Recombinant Human NACHT, LRR and PYD domains-containing protein 9 (NLRP9), partial

What is the domain architecture of human NLRP9 and how does it compare to other NLR family members?

Human NLRP9 belongs to the NLR family and shares the characteristic domain architecture with other family members. It contains a Pyrin Domain (PYD) at the N-terminus, followed by a central NACHT domain, and C-terminal Leucine-Rich Repeats (LRRs). The PYD domain of human NLRP9 assumes an antiparallel six-helical bundle fold that is typical for the death domain superfamily . Unlike some other NLRPs, the human NLRP9 PYD exists as monomers instead of forming higher oligomeric filament structures in solution . This distinctive property may impact how NLRP9 initiates downstream signaling compared to other NLR proteins.

How does NLRP9 contribute to innate immune responses?

NLRP9 contributes to innate immunity primarily through inflammasome formation in response to specific pathogen challenges. Most notably, NLRP9 initiates inflammasome activation during host defense against rotavirus infection . In human cells, full-length NLRP9 interacts with the adapter protein ASC in a rotavirus infection-dependent manner . This interaction is crucial for the formation of the inflammasome complex, which ultimately leads to the processing and release of pro-inflammatory cytokines. The specificity of NLRP9 for rotavirus suggests it plays a specialized role in intestinal immunity, distinguishing it from other inflammasome-forming NLRs with broader pathogen recognition capabilities.

What are the key differences between human NLRP9 and mouse NLRP9 isoforms?

While humans possess only one NLRP9 gene (hNLRP9), mice harbor three distinct isoforms: mNLRP9a, mNLRP9b, and mNLRP9c . This evolutionary divergence suggests species-specific adaptations in NLRP9 function. The three mouse isoforms appear to have functional redundancy in certain contexts, particularly in embryonic development, as demonstrated by knockout studies showing that female mice deficient in all three NLRP9 isoforms exhibit defective blastocyst development and increased lethality in preimplantation embryos . In contrast, mice with only one isoform deleted show varying degrees of developmental delay but can still develop into blastocysts . This functional redundancy among mouse NLRP9 isoforms creates important considerations for researchers using mouse models to study NLRP9 biology applicable to humans.

What is the tissue-specific expression pattern of NLRP9 and what does this suggest about its biological functions?

NLRP9 belongs to a subgroup of reproduction-related NLRPs that are exclusively or mainly expressed in reproductive organs . This expression pattern places NLRP9 alongside NLRP4, NLRP5, NLRP8, and NLRP14 as NLRs with specialized reproductive functions . The human NLRPs with implicated functions in reproductive systems, including NLRP9, are tandemly distributed on chromosome 19, suggesting they arose through a series of tandem duplication events . This genomic organization implies that while these reproductive NLRPs have specialized functions, they may share certain ancestral mechanisms or regulatory elements. The restricted expression pattern explains why NLRP9 mutations may be particularly relevant to reproductive disorders rather than having broad systemic effects.

How does the PYD domain of NLRP9 function in inflammasome assembly compared to other NLR proteins?

The PYD domain of NLRP9 exhibits distinct properties that differentiate it from other NLR family members. Crystallographic studies have revealed that human NLRP9 PYD forms monomers in solution rather than the filamentous structures typical of many inflammasome-forming PYDs . Consistent with this observation, when overexpressed in HEK293T cells, human NLRP9 PYD does not self-polymerize or promote speck formation of ASC . This unusual behavior raises questions about how NLRP9 initiates inflammasome formation despite lacking the typical self-polymerization activity of its PYD domain.

The mechanistic explanation may involve other domains of NLRP9 that help drive PYD polymerization to recruit ASC, or potential binding partners like DHX9 that may license NLRP9 to assemble an inflammasome complex in response to specific stimuli such as rotavirus RNA . Researchers investigating NLRP9-mediated inflammasome assembly should consider these unique properties when designing experiments or interpreting results, as models based on better-characterized NLRPs like NLRP3 may not fully apply to NLRP9.

What mechanisms regulate NLRP9 activation and inhibition?

