Tetrandrine regulates NAADP-mediated calcium signaling through a LIMP-2-dependent and sphingosine-mediated mechanism

Abstract

Tetrandrine (Tet) is a potent inhibitor of Ebola virus replication by blocking NAADP-dependent calcium release through endolysosomal two-pore channels (TPCs) and a moderately potent anti-tumor agent. Using a clickable photoaffinity probe, we identify lysosomal integral membrane protein-2 (LIMP-2) as a direct target of Tet and a key regulator of this calcium signaling. Tet binds LIMP-2's ectodomain, inhibiting lysosomal cholesterol and sphingosine transport, which alters lipid metabolism. Tet treatment and LIMP-2 depletion inhibit NAADP-dependent calcium release, reversible by removing lysosomal cholesterol and sphingosine. Sphingosine triggers lysosomal calcium release via TPCs and restores this signaling in Tet-treated or LIMP-2-deficient cells, revealing a LIMP-2-regulated, sphingosine-dependent lysosomal calcium pathway. At higher doses, Tet induces apoptosis through unfolded protein response activation independently of LIMP-2. These findings highlight Tet as a LIMP-2 inhibitor, elucidate its role in calcium signaling and cell death, and suggest therapeutic potential for Tet and LIMP-2 inhibitors in antiviral treatments.

Fig. 1. Development of alkynyl diazirine derivative of Tet (AD-Tet) as a probe of Tet.

Chemical structure of (A) tetrandrine (Tet), and (B) alkynyl diazirine derivative of tetrandrine (AD-Tet). C Schematic illustration of photoaffinity labeling and click reaction procedures for elucidation of AD-Tet cellular localization and target. Created in BioRender. D Confocal microscopic images of HeLa cells subjected to in situ PAL of AD-Tet, followed by click reaction using BODIPY-azide and DAPI staining. Scale bar, 20 μM. E Left, confocal microscopic images of HeLa cells labeled with AD-Tet, using increasing amounts of Tet as competitor. Scale bar, 20 μM. Right, quantification of cell fluorescence in 80 randomly chosen cells under each treatment condition. Red and black bar represents median and quartiles, respectively. ***, p < 0.001 compares to control (AD-Tet: Tet, 1:0), by one way ANOVA with Tukey multiple comparison test. F Left, confocal microscopic images of GFP-LC3 puncta in HeLa cells induced by the indicated compound for 12 h. Scale bar, 20 μM. Right, Quantification of cellular fluorescence puncta. 30 randomly chosen cells under each treatment were measured. Red and black bar represents median and quartiles respectively. ***, p < 0.001 compares to DMSO, by one way ANOVA with Tukey multiple comparison test. G Co-localization analysis of AD-Tet (ex: 594nm, em: 633nm) and GFP-LC3 (ex: 488nm, em: 512nm). HeLa cells were treated with AD-Tet (5 μM, 12 h), followed by PAL and click chemistry using Alexa Fluor 647-azide. Scale bar, 20 μM. H Confocal microscopic images of HeLa cells showing co-localization of AD-Tet with makers of subcellular organelles. AD-Tet was subjected to PAL and click chemistry using BODIPY-azide. Endoplasmic reticulum (ER), mitochondria, early endosomes, late endosomes, and lysosomes was visualized by immunofluorescence staining of the respective maker. Pearson’s correlation of AD-Tet with each marker is shown on the right. Scale bar, 20 μM. I N-SIM super-resolution microscopic imaging of AD-Tet and LAMP1 in HeLa cells. Scale Bar, 10 μM. For (D), (H), (I), experiment was repeated twice with consistent result. Source data are provided in the Source Data file.

Fig. 2. Identification of LIMP-2 as a putative target of Tet.

A Schematics of the procedures for AD-Tet target identification using SILAC-MS. In forward SILAC, heavy isotope-labeled HeLa cells (Heavy AA) were treated with AD-Tet, and unlabeled HeLa cells (Light AA) were treated with AD-Tet, with Tet as competitor. Treatment was swapped in reverse SILAC. Created in BioRender. B Scatter plot of the proteins identified in SILAC-MS from reciprocal SILAC labeling of HeLa cells. Labels in red represent candidates that fall within the selection criteria (at least four unique peptides identified in both forward and reverse SILCA, and with SILAC ratio >3). C The list of 12 protein candidates identified by SILAC-MS. Lyso, lysosomes; ER, endoplasmic reticulum; Golgi, Golgi complex; Mito, mitochondria. D Violin plot showing fluorescence intensity of AD-Tet puncta in HeLa cells transfected with the indicated siRNA. Cells were transfected with the siRNA for 72 h. AD-Tet (5 μM) was added for 2 h, followed by PAL and click chemistry. For each siRNA, 50 randomly chosen cells from different fields were analysed. Red and black bar represents median and quartiles respectively. ***, P < 0.001 by one-way ANOVA with Tukey post hoc test. E Left, siLIMP-2- and siControl-transfected HeLa cells, and wild type and LIMP-2-KO HeLa cells were incubated with AD-Tet (5 μM) for 2 h, followed by PAL and click chemistry, and immunostaining using anti-LAMP1 antibodies. Right, violin plot of AD-Tet fluorescence in LAMP-1-positive compartment. For each condition, 50 randomly chosen cells were analysed. ***, p < 0.001 compares to siControl by two-tail unpaired T test. F Left, HeLa cells transfected with FLAG (Control) or FLAG-LIMP-2 vector were incubated with AD-Tet (5 μM) for 2 h, subjected to PAL and click chemistry, and immunostaining using anti-FLAG antibodies. Right, quantification of AD-Tet fluorescence in FLAG-positive compartment. For each condition, 50 randomly chosen cells were analysed. ***, p < 0.001 compares to control, by two-tailed unpaired t-tests. Source data are provided in the Source Data file.

