microbe 24, 97 (2018)

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)

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


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