Abstract:In the NLR family, pyrin domain containing 3 (NLRP3) is an intracellular pattern recognition receptor that activates procaspase-1, leading to IL-1β and IL-18 processing and activation in a large complex called the NLRP3 inflammasome. Since various pathogens or endogenous metabolites have been reported to stimulate NLRP3 inflammasome, the interaction between NLRP3 and ASC induced by these stimulants may be an attractive drug target for NLRP3-related diseases, called inflammasomopathies. However, the endogenous … Show more
“…2a . Using wheat germ cell-free system-specific expression plasmids, NLRP3-FL-Btn, FLAG-ASC-FL, and FLAG-ASC-CARD proteins were also synthesized, as described previously [ 5 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…In order to identify directly stimulating endogenous ligands, we previously developed a reconstituted NLRP3 inflammasome in a cell-free system [ 5 , 13 ]. In this study, we successfully prepared Aβ oligomers and Aβ fibrils (Fig.…”
Section: Discussionmentioning
confidence: 99%
“…pDONR221-NLRP3 and pDONR221-ASC were inserted into pEU-E01-GW-bls-STOP and pEU-E01-GW-STOP, respectively, for cell-free protein expression, as previously reported [ 5 ]. The entire cDNA of GFP was derived from pEU-E01-bls-GFP.…”
Section: Methodsmentioning
confidence: 99%
“…We previously developed the NLRP3 inflammasome in a cell-free system to detect intrinsic and extrinsic directly interacting ligands [ 5 ]. The NALP3 inflammasome was reported to be a sensor of phagocytosed Aβ [ 6 ]; however, Aβ did not induce an interaction with NLRP3 or apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in the cell-free system [ 5 ].…”
BackgroundAlzheimer’s disease is a neurodegenerative disease characterized by the interstitial deposition of amyloid β (Aβ) plaque, which is thought to be related to chronic neuroinflammation. Aβ is known to make fibrils via oligomers from monomers. Aβ has been reported to activate the NLRP3 inflammasome in infiltrated macrophages. NLRP3, an intracellular pattern recognition receptor, has been reported to recognize numerous pathogens and/or metabolites and form complexes with adopter protein ASC to make the inflammasome, an interleukin (IL)-1β-processing platform. Although reactive oxygen species from mitochondria have been reported to be involved in the activation of the NLRP3 inflammasome in microglial cells upon the deposition of Aβ, whether Aβ directly or indirectly activates the NLRP3 inflammasome remains unclear.MethodsWe prepared monomers, oligomers, and fibrils of Aβ, which promoted the interaction between NLRP3 and each form of Aβ and analyzed the interaction between NLRP3 and ASC induced by each form of Aβ in a cell-free system with the amplified luminescent proximity homogeneous assay. We also confirmed the physiological relevance in a cell-based assay using human embryonic kidney 293T cells and human peripheral mononuclear cells.ResultsMonomers, oligomers, and fibrils of Aβ were successfully prepared. Aβ oligomers and fibrils interacted with NLRP3. Aβ oligomers and fibrils induced the interaction between NLRP3 and ASC. However, Aβ monomers did not interact with NLRP3 or induce interaction between NLRP3 and ASC in the cell-free system, and IL-1β was not secreted according to the cell-based assay.ConclusionOligomerized Aβ originating from non-toxic Aβ monomers directly interacted with NLRP3, leading to the activation of the NLRP3 inflammasome. This may be an attractive target for the treatment of Alzheimer’s disease.Electronic supplementary materialThe online version of this article (10.1186/s41232-018-0085-6) contains supplementary material, which is available to authorized users.
