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Neuroligin 3 (NLGN3) and neurexins (NRXNs) constitute a canonical transsynaptic cell-adhesion pair, which has been implicated in autism. In autism spectrum disorder (ASD) development of sociality can be impaired. However, the molecular mechanism underlying NLGN3-mediated social development is unclear. Here, we identify non-canonical interactions between NLGN3 and protein tyrosine phosphatase δ (PTPδ) splice variants, competing with NRXN binding. NLGN3-PTPδ complex structure revealed a splicing-dependent interaction mode and competition mechanism between PTPδ and NRXNs. Mice carrying a NLGN3 mutation that selectively impairs NLGN3-NRXN interaction show increased sociability, whereas mice where the NLGN3-PTPδ interaction is impaired exhibit impaired social behavior and enhanced motor learning, with imbalance in excitatory/inhibitory synaptic protein expressions, as reported in the Nlgn3 R451C autism model. At neuronal level, the autism-related Nlgn3 R451C mutation causes selective impairment in the non-canonical pathway. Our findings suggest that canonical and non-canonical NLGN3 pathways compete and regulate the development of sociality. Mutations of Neuroligin 3 (NLGN3) have been associated with autism spectrum disorder (ASD). Here, the authors identify a previously undescribed interaction between NLGN3 and a splice variant of protein tyrosine phosphatase δ (PTP δ) and its role in development of social behaviour in mice.

Leucine-rich repeat transmembrane neuronal proteins (LRRTMs) function as postsynaptic organizers that induce excitatory synapses. Neurexins (Nrxns) and heparan sulfate proteoglycans have been identified as presynaptic ligands for LRRTMs. Specifically, LRRTM1 and LRRTM2 bind to the Nrxn splice variant lacking an insert at the splice site 4 (S4). Here, we report the crystal structure of the Nrxn1β–LRRTM2 complex at 3.4 Å resolution. The Nrxn1β–LRRTM2 interface involves Ca 2+ -mediated interactions and overlaps with the Nrxn–neuroligin interface. Together with structure-based mutational analyses at the molecular and cellular levels, the present structural analysis unveils the mechanism of selective binding between Nrxn and LRRTM1/2 and its modulation by the S4 insertion of Nrxn. Leucine-rich repeat transmembrane neuronal proteins (LRRTMs) function as postsynaptic organizers that induce excitatory synapses. Here authors solve the crystal structure of LRRTM2 in complex with its ligand Nrxn1β and shed light on how selective binding of ligands to LRRTM1/2 is achieved.

Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event 1 – 7 . Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-1 8 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death. Structural, biochemical and mutagenesis studies indicate that, in dying cells, the membrane protein NINJ1 assembles into filaments, disrupting the cell membrane.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response 1 – 3 . The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein 4 – 8 , which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1 −/− macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1 –/– macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1 –/– mice were more susceptible than wild-type mice to infection with Citrobacter rodentium , which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event. The small transmembrane protein NINJ1 promotes plasma membrane rupture in lytic cell death associated with pyroptosis, necrosis and apoptosis.

Lytic cell death culminates in plasma membrane rupture, which releases large intracellular molecules to augment the inflammatory response. Plasma membrane rupture is mediated by the effector membrane protein ninjurin-1 (NINJ1) 1 , which polymerizes and ruptures the membrane via its hydrophilic face 1 , 2 , 3 – 4 . How NINJ1 is restrained under steady-state conditions to ensure cell survival remains unknown. Here we describe the molecular underpinnings of NINJ1 inhibition. Using cryogenic electron microscopy, we determined the structure of inactive-state mouse NINJ1 bound to the newly developed nanobody Nb538. Inactive NINJ1 forms a face-to-face homodimer by adopting a three-helix conformation with unkinked transmembrane helix 1 (TM1), in contrast to the four-helix TM1-kinked active conformation 2 , 3 – 4 . Accordingly, endogenous NINJ1 from primary macrophages is a dimer under steady-state conditions. Inactive dimers sequester the membrane rupture-inducing hydrophilic face of NINJ1 and occlude the binding site for kinked TM1 from neighbouring activated NINJ1 molecules. Mutagenesis studies in cells show that destabilization of inactive face-to-face dimers leads to NINJ1-mediated cell death, whereas stabilization of face-to-face dimers inhibits NINJ1 activity. Moreover, destabilizing mutations prompt spontaneous TM1 kink formation, a hallmark of NINJ1 activation. Collectively, our data demonstrate that dimeric NINJ1 is autoinhibited in trans to prevent unprovoked plasma membrane rupture and cell death. Structure-function studies reveal that the plasma membrane rupture protein NINJ1 homodimerizes through its hydrophilic membrane-rupturing face, thereby rendering NINJ1 inactive in the resting state.

