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Link to original content: http://pubmed.ncbi.nlm.nih.gov/39484379/
Syntaxin11 Deficiency Inhibits CRAC Channel Priming To Suppress Cytotoxicity And Gene Expression In FHLH4 Patient T Lymphocytes - PubMed Skip to main page content
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[Preprint]. 2024 Oct 25:2024.10.25.620144.
doi: 10.1101/2024.10.25.620144.

Syntaxin11 Deficiency Inhibits CRAC Channel Priming To Suppress Cytotoxicity And Gene Expression In FHLH4 Patient T Lymphocytes

Sritama Datta  1 , Abhikarsh Gupta  1 , Kunal Mukesh Jagetiya  1 , Vikas Tiwari  2 , Megumi Yamashita  3 , Sandra Ammann  4   5 , Mohammad Shahrooei  6 , Atharva Rahul Yande  1 , Ramanathan Sowdhamini  2 , Adish Dani  1 , Murali Prakriya  3 , Monika Vig  1
Affiliations

Syntaxin11 Deficiency Inhibits CRAC Channel Priming To Suppress Cytotoxicity And Gene Expression In FHLH4 Patient T Lymphocytes

Sritama Datta et al. bioRxiv. .

Abstract

CRAC channels enable calcium entry from the extracellular space in response to a variety of stimuli and are crucial for gene expression and granule exocytosis in lymphocytes. Here we find that Syntaxin11, a Q-SNARE, associated with FHLH4 disease in human patients, directly binds Orai1, the pore forming subunit of CRAC channels. Syntaxin11 depletion strongly inhibited SOCE, CRAC currents, IL-2 expression and cytotoxicity in cell lines and FHLH4 patient T lymphocytes. Constitutively active H134 Orai1 mutant completely reconstituted calcium entry in Syntaxin11 depleted cells and the defects of granule exocytosis as well as gene expression could be bypassed by ionomycin induced calcium influx in FHLH4 T lymphocytes. Our data reveal a Syntaxin11 induced pre-activation state of Orai which is necessary for its subsequent coupling and gating by the endoplasmic reticulum resident Stim protein. We propose that ion channel regulation by specific SNAREs is a primary and conserved function which may have preceded their role in vesicle fusion.

Keywords: CRAC; CTL; FHLH4; Orai; SNAP; SNARE; SOCE; Stim; Syntaxin11; T lymphocytes; autoimmunity; cytotoxicity; ion channels.

