Borges, J. P. | bioRxiv Science

bioRxiv2023

NINJ1 mediates plasma membrane rupture through formation of nanodisc-like rings

The membrane proteins Ninjurin1 (NINJ1) and Ninjurin2 (NINJ2) are upregulated by nerve injury to increase cell adhesion and promote axonal growth in neurons. NINJ1, but not NINJ2, has also been shown to play an essential role in pyroptosis by promoting plasma membrane rupture downstream of gasdermin D (GSDMD) pore formation, as well as in lytic cell death mediated by other pathways. Recombinant NINJ1 and NINJ2 purified in detergent show irregular rings of various diameters as well as curved filaments. While NINJ1 and NINJ2 both formed ring-like structures when mixed with liposomes, strikingly, only NINJ1, but not NINJ2, ruptures liposome membranes, leading to their dissolution. Because of the better feasibility, we determined the cryo-EM structure of NINJ1 ring segments from detergent by segmenting the irregular rings into shorter fragments. Each NINJ1 subunit contains a transmembrane (TM) helical hairpin (3 and 4) that likely mediates NINJ1 membrane localization, as well as the side-by-side interaction between adjacent subunits. There are two extracellular domain amphipathic helices (1 and 2), among which 1 crosses over to the neighboring subunit at the outside facing surface of the ring, to link NINJ1 subunits together into chains. As such, the inner face of the rings is hydrophobic whereas the outer face of the rings is hydrophilic and should repel membranes. Live cell imaging of NINJ1-deficient THP-1 cells reconstituted with NINJ1-eGFP uncovers the pinching off of NINJ1 rings from the cell surface and the loss of NINJ1 to the culture supernatant in oligomerized forms upon inflammasome activation. Formation of rings is also confirmed by super-resolution imaging of endogenous NINJ1 using anti-NINJ1 antibody. These data suggest that membrane insertion of amphipathic helices and formation of rings with a hydrophilic outer surface underlie the mechanism for NINJ1 to pinch off membranes as if it were a nanodisc-forming amphipathic polymer, leading to membrane rupture and lysis during cell death.

immunology↗

bioRxiv2021

Glycine targets NINJ1-mediated plasma membrane rupture to provide cytoprotection

First recognized 35 years ago, glycine is known to protect cells against plasma membrane rupture from diverse types of tissue injury. This robust and widely observed effect has been speculated to target a late downstream process common to multiple modes of tissue injury. The molecular target of glycine cytoprotection, however, remains entirely elusive. We hypothesized that glycine targets ninjurin-1 (NINJ1), a newly identified executioner of plasma membrane rupture in pyroptosis, necrosis, and post-apoptotic cell death. NINJ1 is thought to cluster within the plasma membrane to cause cell rupture. Here, we first demonstrate that NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection in mouse and human macrophages stimulated to undergo lytic cell death. Next, we show that glycine treatment prevents NINJ1 clustering thereby preserving cellular integrity. By identifying NINJ1 as a glycine target, our data help resolve a long-standing mechanism of glycine cytoprotection. This new understanding will inform the development of cell preservation strategies to counter pathologic lytic cell death pathways. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/471765v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b9c3d9org.highwire.dtl.DTLVardef@1e903f0org.highwire.dtl.DTLVardef@1018498org.highwire.dtl.DTLVardef@a819b9_HPS_FORMAT_FIGEXP M_FIG C_FIG SummaryGlycine is known to protect cells against plasma membrane rupture from diverse types of tissue injury by an unknown mechanism. The authors demonstrate that NINJ1, a newly identified executioner of plasma membrane rupture across lytic cell death pathways, is a glycine target and resolve a longstanding mechanism of glycine cytoprotection.

cell biology↗