While the precise mechanisms regulating NLRP9 activation remain incompletely understood, insights can be drawn from studies of related NLR family members. Based on analogies with NLRP3, post-translational modifications likely play important roles in regulating NLRP9 activity. NLRP3 activity is modulated by phosphorylation at sites like S198 and ubiquitination at sites like K567 . Similar regulatory mechanisms may exist for NLRP9, though specific sites have not been well-characterized.

For inhibition mechanisms, studies of NLRP1 demonstrate that dipeptidyl peptidases DPP8 and DPP9 can suppress inflammasome activation . In the case of NLRP1, DPP9 forms a complex that contains an autoinhibited NLRP1 molecule and an active UPA-CARD fragment, preventing higher-order oligomerization and strengthening autoinhibition . Whether similar protein-protein interactions regulate NLRP9 activity remains an open question worthy of investigation. Researchers studying NLRP9 regulation should consider examining both enzymatic modifications and protein binding partners as potential regulatory mechanisms.

What experimental evidence supports NLRP9's role in embryonic development?

Compelling evidence for NLRP9's role in embryonic development comes from knockout studies in mice. Female mice deficient in all three NLRP9 isoforms demonstrate defective blastocyst development and increased lethality in preimplantation embryos . In vitro culture experiments revealed that fertilized eggs from these triple-knockout females exhibited developmental arrest at the two-cell stage . Interestingly, embryos from females with only two NLRP9 isoforms depleted were able to develop into blastocysts, although they exhibited varying degrees of developmental delay .

These findings establish that NLRP9 isoforms have functionally redundant but essential roles in early embryogenesis in mice. The exact molecular mechanisms by which NLRP9 affects cell division during embryonic development remain to be fully elucidated. Researchers investigating NLRP9's developmental functions should consider designing experiments to determine whether mutations in human NLRP9 are linked to infertility or early pregnancy loss, which would establish clinical relevance for these findings from mouse models.

What is known about NLRP9's role in inflammatory diseases beyond infectious contexts?

NLRP9 has been implicated in several inflammatory conditions beyond its established role in anti-rotavirus defense. In a mouse model of acute lung injury, NLRP9b-deficient mice showed reduced neutrophilic inflammation, better preserved alveolar architecture, and improved survival rates compared to wild-type mice . This suggests NLRP9b plays a detrimental role in acute lung injury pathogenesis, potentially through mechanisms involving elevated proinflammatory cytokines, increased apoptosis, NF-κB activation, and oxidative stress .

What are the optimal expression systems and purification strategies for producing functional recombinant human NLRP9?

When producing recombinant human NLRP9, researchers should consider the following evidence-based approaches:

What cellular and biochemical assays are most appropriate for assessing NLRP9 inflammasome formation and activity?

Several complementary approaches can be employed to assess NLRP9 inflammasome activity:

• ASC Speck Formation Assay: Overexpression of NLRP9 in HEK293T cells can be used to assess interaction with ASC and speck formation using fluorescence microscopy . This provides visual confirmation of inflammasome assembly.

• Cytokine Release Measurements: Quantification of IL-1β and IL-18 release from cells expressing NLRP9 using ELISA provides functional assessment of inflammasome activity .

• Reconstitution in Knockout Cells: Reconstituting NLRP9 expression in NLRP9-deficient cell lines (like THP-1 cells with NLRP9 knockout) allows for clean assessment of NLRP9-specific functions .

• Structural Analysis: For detailed mechanistic studies, structural approaches including cryo-electron microscopy have proven valuable for related NLR proteins and could be applied to NLRP9 complexes.

• Protein-Protein Interaction Studies: Co-immunoprecipitation assays with potential interaction partners like DHX9 or ASC can help elucidate NLRP9's protein interaction network .

Each of these approaches provides distinct and complementary information about NLRP9 function, and combining multiple assays will provide the most comprehensive assessment.

How do the molecular mechanisms of NLRP9 compare to the well-characterized NLRP3 inflammasome?

NLRP9 and NLRP3 exhibit important structural and functional differences despite belonging to the same protein family:

• PYD Domain Properties: Unlike NLRP3, human NLRP9 PYD exists as monomers in solution and does not self-polymerize or promote ASC speck formation when expressed alone . This contrasts with NLRP3 PYD, which readily forms filamentous structures essential for inflammasome assembly.