Fig. 3. Interaction between LIMP-2 and Tet.

A Upper panel, representative in-gel fluorescence scanning SDS PAGE of His₆-LIMP-2_35-430 /AD-Tet (5 μM) adducts, with or without PAL and click reaction, under neutral or acidic pH. Lower panel, coomassie blue staining of His₆-LIMP-2_35-430. B Upper panel, representative in-gel fluorescence scanning of His₆-LIMP-2_35-430/AD-Tet (5 μM) adducts (upper panel) in the presence of excessive Tet. Lower panel, coomassie blue staining (lower panel) of His₆-LIMP-2_35-430. C Representative confocal microscopic images showing AD-Tet fluorescence signals in HeLa cells in response to increasing amount of Tet, SG-005 or SG-094 as competitor. AD-Tet was co-treated with Tet, SG-005, or SG-094 at the indicate mole ratio for 2 h, and subjected to PAL and click chemistry using BODIPY-azide. Scale bar, 20 μM. D Upper panel, representative in-gel fluorescence scanning of His₆-LIMP-2_35-430/AD-Tet adducts, in the presence of increasing amount of SG-094 (upper panel) and SG-005 (lower panel) respectively. Lower panel, coomassie blue staining of His₆-LIMP-2_35-430. E Computational docking of Tet/cholesterol with the ectodomain of LIMP-2. Upper left, cholesterol binding pocket of LIMP-2 highlighting the residues involved in the interaction with cholesterol. Lower left, putative Tet-binding pocket of LIMP-2 highlighting the interacting residues. Right, overlay of cholesterol (green) and Tet (yellow) binding pocket, and the relative positions of the molecules with regard to the orientation of LIMP-2. Cell membrane and the N- and C- fragments: created in BioRender. F Upper panel, representative in-gel fluorescence scanning of His₆-LIMP-2_35-430/AD-Tet adducts, in the presence of increasing amount of cholesterol (upper panel). Lower panel, Coomassie blue staining of His₆-LIMP-2_35-430. G Upper panel, representative in-gel fluorescence scanning of His₆-LIMP-2_35-430/PhotoClick cholesterol adducts, in the presence of increasing amount of Tet. Lower panel, Coomassie blue staining of His₆-LIMP-2_35-430. H Cells were treated with ConA (0.1 μM) and AD-Tet (5 μM) for 2 h, subjected to PAL and click chemistry using BODIPY-azide, followed by immunostaining using anti-LAMP1 antibodies, and visualized by secondary antibodies labeled with Alexa Fluor 647. Scale bar, 20 μM. Source data are provided in the Source Data file.

Fig. 4. Tet altered cellular lipid metabolisms.

A Expression of β-GCase and LIMP-2 in wild type (WT), siControl- and siLIMP-2 transfected, DMSO-treated, and Tet-treated (5 μM) HeLa cells. Cells were transfected with the corresponding siRNA, or treated with Tet for 18 h. B Quantification of cholesterol level in HeLa cells treated with DMSO or Tet (5 μM) for 12 h. Data (N = 4) were expressed as mean +/- SEM. * p = 0.0168, by two-tailed unpaired t-tests. C Coefficients versus VIP plot obtained from OPLS-DA modeling in positive and negative ESI mode. Features with VIP value greater than 1.50 are highlighted in red. D Heat map showing differentially regulated metabolites in DMSO- and Tet-treated (5 μM) HeLa cells. Cells were treated with DMSO (C) or Tet (T) (5 μM) for 12 h before analysis. For each treatment, four independent preparations (C1-C4, T1-T4) were analysed. E Relative signal intensity of sphingosine in DMSO and Tet-treated HeLa cells. Data (N = 4) were expressed as mean +/- SEM. ** p = 0.005, by two-tailed unpaired t-tests. F Left, heat map of different classes of ceramide in cells treated with DMSO (C) and Tet (T) (5 μM). Right, relative signal intensity of sphingosine in HeLa cells treated with DMSO and Tet (5 μM). Data (N = 4) were expressed as mean +/- SEM. ***, p < 0.001, by two-tailed unpaired t-tests. Source data are provided in the Source Data file.

