Reconstituted AIM2 inflammasome in cell-free system

Kaneko, Naoe; Ito, Yuki; Iwasaki, Tomoyuki; Takeda, Hiroyuki; Sawasaki, Tatsuya; Migita, Kiyoshi; Agematsu, Kazunaga; Kawakami, Atsushi; Morikawa, Shinnosuke; Mokuda, Sho; Kurata, Mie; Masumoto, Junya

doi:10.1016/j.jim.2015.08.004

Cited by 10 publications

(15 citation statements)

References 24 publications

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“…Therefore, we employed the wheat germ cell-free protein synthesis system, and Nod2-WT-Btn, Nod2-R334W-Btn, Nod2-N670K-Btn, Nod2-CARDs, FLAG-RICK-WT, and FLAG-RICK-CARD were successfully synthesized ( Figure 1 ). To date, we reported the generation of AIM2-inflammasome in a cell-free system, which is another intracellular pattern recognition receptor with adaptor ASC oligomerization, which was suitable for wheat germ cell-free protein synthesis [ 20 ]. These results suggested that the wheat germ cell-free protein synthesis system has an advantage for reconstituting intracellular pattern recognition receptor complexes for resolving innate immune complex-mediated autoinflammatory diseases.…”

Section: Discussionmentioning

confidence: 99%

Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

Iwasaki

Kaneko

Ito

et al. 2016

The Scientific World Journal

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Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplified luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.

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Section: Discussionmentioning

confidence: 99%

Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

Iwasaki

Kaneko

Ito

et al. 2016

The Scientific World Journal

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“…First, we describe an AIM2 inflammasome in a cell-free system as a prototype [ 56 ] because the AIM2 inflammasome has been well-characterized, and its ligand was reported to be present in poly-deoxyadenylic-deoxythymidylic acid, poly(dA:dT). The direct interaction between AIM2 and poly(dA:dT) was elucidated using the amplified luminescent proximity homogeneous assay (Alpha) [ 14 ].…”

Section: Reconstituted Aim2 Inflammasome In a Cell-free Systemmentioning

confidence: 99%

Applications of reconstituted inflammasomes in a cell-free system to drug discovery and elucidation of the pathogenesis of autoinflammatory diseases

et al. 2017

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The inflammasome, typically consisting of a Nod-like receptor, apoptosis-associated speck-like protein, and pro-caspase-1, has recently been identified as a huge intracellular complex, which plays a crucial role in interleukin-1 maturation or specific physiological functions. Two Nod-like receptors, such as nucleotide-binding oligomerization domains-containing protein (Nod)1 and Nod2, interact with the receptor-interacting protein serine-threonine kinase (RIPK)2 accompanied by Iκ-B kinase (IKK) complexes to construct the nodosome, leading to nuclear factor (NF)-κB activation. The aberrant activation of inflammasomes or nodosomes causes autoinflammatory diseases. Therefore, inflammasomes may be attractive targets to treat autoinflammatory diseases. Our aim is to develop reconstituted inflammasomes in a cell-free system to discover specific molecular-target drugs and elucidate the molecular pathogenesis of autoinflammatory diseases. In this review, we describe reconstituted inflammasomes in a cell-free system.

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“…It has been reported the use of an anti-human ASC monoclonal antibody (clone 23-4, MBL, Nagoya, Japan) for ASC identification, cloning and characterization (Masumoto et al, 2001). Kaneko et al (2015) have studied whether this anti-ASC human antibody would interfere with PYD of ASC and if it could then be considered a therapeutic candidate against disorders due to AIM2-inflammasome dysregulation. Since AIM2 is an intracellular receptor, enforced internalization of both ligands and candidate molecules is necessary for pharmacological screening.…”

Section: Therapeutic Implications Of Aim2 Inflammasomementioning

confidence: 99%

“…Since AIM2 is an intracellular receptor, enforced internalization of both ligands and candidate molecules is necessary for pharmacological screening. Interestingly, the authors developed a reconstituted in vitro AIM2 inflammasome in a cell-free system, which may serve as a useful tool to screen AIM2 inflammasometargeting therapeutics (Kaneko et al, 2015).…”

Section: Therapeutic Implications Of Aim2 Inflammasomementioning

confidence: 99%

The Duality of AIM2 Inflammasome: A Focus on its Role in Autoimmunity and Skin Diseases

Canavese¹

2016

American Journal of Pharmacology and Toxicology

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Understanding the inflammasome biology is one of the most exciting challenges in immuno-pharmacology. The role of the inflammasomes has been recognized in the host defense mechanism against invading pathogens and in the development of several conditions, such as cancer, auto-inflammatory, autoimmune, metabolic and neurodegenerative disorders. DNA recognition by the cells is a crucial immunological step leading to the initiation of an innate immune response. Absent in Melanoma 2 (AIM2) is a cytoplasmic sensor that perceives double-stranded DNA of microbial or host origin. Once the DNA is bound, AIM2 assembles a multiprotein complex named inflammasome, which drives pyroptosis and proteolytic cleavage of pro-IL-1β and pro-IL-18 pro-inflammatory cytokines, leading to a protective inflammasome-mediated host response. However, improper recognition of self-DNA by AIM2 triggers deleterious inflammatory responses, leading to systemic inflammation and several pathological conditions. Therefore, understanding the mechanisms of AIM2-inflammasome-mediated inflammation will provide an essential knowledge base to develop new successful therapeutic strategies to cure the outlined pathologies in which AIM2-inflammasome activation plays a key role, as well as to guide clinical practice. This mini-review provides an overview on the latest research findings on AIM2 inflammasome, with particular focus on its role in autoimmunity and skin disorders. An update on its therapeutic implications has also been documented.

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Reconstituted AIM2 inflammasome in cell-free system

Cited by 10 publications

References 24 publications

Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

Applications of reconstituted inflammasomes in a cell-free system to drug discovery and elucidation of the pathogenesis of autoinflammatory diseases

The Duality of AIM2 Inflammasome: A Focus on its Role in Autoimmunity and Skin Diseases

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