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Figures

Figure 1:
Figure 1:. STX11 is required for SOCE.
(A-F) RNAi mediated depletion of STX11 reduces SOCE. Measurement of thapsigargin (TG) induced SOCE in various cell lines after shRNA mediated depletion of STX11. The traces in panels A,C,E show representative average single cell Fura-2 calcium imaging assays. Bars in panels B,D,F show mean % SOCE ± SE from three independent experiments each where mean SOCE from scramble (scr) shRNA treated group in each experiment was set at 100% and the relative response of STX11 shRNA treated groups was calculated respectively. *P<0.05; **P<0.01; ***P<0.001 using two-tailed Student’s t test. (G) Representative Fura-2 calcium imaging assay showing reconstitution of SOCE in STX11 depleted Jurkat T cells by ectopic expression of STX11. Black (scr shRNA), red (STX11 shRNA), green (STX11 shRNA with STX11 expression). N=2 (H) Ectopic expression of STX11 enhances SOCE. Representative Fura-2 calcium imaging assay showing SOCE in HEK293 cells expressing STX11 (red) or empty vector (EV) (black). (N=3) (I) STX11 mediated enhancement of SOCE is dependent on Orai1. A representative Fura 2 calcium imaging assay showing measurement of thapsigargin (TG) induced SOCE in HEK293 cells where Orai1 expression was depleted using shRNA and STX11 was over-expressed. (N=2)
Figure 2:
Figure 2:. STX11 depletion suppresses ICRAC, downstream signaling and gene expression in Jurkat T cells.
ICRAC was recorded from Jurkat T-cells in the whole-cell recording configuration in 20 mM extracellular Ca2+ Ringer’s solution. ICRAC was induced by passive depletion of intracellular Ca2+ stores by dialyzing 8 mM BAPTA into the cell via the patch-pipette. (A) Representative current at −100 mV in Jurkat T cell transfected with scr shRNA construct. The current is blocked by extracellular La3+ (100 μM) and replacing the 20 mM Ca2+ Ringer’s solution with a divalent free solution (DVF) evokes a large Na+ current which depotentiates over tens of seconds. The current-voltage (I-V) relationship of the Ca2+ and DVF currents are shown on the right. (B) ICRAC from a Jurkat T cell transfected with STX11 shRNA. Both Ca2+ and Na+ current amplitudes are reduced relative to control cells. The I-V relationships (right plots) show no change in ion selectivity. (C-D) Summary of the current amplitudes of Ca2+ and Na+ currents and current reversal potentials in scr and STX11 knockdown cells. (E&F) Estimation of nuclear translocation of NFAT. (E) Western Blot showing nuclear translocation of NFAT in Jurkat T cells treated with scr or STX11 shRNA for 4 days and stimulated with PMA+TG for 30min prior to the preparation of nuclear and cytoplasmic extracts. (N=3) (F) Representative confocal images of Jurkat T cells treated with scr or STX11 shRNA for 4 days and stimulated with 10ug/ml anti-CD3 for 1 hour. Following stimulation, cells were fixed, permeabilized and stained using anti-NFAT primary antibody, followed by donkey anti-rabbit AF647 secondary antibody, and counter-stained with DAPI to mark the nuclei. (N=2) (G) Box and whisker plot showing percent nuclear NFAT in Jurkat T cells quantified from 40-50 cells populating 10 randomly chosen fields per group in (F). Boundaries of the box plots represent 25th and 75th percentile values, horizontal line represents mean, white circle represents median and whiskers denote the outliers. (H) Quantitative PCR to assess IL-2 transcription in anti-CD3 stimulated Jurkat T cells. Jurkat T cells were treated with scr or STX11 shRNA for 4 days and stimulated with 5ug/ml anti-CD3 for 3 hours. Total RNA was extracted from cells and subjected to QPCR analysis using Taqman probes for IL-2 and beta-actin. The bars show relative IL-2 mRNA expression levels with the scr shRNA treated group set at 100%. Shown here are mean ± SE. (N=3) (F-H) *P<0.05; **P<0.01; ***P<0.001 using two-tailed Student’s t test.
Figure 3:
Figure 3:. STX11 directly binds resting Orai1 in the plasma membrane.
