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Updated: Jul 8, 2026
10:28
A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
Published on: August 17, 2019
ATP binding by monarch-1/NLRP12 is critical for its inhibitory function
Zhengmao Ye1, John D Lich, Chris B Moore
1Department of Microbiology and Immunology, University of North Carolina, CB7295, 450 West St., Chapel Hill, NC 27599, USA.
Molecular and Cellular Biology
|December 28, 2007
Summary
Purified Monarch-1/NLRP12 protein binds and hydrolyzes ATP, a function requiring intact Walker A/B motifs. This ATP binding regulates Monarch-1
Area of Science:
- Molecular Biology
- Immunology
- Biochemistry
Background:
- Nucleotide-binding domain-leucine-rich repeat (NLR) proteins are crucial in innate immunity and linked to human disorders.
- The nucleotide-binding domain (NBD) is a defining feature of NLRs but remains understudied in its purified form.
- Monarch-1/NLRP12 is an NLR protein that negatively regulates NF-kappaB signaling.
Purpose of the Study:
- To investigate the biochemical properties of purified Monarch-1/NLRP12, focusing on its nucleotide-binding domain.
- To elucidate the role of ATP binding and hydrolysis in Monarch-1/NLRP12 function and regulation of NF-kappaB signaling.
Main Methods:
- Purification of Monarch-1/NLRP12 protein.
- In vitro assays to assess ATP binding and hydrolysis activity.
- Site-directed mutagenesis to create Walker A/B motif mutants.
- Cell-based assays using THP-1 monocytes to evaluate Monarch-1/NLRP12 function in NF-kappaB signaling and cytokine production.
Main Results:
- Purified Monarch-1/NLRP12 specifically binds ATP and exhibits ATP hydrolysis activity.
- Intact Walker A/B motifs are essential for Monarch-1/NLRP12's ATP hydrolysis, self-oligomerization, and interaction with signaling molecules (NIK, IRAK-1).
- A Walker A/B mutant Monarch-1 protein leads to increased pro-inflammatory cytokine and chemokine production, mimicking Monarch-1 silencing.
Conclusions:
- ATP binding and hydrolysis by the Walker A/B motifs are critical for Monarch-1/NLRP12's anti-inflammatory function.
- These findings support a model where ATP binding regulates Monarch-1/NLRP12's inhibitory activity on NF-kappaB signaling.
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