Syntaxin11 Deficiency Inhibits CRAC Channel Priming to Suppress Cytotoxicity and Gene Expression in T Lymphocytes

Reviewer #1 (Public review):

Summary:

Patients with STX11 mutations develop familial hemophagocytic lymphohistiocytosis Type 4, a fatal immune disorder marked by defective T and NK cell cytotoxicity and cytokine storm. The conventional explanation attributes this to impaired cytotoxic granule release, but this has never fully accounted for the broader disease picture. This study proposes an alternative mechanism. The authors show that STX11 is required for store-operated calcium entry through ORAI1 channels, which are essential for both cytotoxic killing and NFAT-driven gene expression in T cells. In STX11-deficient cells, ORAI1 currents drop, NFAT nuclear translocation fails, IL-2 expression is suppressed, and degranulation is impaired. These defects are largely rescued by ionomycin or a constitutively active ORAI1 mutant, placing the primary lesion at calcium signaling rather than the fusion machinery. Mechanistically, STX11 binds the C-terminal tail of ORAI1 via its Habc domain and maintains ORAI1 in a state competent for productive assembly prior to STIM1-dependent gating, a step the authors call "priming."

Strengths:

The paper identifies a novel and disease-relevant role for STX11 in calcium channel regulation and raises the possibility of using channel agonists as a therapeutic strategy in the disease. The biochemical and functional data are of high quality and generally consistent with the interpretation. The proposal that a non-conventional syntaxin directly interacts with ion channels to prime its activation is novel and interesting.

Weaknesses:

For readers to appreciate the value of patient experiments derived from a single individual, the authors should quote prior studies showing that STX11 protein levels are abolished in all known human STX11 mutations. The priming model, while functionally well-supported, rests on indirect structural evidence, and the precise conformational transition involved remains to be defined. These are acknowledged limitations, but alternate mechanisms have not been explored and formally excluded. More direct evidence should be provided to exclude the possibility that STX11 could act as a conventional SNARE and sustain calcium fluxes by promoting the delivery of additional ORAI1 channels from vesicles.

Reviewer #2 (Public review):

Summary:

Vig's lab delineates a critical role for STX11 in CRAC channel function, particularly in the context of the fatal immune disorder familial hemophagocytic lymphohistiocytosis type 4 (FHL4). They demonstrate that Syntaxin 11 directly binds and regulates Orai1, and that STX11 depletion abolishes CRAC currents and downstream signaling. Loss of STX11 reduces IL2 gene expression and impairs degranulation, both of which are rescued by the constitutively active Orai1 mutant H134S, whereas a gain‑of‑function mutant targeting the C‑terminus fails to restore these defects. The authors conclude that STX11 primes Orai1 for optimal local assembly that is independent of STIM1 yet required for CRAC channel gating.

Strengths:

This study is firmly grounded in disease biology and demonstrates that STX11 downregulation leads to profound functional defects. Using a comprehensive suite of methods and analyses, the authors interrogate the co-regulation of STX11 and Orai1 and present a near-complete view of STX11's modulatory role in CRAC channel function and downstream signaling pathways. The figures are clear, and the statistical analyses are rigorous and convincing.

Weaknesses:

The authors conclude that Syntaxin 11 directly binds Orai1. This conclusion is well supported by a multifaceted approach, including co-immunoprecipitation (co-IP), molecular dynamics simulations, co-localization/FRET assays, and targeted mutational analysis-all of which are thoroughly executed. While the interaction appears reasonably strong in co-IP experiments, the STX11-Orai1 interaction is comparatively weaker in pull-down assays, which the authors attribute to instability of the purified His-STX11 protein. A remaining gap is direct evidence of interaction in live cells; this is understandably challenging given that fluorescent tagging of STX11 is not feasible. Fully resolving this question lies beyond the scope of the present study and will require more advanced approaches to capture STX11 binding dynamics.

Author response:

eLife Assessment

This study reports a novel function for syntaxin 11, a specialized SNARE protein critical for the immune system whose mutations cause familial hemophagocytic lymphohistiocytosis type 4. The data convincingly show that depletion of STX11 impairs store-operated calcium entry in Jurkat T cells and that this defect is recapitulated in primary cells from a patient suffering from the disease; the authors further show that the syntaxin interacts with the pore subunit of the ORAI1 channel and propose that it primes the channel by promoting the assembly of multimers before activation by its endogenous ligand, the ER Ca2+ sensing protein STIM1. This is a conceptually important claim that challenges the prevailing view that all structural transitions in ORAI1 are STIM-driven. The data are high-quality and broadly consistent with the interpretation, but alternative mechanisms for the defects are not considered; additional work should rule out vesicular trafficking, discuss other mechanisms, and address methodological issues.

