Tailoring NINJ1 for the Control of Plasma Membrane Rupture in Regulated Cell Death 2260243

Introduction Cell death enables host cells to eliminate pathogens and maintain tissue homeostasis. Consequently, dysregulation of cell death pathways contributes to a wide range of pathologies, including cancer, autoimmune diseases, and aging. Most of the cell death pathways culminate in plasma membrane rupture (PMR), a process actively executed by NINJ1. In resting cells, inactive NINJ1 exists as homodimers, but upon activation of upstream cell death triggers, NINJ1 oligomerize to induce PMR. Previous studies have identified key functional residues within NINJ1 but the mechanism underlying PMR require further elucidation. Methods To better understand the functional importance of NINJ1 in lytic cell death, we engineered NINJ1 knockout (KO) macrophages by CRISPR-Cas9. To gain insight into the mechanisms regulating NINJ1 activity, we evaluated the ability of specific ectopically expressed NINJ1 point mutants to mediate lytic death. Results Upon LPS and Nigericin treatment, NINJ1 was shown to be essential for LDH release, but dispensable for NLRP3 expression, Gasdermin D (GSDMD) cleavage, and propidium iodide (PI) uptake. These findings corroborate that NINJ1 is not required for initial PM permeabilization but is crucial for the subsequent PMR during lytic cell death. When overexpressed in HEK 293T cells K45Q, D53A, Q91A and G95L mutations resulted in a pronounced loss-of-function effect. When co-expressed in an overexpression model of auto-activation of wild-type (WT) NINJ1, these mutants markedly attenuated the PMR activity of WT NINJ1, suggesting a true dominant-negative effect as opposed to just loss-of-function. A dose-response analysis revealed that these mutants inhibit WT NINJ1 activity at ratios below 1:1, likely pointing to additional mechanisms for NINJ1 regulation beyond stabilization of homodimerization. Conclusion Our results confirm the central role of NINJ1 in PMR and highlight the functional significance of specific residues, advancing our mechanistic understanding of NINJ1 inhibition. Funding Source G.G.R supporteed by the Ramón Areces Foundation Research Fellowship. C.L.E. supported by the Ragon Early Independence Fellowship, Karin Grunebaum Cancer Research Foundation Faculty Research Fellowship, and NIH grant R35GM159938.