• Activation Triggers: NLRP3 responds to a wide range of stimuli including nigericin, ATP, and various cellular stress signals , whereas NLRP9 appears more specialized, with established activation primarily in response to rotavirus infection .

• Regulatory Mechanisms: NLRP3 is regulated by numerous post-translational modifications, including phosphorylation at S198 and ubiquitination at K567 . While similar modifications likely regulate NLRP9, the specific sites and modifying enzymes remain to be identified.

• Inhibition Susceptibility: NLRP3 can be inhibited by small molecules like MCC950, with various amino acid substitutions affecting inhibitor sensitivity . Equivalent inhibitors specific for NLRP9 have not been well-characterized.

• Tissue Distribution: NLRP3 is broadly expressed in immune cells, whereas NLRP9 exhibits a more restricted expression pattern with prominence in reproductive tissues , suggesting more specialized biological functions.

Understanding these differences is crucial for researchers developing experimental approaches or therapeutic strategies targeting specific NLR family members.

What can be learned about NLRP9 structure-function relationships from studies of NLRP1 and DPP9 interactions?

Studies of NLRP1 and its interaction with DPP9 provide valuable insights that may apply to NLRP9:

• Complex Formation Principles: NLRP1 forms a 2:1 complex with DPP9, containing an autoinhibited NLRP1 molecule and an active UPA-CARD fragment . This suggests that NLR proteins can form complex stoichiometric relationships with regulatory partners.

• Domain-Specific Interactions: In the NLRP1-DPP9 complex, specific domains mediate key interactions - the ZU5 domain binds to the β-propeller domain of DPP9, while an N-terminal loop from the UPA domain inserts into the DPP9 substrate-binding channel . Similar domain-specific interaction patterns might exist for NLRP9 with its regulatory partners.

• Dual Mechanisms of Inhibition: DPP9 inhibits NLRP1 through two mechanisms: preventing higher-order oligomerization of active fragments and strengthening autoinhibition . NLRP9 may be similarly regulated through multiple concurrent mechanisms.

• Enzymatic Activity Requirements: Both binding and enzymatic activity of DPP9 are required for inhibition of NLRP1 . This suggests that when investigating NLRP9 regulatory interactions, researchers should consider both physical binding and enzymatic activities of potential regulatory partners.

These principles from NLRP1 studies provide a valuable framework for designing experiments to investigate NLRP9 regulation and activation mechanisms.

What are the most promising approaches for determining the complete structure of human NLRP9?

Determining the complete structure of human NLRP9 presents significant challenges, but several approaches show promise:

Despite technological advances, obtaining the structure of full-length NLRs remains challenging . Researchers should consider multiple complementary approaches rather than relying on a single method.

What genetic approaches would be most informative for understanding NLRP9's role in human reproductive disorders?

Several genetic approaches would advance our understanding of NLRP9's role in human reproductive disorders:

• Targeted Sequencing Studies: Sequencing NLRP9 in cohorts with unexplained infertility, recurrent early pregnancy loss, or preimplantation embryonic development failures could identify potential pathogenic variants.

• Functional Validation in Mouse Models: Creating knock-in mice harboring human NLRP9 variants identified in patients could validate their pathogenicity and elucidate mechanisms of reproductive dysfunction.

• Single-Cell Transcriptomics: Analyzing NLRP9 expression patterns in human oocytes and early embryos at different developmental stages would clarify its temporal importance in human development.

• CRISPR/Cas9 Editing in Human Embryonic Stem Cells: Creating NLRP9 mutations in human embryonic stem cells and studying their differentiation into germ cells could reveal human-specific functions.

• Protein Interaction Studies: Identifying NLRP9 binding partners in reproductive tissues through approaches like proximity labeling would illuminate the protein networks through which NLRP9 influences reproduction.

Given the evidence from mouse studies showing embryonic lethality with complete NLRP9 deficiency , human studies should focus on hypomorphic variants rather than complete loss-of-function to identify clinically relevant phenotypes.