Fig. 5. Tet differentially regulates cell cholesterol homeostasis and cell death.

A Upper, LIMP-2 expression in HeLa cells under different treatment. Bottom, Quantification of filipin signal in LAMP1-positive vesicles under different treatment. Cells were transfected with siRNA (72 h), or treated with Tet or AD-Tet (18 h), before subjected to filipin staining and immunofluorescence staining using LAMP-1 antibodies. 30 randomly chosen cells were quantified for filipin level in LAMP-1-positive compartments. ***, p < 0.001 by one-way ANOVA with Tukey post hoc test. B Upper, Illustration of experiment measuring lysosomal cholesterol egress. Created in BioRender. Lower, quantification of BD-chol level in Lysotracker-positive compartments of HeLa cells pre-loaded with BD-chol/Dil-LDL in cholesterol-free medium. Cells were replenished with fresh medium and subjected to Lysotracker staining at regular intervals. Data (N = 3) were expressed as mean +/- SEM. * p= 0.0159 (Tet, 5 μM), and * p= 0.0170, ** p= 0.0020 (siLIMP-2), compared with T=0, by two-way ANOVA with Tukey post hoc test. C SREBP2 and LIMP-2 expression in HeLa cells subjected to the indicated treatment. n-SREBP2, cleaved SREBP2. Cells transfected with siRNA were analysed at 72 h post-transfection. Cells treated with Tet / AD-Tet were analysed after 18 h. D DEG comparison (> 2-fold, q-value<0.05) of HeLa cells treated with DMSO versus Tet, or DMSO versus AD-Tet. E, F GO analysis using DEGs in HeLa cells induced by 5 μM (E) and 15 μM (F) of Tet and AD-Tet respectively. Arrows indicate cholesterol-related (E) and ER stress-related (F) process elicited by both compounds. G Western blots (upper panel) of Tet-treated and siLIMP-2-transfected HeLa cells showing activation of eIF2α (p-eIF2α), and RT-PCR analysis (lower panel) showing splicing of XBP1 gene. TG, Thapsigargin (500nM). H Western blots analysis of apoptotic marker (cleaved PARP) in HeLa cells treated with Tet (5, and 15 μM) for 72 h, or transfected with siLIMP-2. I FACS analysis showing apoptosis of HeLa cells treated with Tet (5 and 15 μM) for 72 h. Data (N = 3) were expressed as mean +/- SEM. ***, p < 0.001 by one-way ANOVA with Tukey post hoc test. Source data are provided in the Source Data file.

Fig. 6. Pharmacological activity of Tet in vivo.

A Serum cholesterol level in mice treated with Tet. Mice were fed by oral gavage daily with H₂O or Tet at the indicated dose, for 10 days consecutively. At day 0, 3, 5, and 10, serum were taken for cholesterol measurement. Data (N = 5 in each group) were expressed as mean +/- SEM. ** p = 0.003, *** p < 0.001, by two way ANOVA with Tukey post hoc test. B Serum LDL level in mice treated with Tet. Serum obtained from mice (n = 5 in each group) at day 10 after feeding with Tet were subjected to LDL measurement. Data were expressed as mean +/- SEM. * p= 0.02, ** p = 0.005, by one way ANOVA with Tukey post hoc test. C Expression of n-SREBP2 in livers of the control (C) and Tet-treated (L: 60mg/kg/day; H: 150mg/kg/day) mice. Livers were harvested at the last day of treatment. D Macroscopic analysis of livers obtained from mice fed with Tet at day 10. E, F Relative level of cholesterol (E) and sphingosine (F) in the livers of mice from different treatments. Livers were harvested at the last day of treatment. Data were expressed as mean +/- SEM. E, ** P = 0.005 compare with H₂O. (F), ** P = 0.002 and *** P < 0.001 compare with H₂O, by one way ANOVA with Tukey post hoc test. G H&E staining of the livers from mice fed with Tet (60 and 150mg/kg/day) showing the presence of small lipid vesicles in hepatocytes. Arrow indicates bubbly cytoplasm of stellate cells. H Serum ALT and AST level from mice (n = 5 in each group) at day 10 after feeding with Tet. Data were expressed as mean +/- SEM. ** p = 0.0024, ***, p < 0.001, by one way ANOVA with Tukey post hoc test. Source data are provided in the Source Data file.

Fig. 7. Role of LIMP-2 in NAADP-mediated Ca²⁺ signaling.