(A) STX11 localizes in the plasma membrane. Representative confocal images of HEK293 cells expressing internal-HA-tagged STX11, stained using anti-HA antibody followed by alpaca VHH anti-rabbit secondary nanobody. Scale bar 15um. (N=3) (B) Co-localization of STX11 and Orai1 in the plasma membrane. Representative confocal images of HEK293 cells expressing Orai1-YFP and STX11, stained using anti-STX11 antibody followed by donkey anti-rabbit secondary antibody. Scale bar 10um. (N=3). (C&D) Co-immunoprecipitation of STX11 with Orai1. Whole cell lysates of resting and store-depleted HEK293 cells expressing either Flag-Orai1 and STX11 (C) or STX11 and Orai1-Myc-His (D) were subjected to immunoprecipitation and Western Blot using anti-Myc, anti-Flag or anti-STX11 antibodies, as indicated. (N=3) (E) Schematic showing key domains of STX11 and Orai1 used for in vitro pulldown assays. (F) Pull-down assay showing in vitro binding of His-tagged full length STX11 to MBP-tagged cytosolic domains of Orai1. MBP-tagged Orai1 fragments, expressed in E. Coli and immobilized on the amylose resin, were incubated with purified His-tagged STX11 protein. Post incubation, beads were washed, boiled and subjected to Western Blot analysis using anti-STX11 antibody. (Top panel) Ponceau S staining showing the input of MBP alone or MBP-tagged fragments. (Bottom panel) Western Blot using anti-STX11 antibody. (N=3) (G) Pull-down assay showing in vitro binding of His-tagged Habc domain of STX11 to MBP-tagged Orai1 N- and C-termini performed as described above. (Top panel) Ponceau S staining showing the input of MBP alone or MBP-tagged Orai1 cytosolic tails. (Bottom panel) Western Blot using anti-His antibody. (N=3). (H-I) Representative structure of STX11 Habc and Orai1 C-terminus complex after MD simulation (H) Sphere representation of STX11 Habc (green) and Orai1 C-terminus (cyan) complex. The N-termini are highlighted in blue, and C-termini are highlighted in red (I) Cartoon representation of STX11-Habc (green) and Orai1 C-terminus (cyan) complex highlighting the CA of terminal residues as spheres (J) Protein-protein interactions between the STX11 Habc and the Orai1 C-terminus.
Figure 4:
Figure 4:. STX11 depletion compromises the functional assembly of Orai1 with Stim1 in ER-PM junctions.
(A-B) Representative confocal images of resting (A) and store-depleted (B) scr and STX11 shRNA treated HEK293 cells expressing N-terminal CFP tagged Orai1 (CFP-Orai1) and C-terminal YFP tagged Stim1 (Stim1-YFP). (C-H) Box and whisker plots showing (C) Quantification of Stim1-YFP intensities inside Stim:Orai clusters of scr and STX11 shRNA treated HEK293 cells, ~8 min post store-depletion. (D-E) Quantification of CFP-Orai1 intensities inside (D) and outside (E) Stim1-YFP clusters of scr and STX11 shRNA treated cells, ~8 minute post store-depletion. N=3. Boundaries of the box plots represent 25th and 75th percentile values, horizontal line represents mean, white circle median and whiskers denote the outliers. (F) Quantification of total Orai1 levels in the plasma membrane of STX11 depleted cells. U2OS cells stably expressing Orai1-BBS-YFP were transduced with scr (black) or STX11 (red) shRNA, stimulated with 1uM TG, incubated with alpha-bungarotoxin alexa 647 (BTX-A647) and washed. BTX binding to surface Orai1 was measured using FACS, where binding to wildtype HEK293 cells was used as control. (N=3). (G-J) Quantification of C-term tagged Orai1 (Orai1-YFP) and N-term tagged Stim1 (CFP-Stim1) inside puncta in control and STX11 depleted cells, ~6 minute post store-depletion. (G-H) Quantification of mean intensities of Orai1-YFP (G) and CFP-Stim1 (H) inside puncta. n=8. Quantification of mean area of Orai1-YFP (I) and CFP-Stim1 (J) puncta. The continuous and dotted lines represent mean and median respectively. *P<0.05, **P<0.01 and ***P<0.001 using two-tailed Student’s t test. (K) Representative Fura-2 calcium imaging assay to measure Thapsigargin induced SOCE in Orai1-YFP and CFP-Stim1 expressing HEK293 cells treated with scr or STX11 shRNA.
Figure 5:
Figure 5:. STX11 binding to resting Orai1 allows successful gating by Stim1.