The Fasciclin 1 (FAS1) domain is an ancient structural motif in extracellular proteins present in all kingdoms of life and particularly abundant in plants. The FAS1 domain accommodates multiple interaction surfaces, enabling it to bind different ligands. The frequently observed tandem FAS1 arrangement might both positively and negatively regulate ligand binding. Additional protein domains and post-translational modifications are partially conserved between different evolutionary clades. Human FAS1 family members are associated with multiple aspects of health and disease. At the cellular level, mammalian FAS1 proteins are implicated in extracellular matrix structure, cell to extracellular matrix and cell to cell adhesion, paracrine signaling, intracellular trafficking and endocytosis. Mammalian FAS1 proteins bind to the integrin family of receptors and to protein and carbohydrate components of the extracellular matrix. FAS1 protein encoding plant genes exert effects on cellulosic and non-cellulosic cell wall structure and cellular signaling but to establish the modes of action for any plant FAS1 protein still requires biochemical experimentation. In fungi, eubacteria and archaea, the differential presence of FAS1 proteins in closely related organisms and isolated biochemical data suggest functions in pathogenicity and symbiosis. The inter-kingdom comparison of FAS1 proteins suggests that molecular mechanisms mediating interactions between cells and their environment may have evolved at the earliest known stages of evolution.

Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1α, Nrxn1β, Nrxn2β, Nrxn3α, and Nrxn3β mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1α and Nrxn3α (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-α, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that α-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing.

Plasma membrane rupture (PMR), once considered a passive event in necrosis, is actively executed by ninjurin-1 (NINJ1). Although PMR is a well-documented phenomenon across animal species, its molecular mechanism and immunological function in early diverging jawed vertebrates remain unclear. Herein, we identify a functional homolog of NINJ1 in turbot. Ectopic expression of turbot NINJ1 effectively lyses the plasma membrane, leading to rapid PMR and massive release of cellular contents. Given that turbot NINJ1 has two transmembrane helices, it could distribute within membrane-bound organelles and the plasma membrane as PMR progresses. Co-expression with turbot gasdermin significantly amplifies NINJ1-mediated PMR, even when both proteins are present at non-cytotoxic levels. Structural analysis reveals the importance of its extracellular and transmembrane helices, with specific functional residues crucial for its PMR-inducing activity. Bacterial infection upregulates NINJ1 and activates pyroptosis signaling pathway, and provokes strong proinflammatory responses. Inhibiting NINJ1 improves fish survival, whereas blocking pyroptosis actually dampens host antimicrobial immunity. Collectively, the present findings delineate a mechanistically distinct role of NINJ1 in regulating programmed cell death in turbot, highlighting the complex and bifunctional nature of cell death in teleost immunity. Highlights • Turbot NINJ1 effectively lyses the plasma membrane and causes rapid release of intracellular contents. • Turbot NINJ1 synergistically cooperates with gasdermin to promote lytic cell death. • Functional residues in extracellular and transmembrane helices are essential for NINJ1-mediated plasma membrane lysis. • NINJ1 orchestrates antimicrobial immunity in conjunction with pyroptosis against bacterial infection.

Ninjurin1 (NINJ1) is initially identified as a nerve injury-induced adhesion molecule that facilitates axon growth. It is initially characterized to promote nerve regeneration and mediate the transendothelial transport of monocytes/macrophages associated with neuroinflammation. Recent evidence indicates that NINJ1 mediates plasma membrane rupture (PMR) in lytic cell death. The absence or inhibition of NINJ1 can delay PMR, thereby mitigating the spread of inflammation resulting from cell lysis and preventing the progression of various cell death-related pathologies, suggesting a conserved regulatory mechanism across these processes. Further research elucidated the structural basis and mechanism of NINJ1-mediated PMR. Although the role of NINJ1 in PMR is established, the identity of its activating factors and its implications in diseases remain to be fully explored. This review synthesizes current knowledge regarding the structural basis and mechanism of NINJ1-mediated PMR and discusses its significance and therapeutic targeting potential in inflammatory diseases, neurological disorders, cancer, and vascular injuries.

Human leukocyte antigen E (HLA-E) communicates cellular health to natural killer (NK) cells through presentation of peptides derived from the leader sequence of classical major histocompatibility complex class I (MHC-I), inhibiting NK cell activation and lysis of healthy cells. Besides this canonical role, HLA-E can also present peptides from pathogens such as Mycobacterium tuberculosis (Mtb) to T cells and can inhibit phagocytosis by engaging with LILRB1/2. To identify additional HLA-E binding surface molecules, we utilized a CRISPR/Cas9 activation screen with HLA-E tetramers, which identified Stabilin (STAB)1 and STAB2 as novel interactors. This interaction depended on the nature of the peptide/HLA-E complex, whereby high affinity peptides did not permit the interaction while low affinity peptides did. Functionally, expression of STAB1 or STAB2 on THP-1 monocytes increased phagocytic uptake of HLA-E coated microbeads. These results provide the first evidence of an interaction between Stabilin receptors and specific HLA-E conformations.