A Quantification of Ca²⁺ release in HEK293 cells by the indicated treatment. Cells were pre-loaded with Calbryte 520, followed by captured of microscope images every 3s for 5mins. Thirty seconds after starting of imaging, cells were treated with NAADP (1 μM), liposomes (10μl), NAADP-liposomes (10μl), and TPC2-A1-N (10 μM), respectively. ***, P < 0.001 compared to untreated cells by one-way ANOVA with Tukey post hoc test. B Quantification of NAADP-induced Ca²⁺ release in the presence of the putative TPCs inhibitors. HEK293 cells were treated with DMSO, Tet (5 μM), AD-Tet (5 μM), SG-005 (5 μM), or SG-094 (5 μM), respectively, for 30minutes before the addition of NAADP-liposomes. ***, P < 0.001 compared to DMSO-treated cells by one-way ANOVA with Tukey post hoc test. C Quantification of NAADP-induced Ca²⁺ release in cells transfected with the indicated siRNAs and in LIMP-2 knockout (LIMP-2-KO) HEK293 cells. HEK293 cells were transfected with the indicated siRNA for 72 h before the experiment. ***, P < 0.001, by one-way ANOVA with Tukey post hoc test. D Relative filipin intensity in LAMP1-positive vesicles of HeLa cells treated with Tet or transfected with siLIMP-2, with or without MβCD (1mM) treatment for 12 h. Cells were then immune-stained with LAMP1 antibody, followed by filipin staining. In each condition, fluorescence intensity of 20 randomly chosen cells were measured. ** p = (DMSO vs. Tet, 5 μM), ** p = 0.008 (Tet, 5  μM vs. Tet, 5 μM+MβCD), *** p < 0.001, by one-way ANOVA with Tukey post hoc test. E, F Quantification of NAADP-induced Ca²⁺ release in HEK293 cells treated with Tet or transfected with siLIMP-2, with or without addition of MβCD (1mM) (E) or ectopic expression of NPC1 (F). Tet (5 μM) was added to the cells for 30min before Ca²⁺ measurement. MβCD was added at to the cells 6 h before measurement. ***, p < 0.001 by one-way ANOVA with Tukey post hoc test. Ca²⁺ level was presented as change in maximum fluorescence intensity [(F_max-F₀)/F₀]. In each condition, the fluorescence intensity of 40 randomly chosen cells were measured. Source data are provided in the Source Data file.

Fig. 8. Role of sphingosine in the regulation of NAADP-mediated Ca²⁺ signaling.

A Quantification of NAADP-induced Ca²⁺ release in HEK293 cells treated with NAADP-liposomes or sphingosine at the indicated concentration. NAADP-liposomes or sphingosine was added to the cells immediately before Ca²⁺ measurement. ***, p < 0.001 by one-way ANOVA with Tukey post hoc test. B Representative images of calcium signals induced by NAADP-liposomes and sphingosine. C Quantification of sphingosine-induced Ca²⁺ release in HEK293 cells transfected with TPC siRNAs or treated with sphingosine inhibitor PF543. Cells were transfected with the indicated siRNA for 72 h before the experiment. PF543 (10 μM) was added to the cells 1 h before the experiment. Sphingosine (Sph) was added to the cells at the indicated time (arrow) during the measurement immediately before the measurement. ***, P < 0.001 compared to DMSO-treated cells by one-way ANOVA with Tukey post hoc test. D, E Quantification of NAADP- and sphingosine-induced Ca²⁺ release in HEK293 cells treated with Tet or transfected with siLIMP-2. In Tet treatment group, cells were pretreated with Tet (5 μM) for 30min before the experiment. In the siLIMP-2 transfection group, cells were transfected with siLIMP2 for 72 h before the experiment. Arrow indicates the addition of NAADP-liposomes (NAADP) or/and sphingosine (Sph) (0.05 μM or 0.5 μM) to the cells. ***, P < 0.001 compared to DMSO-treated cells by one-way ANOVA with Tukey post hoc test. For all experiments involving Ca²⁺ measurement, Ca²⁺ level was presented as change in maximum fluorescence intensity [(F_max-F₀)/F₀]. In each condition, the fluorescence intensity of 40 randomly chosen cells were measured. Source data are provided in the Source Data file.

Fig. 9. Schematic illustration of the proposed mechanism of Tet-mediated inhibition of NAADP-mediated calcium signaling.

A Under control condition, cholesterol and sphingosine undergo lysosomal efflux mediated by LIMP-2 and TPC2, respectively. TPCs mediated Ca²⁺ efflux in response to NAADP. B In the presence of Tet, cholesterol, and sphingosine efflux through LIMP-2 was blocked, TPCs become insensitive to NAADP activation. C In the presence of Tet, the addition of sphingosine (low concentration) and NAADP, or sphingosine (high concentration) alone restored TPCs-mediated Ca²⁺ efflux. Created in BioRender.