(A) Representative Fura-2 calcium imaging assay to measure constitutive calcium influx in Orai1-S-S-GFP expressing control or STX11 depleted HEK293 cells. Cells were imaged in Ringer’s buffer containing 0mM followed by 2mM extracellular Ca2+. (B) Box and whisker plot showing quantification of constitutive calcium influx across experiments as shown in (A). N=3. (C) Representative images of YFP-CAD localization in Orai1-CFP expressing, control and STX11 depleted HEK293 cells. (D) Quantification of YFP-CAD in the plasma membrane of Orai1-CFP expressing HEK293 cells represented as % of total YFP-CAD in respective cells. (E) Representative Fura-2 calcium imaging assay to measure constitutive calcium influx in Orai1-CFP and YFP-CAD expressing control or STX11 depleted HEK293 cells. (F-G) Quantification of constitutive calcium influx in Orai1-CFP and YFP-CAD expressing cells described in E, at 0mM (F) and 2mM (G) extracellular Ca2+. N=3. (H) Representative Fura-2 calcium imaging assay to measure constitutive calcium influx in Orai1-H134S mutant expressing control and STX11 depleted HEK293 cells as described in (A). (I) Quantification of constitutive calcium influx across experiments as shown in (H). N=3. Boundaries of the box plots in B,D,F,G&I represent 25th and 75th percentile values, horizontal line represents mean, white circle median and whiskers denote the outliers. *P<0.05, **P<0.01 and ***P<0.001 in (C-D,F-G,I) using two-tailed Student’s t test.
Figure 6:
Figure 6:. Ionomycin rescues the cytotoxicity and gene expression defects in STX11 deficient FHLH4 patient T lymphocytes.
(A) Sanger sequencing of the patient DNA showing deletion of a single of Adenine at the 752nd position and the resulting frameshift. (B) Western Blot of whole cell lysates prepared from healthy human control and FHLH4 patient PBMCs showing the relative molecular weight and abundance of the wildtype and mutant STX11 bands. (C) Fura-2 calcium imaging assay measuring anti-CD3 induced SOCE in wildtype and FHLH4 PBMCs. (D) Fura-2 calcium imaging assay measuring SOCE in wildtype and FHLH4 PBMCs expressing either empty vector (EV) or wildtype human STX11, respectively. (E) Representative Fura-2 calcium imaging assay measuring SOCE in wildtype PBMCs with or without ectopic expression of wildtype STX11. (F) Bar plot showing quantification of SOCE across multiple experiments in (E). N=3. (G) Granule release assay performed on the in vitro cultured healthy human control and FHLH4 patient CD8 T cells. PBMCs isolated from control and FHLH4 patient blood were stimulated with (2ug/ml) PHA for 48 hours. On the day of assay, cells were washed and either left unstimulated or restimulated with plate coated anti-CD3 + soluble anti-CD28 or PMA + Ionomycin in the presence of CD107a-PE antibody, stained with anti-CD8 antibody and analyzed. (N=2) (H) Quantification of the granule release assays as shown in (G). (I) Analysis of IL-2 expression in control and FHLH4 T cells. In vitro cultured healthy human control and FHLH4 patient T cells were rested and stimulated with either plate coated anti-CD3 + soluble anti-CD28 or PMA + Ionomycin for 6 hours. Total RNA was extracted and subjected to quantitative PCR analysis using Taqman probes for IL-2 and beta-actin in triplicates. *P<0.05; **P<0.01; ***P<0.001 using two-tailed Student’s t-test.
Figure 7:
Figure 7:. Hypothetical model showing Orai1 bound to STX11 for its priming.
Blue ribbons depict Orai1 in plasma membrane (PM). Green and Orange ribbons represent Habc and SNARE domains of STX11, respectively. Also shown are the C-terminal cysteines of STX11 which facilitate membrane attachment of the protein.
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References

    1. Clapham D.E. Calcium signaling. Cell 131, 1047–1058 (2007). - PubMed
    1. Vig M. & Kinet J.P. The long and arduous road to CRAC. Cell Calcium 42, 157–162 (2007). - PMC - PubMed
    1. Vig M. et al. Defective mast cell effector functions in mice lacking the CRACM1 pore subunit of store-operated calcium release-activated calcium channels. Nat Immunol 9, 89–96 (2008). - PMC - PubMed
    1. Vig M. & Kinet J.P. Calcium signaling in immune cells. Nat Immunol 10, 21–27 (2009). - PMC - PubMed
    1. Maul-Pavicic A. et al. ORAI1-mediated calcium influx is required for human cytotoxic lymphocyte degranulation and target cell lysis. Proc Natl Acad Sci U S A 108, 3324–3329 (2011). - PMC - PubMed

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