We thank the editor and reviewers for assessing our work. Although significant amount of data in this paper already rule out any potential defects in the vesicular trafficking of Orai1 in cells lacking STX11, we will still include the additional suggested experiments. In the revised version, we will include the various experiments that we had already performed to measure vesicular trafficking and ER-PM junctions in STX11 depleted cells. We will discuss any remaining alternate explanations, include missing methods, quantifications and calibrations, where applicable, and provide response to each of the reviewer’s comments.

Public Reviews:

Reviewer #1 (Public review):

Weaknesses:

For readers to appreciate the value of patient experiments derived from a single individual, the authors should quote prior studies showing that STX11 protein levels are abolished in all known human STX11 mutations. The priming model, while functionally well-supported, rests on indirect structural evidence, and the precise conformational transition involved remains to be defined. These are acknowledged limitations, but alternate mechanisms have not been explored and formally excluded. More direct evidence should be provided to exclude the possibility that STX11 could act as a conventional SNARE and sustain calcium fluxes by promoting the delivery of additional ORAI1 channels from vesicles.

In the revised version, we will include references for the prior STX11 human mutations that have been biochemically characterized till date (Bryceson, Rudd et al. 2007);(Muller, Chiang et al. 2014);(Macartney, Weitzman et al. 2011);(Marsh, Satake et al. 2010). As the reviewer has correctly pointed out, the STX11 protein levels were almost completely abolished in these studies. Therefore, the prior mutations are essentially comparable to the frameshift mutation characterized in this study, in terms of the mechanisms underlying the phenotypic defects reported here versus earlier. From a mechanistic point of view, we believe that our data from even a single FHLH4 patient, where STX11 levels were severely depleted, and additional knockdown studies across three different cell lines, are representative of all STX11 patients that have been reported thus far.

Regarding the Reviewers’ concern that absence of STX11 as a conventional SNARE could affect Orai1 channel delivery from intracellular vesicles. We would like to point out the following:

(1) In Miao et al. 2013 (Miao, Miner et al. 2013), Figure 3C-D, we conclusively showed that expression of a dominant negative mutant of NSF, a non-redundant protein in vesicle trafficking, impaired vesicle trafficking but did not impair SOCE. This experiment had essentially ruled out a role for vesicle trafficking in SOCE. In the same paper, we had also shown that Orai1 levels in the PM do not increase post-store depletion (Figure 3, figure supplement 2).

(2) In this manuscript (Supplementary Figure 3B), we have shown that U2OS cells stably expressing Orai1-BBS-YFP have identical levels of Orai1 in the PM with and without STX11 depletion. This shows that the biosynthesis or delivery of Orai1 to the PM is not affected by STX11 depletion, another broadly classified member of the vesicle trafficking. The levels were also assessed in store-depleted U2OS cells but not included here because in Miao et al. 2013 we had already shown that levels of PM Orai1 are essentially equal in resting and store-depleted cells. In our revised submission, we will include the data from store-depleted cells in U2OS and also repeat this experiment in the other cell types used in this paper. In addition, in our revised submission, we will include three different vesicle trafficking assays performed in STX11 depleted cells.

(3) Most importantly, in Figure 7I-J of this manuscript, we showed that calcium influx from a constitutively active mutant Orai1 (Orai H134S) is identical between STX11 depleted and scramble control cells. If wildtype Orai1 was indeed stuck in vesicles in STX11 depleted cells, then how would H134S Orai1 be able to rescue the defect in SOCE? In fact, the Orai1 mutant calcium flux assays were done using a 20X water objective, to visualize and confirm whether the expression of mutant and WT Orai1 was comparable in the PM. We will include the quantification of PM levels of Orai1 mutants w.r.t WT Orai1 in the revised paper.

(4) We have generated and been using HEK293, U2OS and Jurkat cell lines that stably express fluorescently tagged Orai1 for most of our experiments (Miao, Miner et al. 2013); (Li, Miao et al. 2016);(Ramanagoudr-Bhojappa, Miao et al. 2021). In each case, we have never observed Orai1 in intracellular vesicles with or without store depletion. In all cases, it is constitutively and stably expressed in the PM.

In summary, significant amount of data in this paper already rule out any potential reduction in the PM levels of Orai1 in cells lacking STX11. We will still do the additional experiments suggested by the Reviewer 1.

Regarding STX11 induced precise conformational transition, we are trying to setup collaborations with scientists who might be able to visualize this in vivo.

