NEK7 recombinant protein: a key regulator of NLRP3 inflammasome assembly and activation

Author: Senior Product Manager of UA Views：6

This article systematically elaborates on the molecular characteristics and immune functions of NIMA-related kinase 7 (NEK7), focusing on the discovery process and validation evidence establishing it as a key regulatory factor in NLRP3 inflammasome assembly and activation. It analyzes the protein domain basis of the NEK7–NLRP3 interaction and its kinase activity-independent binding properties, and explores the selective blocking effect of NEK7 deficiency on NLRP3-specific inflammatory responses as well as the regulatory mechanism of extracellular potassium ion concentration on this interaction.

Recent Advances
Product Information

Recent Advances

NEK7 Recombinant Protein: A Key Regulatory Factor in NLRP3 Inflammasome Assembly and Activation

Summary: This article focuses on the molecular characteristics and immune functions of NIMA-related kinase 7 (NEK7), systematically elaborating on the discovery process and validation evidence of its role as a key regulatory factor in NLRP3 inflammasome assembly and activation, analyzing the protein domain basis of the interaction between NEK7 and NLRP3 and its kinase-activity-independent binding properties, and discussing the selective blocking effect of NEK7 deficiency on NLRP3-specific inflammatory responses as well as the regulatory mechanism of extracellular potassium ion concentration on this interaction.

I. Biological Background of Inflammasomes and Unsolved Questions in NLRP3 Activation.

Inflammasomes are intracellular protein complexes capable of activating inflammatory caspase protein kinases, playing an important regulatory role in the occurrence and control of inflammation in the body. To date, four types of inflammasomes have been discovered: NLRP1, NLRP3, NLRC4, and AIM2. Mutations in NLRP3 can trigger many inflammatory diseases, including cryopyrin-associated periodic syndromes. It is currently known that potassium ion efflux plays an important role in NLRP3 activation, but how NLRP3 responds to potassium ion efflux remains unclear. To address this question, the research group led by Gabriel Núñez at the University of Michigan Medical School focused on identifying other proteins that interact with NLRP3, and their findings were published in the journal Nature. This research background reveals a core scientific question: whether there exists a key adaptor protein between the upstream signal of potassium ion efflux and the downstream event of NLRP3 inflammasome assembly.

II. Identification of NEK7 as an NLRP3-Interacting Protein.

The researchers first identified a class of protein kinases called NIMA-related kinase 7 through liquid chromatography-mass spectrometry. Through in vitro overexpression Pull-Down and co-immunoprecipitation approaches, the authors also demonstrated that stimulation of macrophages with LPS plus ATP can trigger the binding of NEK7 to NLRP3. This research path from unbiased mass spectrometry screening to targeted validation provides direct evidence for the identity of NEK7 as an NLRP3 binding partner. Notably, the binding of NEK7 to NLRP3 is stimulus-induced rather than constitutive, suggesting that this interaction itself may be a regulated step in the inflammasome activation process.

III. Domain Basis of the NEK7-NLRP3 Interaction.

To identify the interaction sites between the two proteins, the authors used HEK293T cells and transfected exogenous wild-type NLRP3 and mutant NLRP3, ultimately using the Pull-Down method to detect whether interaction would occur. The results showed that in HEK293T cells, wild-type full-length NLRP3 was able to interact with NEK7, NLRP3 mutants lacking the Pyrin domain were also able to interact, while mutants lacking the LRR domain were unable to interact with NEK7; furthermore, the LRR alone was also unable to interact with NEK7. These results indicate that the LRR and NOD domains of the NLRP3 protein play key roles in the interaction between the two. Similarly, the authors transfected wild-type and mutant NEK7 into HEK293T cells and found that the key region was a stretch of amino acid sequence located in the middle, from positions 33 to 212. Interestingly, through previous studies, the authors mutated several amino acid sites of NEK7 with enzymatic activity and found that these mutations did not affect its normal binding to NLRP3. This indicates that the interaction between the two does not depend on NEK7's enzymatic activity; NEK7's role in this process is more akin to a structural scaffold or adaptor molecule rather than exerting catalytic function through phosphorylation of substrates.

