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  1. eLife assessment

    It's been widely known that the amino acid Glycine can work as a cytoprotectant and inhibit pyroptosis-associated plasma membrane rupture. However, a long-standing question has been: how does Glycine cytoprotection work? The authors observed that Glycine treatment phenocopied deficiency of NINJ1 (a recently reported cell surface molecule critical for plasma membrane rupture), and can inhibit aggregation of NINJ1. Understanding the intrinsic mechanism by which Glycine affects NINJ1 could provide a significant advance in the cell death field.

  2. Reviewer #1 (Public Review):

    In the submitted manuscript, the authors observed that Glycine treatment could phenocopy deficiency of NINJ1, a recently discovered cell surface molecule critical for plasma membrane rupture, and also inhibit the aggregation of NINJ1. However, whether Glycine directly inhibits NINJ1 was not examined, and thus, the manuscript falls short of having a significant impact in the field.

    Strengths of the manuscript:

    1. Timely. There is great interest in understanding the mechanism of plasma membrane rupture.
    2. The data provided using several mouse and human cell culture systems overall support the conclusion that Glycine targets NINJ1-mediated plasma membrane rupture (as the title says).

    However, most of the presented data is predictable from previous publications. Direct evidence of the mechanism by which NINJ1 is inhibited by Glycine, or in other words, NINJ1 as the direct target of Glycine, was not provided in this manuscript. It is therefore still possible that Glycine acts indirectly upstream of NINJ1. This possible indirect mechanism can be inferred from previous reports where other amino acids such as Serine also could inhibit cell lysis (reviewed in PMID: 27066896).

  3. Reviewer #2 (Public Review):

    In this manuscript, the authors investigated the mechanism by which glycine prevents cell membrane rupture. They found that deficiency of NINJ1 (the key executioner of plasma membrane rupture by forming oligomers) phenocopies the cytoprotection of glycine during lytic cell death, and glycine treatment inhibits the oligomerization of NINJ1. Based on these observations, they claimed that glycine executes its inhibitory effect on cell lysis by targeting and inactivating NINJ1. This study addresses an important subject, because how glycine prevents cell membrane rupture is not understood and the literature is full of the implausible conclusion that it works as an osmoprotectant and that pyroptotic cell rupture is secondary to osmotic changes in cells undergoing pyroptosis, even though the gasdermin pore is very large and should allow the free passage of ions and many small proteins.

  4. Reviewer #3 (Public Review):

    This manuscript investigates the basis for the cytoprotective effect of exogenous glycine, which has been known to limit cell lysis in response to various stimuli. The authors propose Ninjurin 1 (NINJ1) as a possible regulator or target of glycine-induced blockade of cell lysis, which is an attractive model, given the recently-described role of NINJ1 in inducing membrane rupture downstream of gasdermin cleavage in response to apoptotic and pyroptotic stimuli. The data that support the conclusion are that the authors report that glycine treatment phenocopies NINJ1 deficiency. They go on to conclude, using both native gel western electrophoresis and fluorescence microscopy to assay NINJ1 aggregation, that glycine treatment prevents higher order NINJ1 oligomerization. The authors test these observations in primary human and mouse cells as well as in human and murine macrophage cell lines. The analysis of the role of glycine in both human and murine cells is a strength of the work. This topic is of broad importance, as the mechanism and manner by which cells die impacts host defense against infection, cancer, and autoinflammatory disease. The mechanisms of terminal cell lysis remain surprisingly unclear as recent studies have found that gasdermin cleavage and oligomerization are not sufficient to mediate cell lysis and that cells can survive in the presence of functional gasdermin D pores. Previous studies have reported that glycine treatment limits the release of some cytoplasmic contents during the activation of pyroptosis, but does not affect the secretion of IL-1 cytokines. This property of glycine phenocopies NINJ1 deficiency, suggesting a possible link between the two. This work, therefore, has the potential to shed further light on the regulation of cell lysis, if the studies can be made more definitive with better quantification and more robust controls, which are currently missing for a large portion of the data.

    Overall, the area and topic being investigated are of broad interest. While the manuscript attempts to make inroads into how glycine functions as a cytoprotectant, in its current form, the manuscript does not provide definitive evidence that glycine functions through NINJ1, and the data that are currently provided require substantial development, including the addition of key controls and better quantification of microscopy in order for the authors to robustly make the conclusions that they would like to make.