The readers should note that purification of isolated pore subunits of ion channels followed by crystallization or expression in membranes for cryo-EM is currently considered a gold standard in the analysis of ion channel pore subunits. However, we have shown that ion channels are dynamic macromolecular complexes, in vivo (Li, Miao et al. 2016), where synaptic proteins dynamically bind to induce conformational changes and affect their stoichiometry (Li, Miao et al. 2016). Please also see (Chorev, Baker et al. 2018) and (Dorwart, Wray et al. 2010). More advanced in vivo approaches therefore need to be developed to enable visualization of the dynamics of ion channel macromolecular complexes in the native environment. In the absence of such approaches, the structural insights obtained from detergent purified subunits will remain incomplete and biased.

Reviewer #2 (Public review):

Weaknesses:

The authors conclude that Syntaxin 11 directly binds Orai1. This conclusion is well supported by a multifaceted approach, including co-immunoprecipitation (co-IP), molecular dynamics simulations, co-localization/FRET assays, and targeted mutational analysis-all of which are thoroughly executed. While the interaction appears reasonably strong in co-IP experiments, the STX11-Orai1 interaction is comparatively weaker in pull-down assays, which the authors attribute to instability of the purified His-STX11 protein. A remaining gap is direct evidence of interaction in live cells; this is understandably challenging given that fluorescent tagging of STX11 is not feasible. Fully resolving this question lies beyond the scope of the present study and will require more advanced approaches to capture STX11 binding dynamics.

We thank the reviewer for acknowledging that the above studies will require standardization of advanced techniques which are beyond the scope of the present study. We plan to continue developing methods that will allow us to visualize the binding and unbinding of STX11 to Orai1 in vivo.

References:

Bryceson, Y. T., E. Rudd, C. Zheng, J. Edner, D. Ma, S. M. Wood, A. G. Bechensteen, J. J. Boelens, T. Celkan, R. A. Farah, K. Hultenby, J. Winiarski, P. A. Roche, M. Nordenskjold, J. I. Henter, E. O. Long and H. G. Ljunggren (2007). "Defective cytotoxic lymphocyte degranulation in syntaxin-11 deficient familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients." Blood 110(6): 1906-1915.

Chorev, D. S., L. A. Baker, D. Wu, V. Beilsten-Edmands, S. L. Rouse, T. Zeev-Ben-Mordehai, C. Jiko, F. Samsudin, C. Gerle, S. Khalid, A. G. Stewart, S. J. Matthews, K. Grunewald and C. V. Robinson (2018). "Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry." Science 362(6416): 829-834.

Dorwart, M. R., R. Wray, C. A. Brautigam, Y. Jiang and P. Blount (2010). "S. aureus MscL is a pentamer in vivo but of variable stoichiometries in vitro: implications for detergent-solubilized membrane proteins." PLoS Biol 8(12): e1000555.

Li, P., Y. Miao, A. Dani and M. Vig (2016). "alpha-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels." Mol Biol Cell 27(16): 2542-2553.

Macartney, C. A., S. Weitzman, S. M. Wood, D. Bansal, M. Steele, M. Meeths, M. Abdelhaleem and Y. T. Bryceson (2011). "Unusual functional manifestations of a novel STX11 frameshift mutation in two infants with familial hemophagocytic lymphohistiocytosis type 4 (FHL4)." Pediatr Blood Cancer 56(4): 654-657.

Marsh, R. A., N. Satake, J. Biroschak, T. Jacobs, J. Johnson, M. B. Jordan, J. J. Bleesing, A. H. Filipovich and K. Zhang (2010). "STX11 mutations and clinical phenotypes of familial hemophagocytic lymphohistiocytosis in North America." Pediatr Blood Cancer 55(1): 134-140.

Miao, Y., C. Miner, L. Zhang, P. I. Hanson, A. Dani and M. Vig (2013). "An essential and NSF independent role for alpha-SNAP in store-operated calcium entry." Elife 2: e00802.

Muller, M. L., S. C. Chiang, M. Meeths, B. Tesi, M. Entesarian, D. Nilsson, S. M. Wood, M. Nordenskjold, J. I. Henter, A. Naqvi and Y. T. Bryceson (2014). "An N-Terminal Missense Mutation in STX11 Causative of FHL4 Abrogates Syntaxin-11 Binding to Munc18-2." Front Immunol 4: 515.

Ramanagoudr-Bhojappa, R., Y. Miao and M. Vig (2021). "High affinity associations with alpha-SNAP enable calcium entry via Orai1 channels." PLoS One 16(10): e0258670.