“…2a . Using wheat germ cell-free system-specific expression plasmids, NLRP3-FL-Btn, FLAG-ASC-FL, and FLAG-ASC-CARD proteins were also synthesized, as described previously [ 5 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…In order to identify directly stimulating endogenous ligands, we previously developed a reconstituted NLRP3 inflammasome in a cell-free system [ 5 , 13 ]. In this study, we successfully prepared Aβ oligomers and Aβ fibrils (Fig.…”
Section: Discussionmentioning
confidence: 99%
“…pDONR221-NLRP3 and pDONR221-ASC were inserted into pEU-E01-GW-bls-STOP and pEU-E01-GW-STOP, respectively, for cell-free protein expression, as previously reported [ 5 ]. The entire cDNA of GFP was derived from pEU-E01-bls-GFP.…”
Section: Methodsmentioning
confidence: 99%
“…We previously developed the NLRP3 inflammasome in a cell-free system to detect intrinsic and extrinsic directly interacting ligands [ 5 ]. The NALP3 inflammasome was reported to be a sensor of phagocytosed Aβ [ 6 ]; however, Aβ did not induce an interaction with NLRP3 or apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in the cell-free system [ 5 ].…”
BackgroundAlzheimer’s disease is a neurodegenerative disease characterized by the interstitial deposition of amyloid β (Aβ) plaque, which is thought to be related to chronic neuroinflammation. Aβ is known to make fibrils via oligomers from monomers. Aβ has been reported to activate the NLRP3 inflammasome in infiltrated macrophages. NLRP3, an intracellular pattern recognition receptor, has been reported to recognize numerous pathogens and/or metabolites and form complexes with adopter protein ASC to make the inflammasome, an interleukin (IL)-1β-processing platform. Although reactive oxygen species from mitochondria have been reported to be involved in the activation of the NLRP3 inflammasome in microglial cells upon the deposition of Aβ, whether Aβ directly or indirectly activates the NLRP3 inflammasome remains unclear.MethodsWe prepared monomers, oligomers, and fibrils of Aβ, which promoted the interaction between NLRP3 and each form of Aβ and analyzed the interaction between NLRP3 and ASC induced by each form of Aβ in a cell-free system with the amplified luminescent proximity homogeneous assay. We also confirmed the physiological relevance in a cell-based assay using human embryonic kidney 293T cells and human peripheral mononuclear cells.ResultsMonomers, oligomers, and fibrils of Aβ were successfully prepared. Aβ oligomers and fibrils interacted with NLRP3. Aβ oligomers and fibrils induced the interaction between NLRP3 and ASC. However, Aβ monomers did not interact with NLRP3 or induce interaction between NLRP3 and ASC in the cell-free system, and IL-1β was not secreted according to the cell-based assay.ConclusionOligomerized Aβ originating from non-toxic Aβ monomers directly interacted with NLRP3, leading to the activation of the NLRP3 inflammasome. This may be an attractive target for the treatment of Alzheimer’s disease.Electronic supplementary materialThe online version of this article (10.1186/s41232-018-0085-6) contains supplementary material, which is available to authorized users.
“…The NLRP3 entry clones, pDONR221-NLRP3-FL, DONR221-NLRP3-(Exons 1-2), pDONR221-NLRP3-(Exons 3-9), pDONR221-NLRP3-(Exons 4-9), and pDONR221-ASC, were constructed as previously reported. 16 The constructed plasmids were used to synthesize specific proteins with the WEPRO1240 Expression Kit (Cell-free, Inc., Matsuyama, Japan) followed by Western blotting.…”
Recent findings revealed that type 2 diabetes mellitus (T2D) is a chronic
inflammatory disease and an islet amyloid polypeptide (IAPP)/amylin, is
deposited within pancreatic islets. IAPP/amylin has been reported to activate
NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome in
infiltrated macrophages. NLRP3, an intracellular pattern recognition receptor,
has been shown to recognize pathogens and/or metabolites and complexes with the
adopter protein apoptosis-associated speck-like protein containing a
caspase-recruitment domain ASC to form a huge complex, called an inflammasome,
an interleukin (IL)-1β-processing platform. Although reactive oxygen species
(ROS) were reported to be involved in activation of NLRP3 inflammasome, we were
hypothesized that IAPP could directly activate NLRP3 inflammasome, leading to
islets β-cell death. We analyzed expression of the inflammasome components ASC,
NLRP3, caspase-1, IL-1β, IAPP/amylin, and insulin immunohistochemically in
Langerhans’ islets of autopsy cases. The initial event of NLRP3 inflammasome
activation was assessed using a cell-free system consisting of NLRP3 and ASC
with the amplified luminescent proximity homogeneous assay. IAPP/amylin
deposition in Langerhans’ islets was detected and significantly correlated with
expressions of IL-1β and ASC. IAPP/amylin directly interacted with NLRP3 and
initiated an interaction between NLRP3 and ASC in a cell-free system. The
deposition of IAPP/amylin in β-cells of Langerhans’ islets may act together with
the expression level of an inflammasome component, ASC, to regulate IL-1β
processing, and directly lead to the dysfunction of β-cells. The interaction
between IAPP/amylin and NLRP3 could be an attractive drug target to avoid both
inflammation and β-cell death for T2D therapy.
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