IV. Selective Blocking of NLRP3 Inflammasome Activation by NEK7 Deficiency.

Because homozygous NEK7 knockout mice are lethal, the authors could not obtain NEK7 knockout mice through conventional means. Therefore, the authors used irradiation to kill lymphocytes in mice and constructed chimeras through fetal liver cell transplantation technology. At this point, based on the phenotype of the transplanted cells, wild-type or NEK7-deficient bone marrow-derived macrophages could be obtained from the chimeric mice. On this basis, the authors subjected the obtained bone marrow-derived macrophages to different types of stimulation. The results showed that stimulation with ATP, Nigericin, and Gramcidin could trigger caspase-1 cleavage and IL-1β release, while in NEK7-deficient bone marrow-derived macrophages, caspase-1 cleavage or IL-1β release was inhibited after the above stimulations. In contrast, stimulation with Poly(dA:dT) and salmonella could trigger caspase-1 cleavage and IL-1β release in both wild-type bone marrow-derived macrophages and mutant bone marrow-derived macrophages. In addition, the authors also confirmed the above phenomena using shRNA approaches. These results indicate that NEK7 deficiency can specifically block NLRP3 inflammasome activation without affecting the functions of AIM2 and NLRC4 inflammasomes, and this selectivity is key evidence for NEK7 as an NLRP3-specific regulatory factor.

V. Mechanistic Validation of NEK7's Involvement in Inflammasome Complex Aggregation.

Next, the authors investigated whether NEK7 participates in the aggregation process of the inflammasome protein complex. First, the authors compared ASC aggregation inside cells before and after stimulation in wild-type bone marrow-derived macrophages and mutant bone marrow-derived macrophages using intracellular staining immunofluorescence experiments. The results showed that under ATP and Nigericin stimulation, obvious ASC aggregates appeared inside wild-type bone marrow-derived macrophages, while this phenomenon was absent in mutant bone marrow-derived macrophages. In contrast, stimulation with Poly(dA:dT) and salmonella both induced the production of ASC aggregates. In addition, the authors verified the above differences in ASC aggregate formation through cross-linking and biochemical analysis methods. Subsequently, the authors also used cross-linking biochemical detection methods to demonstrate that the aggregation of NLRP3 and NEK7 themselves is also affected by the interaction between NEK7 and NLRP3. Furthermore, the authors found that the interaction between NLRP3 and NEK7 is also affected by extracellular potassium ion concentration, namely that when the extracellular potassium ion concentration exceeds 50 mM, the interaction between the two disappears. This finding directly links the upstream signal of potassium ion efflux with the molecular event of NEK7-NLRP3 binding, providing a mechanistic explanation for understanding the early steps of NLRP3 activation.

VI. Conclusion.

In NEK7-related basic research and inflammasome mechanism exploration, high-quality recombinant NEK7 protein is a core tool for dissecting its binding mechanism with NLRP3 and evaluating inflammasome regulatory strategies. The GST tag facilitates efficient purification through glutathione affinity chromatography and can serve as a detection tag for analyses such as enzyme-linked immunosorbent assay and surface plasmon resonance. To meet the needs of NEK7 functional research and inflammasome activation mechanism exploration, Ulab provides NEK7 GST Tag Protein, Human. This product is suitable for application scenarios such as binding activity analysis of NEK7 and NLRP3, research on NEK7-mediated NLRP3 inflammasome assembly mechanisms, in vitro evaluation of the effects of potassium ion concentration on the NEK7-NLRP3 interaction, and screening of anti-inflammatory drugs targeting the NEK7-NLRP3 interface.

This article is reviewed and published by the technical expert team of UA

Disclaimer: This article partially utilizes artificial intelligence assistance in its creation. If any content involves copyright or intellectual property issues, please let us know and we promise to verify and remove it as soon as possible.

Purchase recombinant protein, choose

UA protein focuses on providing various protein reagents, raw materials, and services required for drug research and development, cell therapy, gene therapy, and basic scientific research, including drug target proteins, immune checkpoint proteins, cytokines, tool enzymes, customized protein expression, and full-length transmembrane protein development. Nanjing Univ is committed to providing customers with high-quality products and professional services, and building a High-tech Biological Enterprise with International Competitiveness.

Buy antibodiesFind UA www.ua-bio.com | 15 years of protein development experience

Nanjing UNIV Biotechnology Co., Ltd. Email：order@ua-bio.com Phone：+86-25-56221161

more products

Product Information

The Last The Next