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Evaluation of Caspase Activation to Assess Innate Immune Cell Death
Published on: January 20, 2023
A Disease-associated Mutant of NLRC4 Shows Enhanced Interaction with SUG1 Leading to Constitutive FADD-dependent
Akhouri Kishore Raghawan1, Anand Sripada1, Gayathri Gopinath1
1From the CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500007, India.
Abstract:
Nod-like receptor family card containing 4 (NLRC4)/Ipaf is involved in recognition of pathogen-associated molecular patterns leading to caspase-1 activation and cytokine release, which mediate protective innate immune response. Point mutations in NLRC4 cause autoinflammatory syndromes. Although all the mutations result in constitutive caspase-1 activation, their phenotypic presentations are different, implying that these mutations cause different alterations in properties of NLRC4. NLRC4 interacts with SUG1 and induces caspase-8-mediated cell death. Here, we show that one of the autoinflammatory syndrome-causing mutants of NLRC4, H443P, but not T337A and V341A, constitutively activates caspase-8 and induces apoptotic cell death in human lung epithelial cells. Compared with wild type NLRC4, the H443P mutant shows stronger interaction with SUG1 and with ubiquitinated cellular proteins. Phosphorylation of NLRC4 at Ser533 plays a crucial role in caspase-8 activation and cell death. However, H443P mutant does not require Ser533 phosphorylation for caspase-8 activation and cell death. Caspase-8 activation by NLRC4 and its H443P mutant are dependent on the adaptor protein FADD. A phosphomimicking mutant of NLRC4, S533D does not require SUG1 activity for inducing cell death. Ubiquitin-tagged NLRC4 could induce cell death and activate caspase-8 independent of Ser533 phosphorylation. Our work suggests that SUG1-mediated signaling results in enhanced ubiquitination and regulates FADD-dependent caspase-8 activation by NLRC4. We show that the autoinflammation-associated H443P mutant is altered in interaction with SUG1 and ubiquitinated proteins, triggering constitutive caspase-8-mediated cell death dependent on FADD but independent of Ser533 phosphorylation.
Insights
Nod-like receptor family card containing 4 (NLRC4) mutations can cause autoinflammatory syndromes. The H443P NLRC4 mutant constitutively activates caspase-8 and cell death, independent of Ser533 phosphorylation, by interacting with SUG1 and ubiquitinated proteins.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Nod-like receptor family card containing 4 (NLRC4) recognizes pathogen-associated molecular patterns, activating caspase-1 for innate immune responses.
- Point mutations in NLRC4 lead to autoinflammatory syndromes, characterized by constitutive caspase-1 activation but varying phenotypes.
- NLRC4 interacts with SUG1 and can induce caspase-8-mediated cell death.
Purpose of the Study:
- To investigate the mechanism by which the NLRC4 H443P autoinflammatory mutant induces caspase-8-mediated cell death.
- To determine the role of Ser533 phosphorylation and SUG1 interaction in NLRC4-induced cell death.
- To elucidate the signaling pathway involving SUG1, ubiquitination, and FADD in NLRC4-mediated apoptosis.
Main Methods:
- Comparison of wild-type NLRC4 with H443P, T337A, and V341A mutants in human lung epithelial cells.
- Assessment of caspase-8 activation and apoptotic cell death.
- Analysis of protein interactions with SUG1 and ubiquitinated proteins.
- Investigation of the role of Ser533 phosphorylation and FADD adaptor protein.
Main Results:
- The H443P NLRC4 mutant, unlike T337A and V341A, constitutively activates caspase-8 and induces apoptosis.
- H443P exhibits enhanced interaction with SUG1 and ubiquitinated proteins compared to wild-type NLRC4.
- Caspase-8 activation and cell death by H443P are independent of Ser533 phosphorylation but dependent on FADD.
- SUG1 activity is not required for cell death induced by a phosphomimicking S533D NLRC4 mutant.
Conclusions:
- The autoinflammation-associated H443P NLRC4 mutant triggers constitutive caspase-8-mediated cell death.
- This process is regulated by enhanced interaction with SUG1 and ubiquitinated proteins, leading to FADD-dependent activation of caspase-8.
- The H443P mutant bypasses the requirement for Ser533 phosphorylation in caspase-8 activation and cell death, highlighting a distinct pathogenic mechanism.
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