Journal: bioRxiv

Article Title: Sialin2 Functions as a Mammalian Nitrate Sensor to Sustain Mitochondrial Homeostasis

doi: 10.1101/2025.05.04.652104

Figure Lengend Snippet: Nitrate-Sialin2 signaling promotes LKB1-mitochondrial recruitment to activate AMPK phosphorylation a, Schematic workflow of phosphoproteomic analysis. b, Top enriched KEGG pathways in HEK293T cells treated with 4 mM nitrate or control vehicle for 4 h listed by the rank of P value based on DAVID analysis. Data represent three biological replicates per condition. c, IF staining images (left) and quantification (right) of pAMPK T172 in control and SLC17A5 knockout (sg SLC17A5 ) NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. d, Quantification of pAMPK T172 in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA), Sialin, and Sialin2. N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 8e. e, Schematic diagram of subcellular compartment-specific biosensor osABKAR. f, Quantification of FRET/CFP ratio of Mito-ABKAR in control and sg SLC17A5 NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 9d. g, h, IF staining images (upper, representative YFP images, lower, representative pseudocolor images of FRET/CFP ratio show the FRET response; g) and quantification (h) of FRET/CFP ratio of Mito-ABKAR in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA), Sialin, and Sialin2. N = 30 cells from representative experiments of three repeats. i, Quantification of FRET/CFP ratio of Lyso-ExRai-ABKAR in control and sg SLC17A5 NRK cells treated with metformin (Met, 10 mM) for 4 h. N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 9h. j, k, PLA of Sialin/Sialin2-LKB1 in NRK (j) or HEK293T (k) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images of HEK293T cells in Extended Data Fig. 10c. l, PLA of Sialin/Sialin2-CaMKK2 in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 10d. m, Quantification of FRET/CFP ratio of Mito-ABKAR in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 10e. n, o, IF staining images of LKB1 and MitoTracker in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA), Sialin, and Sialin2 (n). Colocalization quantified by Pearson’s correlation coefficient (o). N = 30 cells from representative experiments of three repeats. p, Immunoblot analysis of pAMPK T172 , AMPK, Flag, and Sialin2 in control and sg SLC17A5 HEK293T cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). Representative images of n = 3 independent experiments were shown. q, r, IF staining images (upper, representative YFP images, lower, representative pseudocolor images of FRET/CFP ratio show the FRET response; q) and quantification (r) of FRET/CFP ratio of Mito-ABKAR in control and sg SLC17A5 HEK293T cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). N = 30 cells from representative experiments of three repeats. s, Schematic illustration of nitrate enhances Sialin2-LKB1 interaction, promotes the recruitment of LKB1 to mitochondria, which in turn promotes the phosphorylation and activation of AMPK. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Monoclonal antibodies against CTSB (Santa Cruz Biotechnology, clone H-5, #sc- 365558), Tomm20 (Cell Signaling, clone D8T4N, #42406), LAMP1 (Invitrogen, clone LY1C6, #MA1-164), pAMPK T172 (Cell Signaling, clone 40H9, #2535), AMPK (Cell Signaling, clone D5A2, #5831), LKB1 (Santa Cruz Biotechnology, clone G-12, #sc- 374300), CAMKK2 (Cell Signaling, clone 6G9, #50049), Cox IV (Cell Signaling, clone 3E11, #4850), PGC1α (Santa Cruz Biotechnology, clone D-5, #sc-518025), HA- tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), GAPDH (Cell Signaling, clone 14C10, #2118), ACTB (Proteintech, clone 2D4H5, #66009), HSP90 (Cell Signaling, clone C45G5, #4877), and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), pAMPK T172 (Invitrogen, #PA5-37821), LKB1 (Proteintech, #10746), MT-ND5 (Proteintech, #55410), CYTB (Proteintech, #55090), MT-CO2 (Proteintech, #55070), ATP8 (Proteintech, #26723), TFAM (Proteintech, #22586), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Phospho-proteomics, Control, Staining, Knock-Out, Western Blot, Plasmid Preparation, Activation Assay

Journal: bioRxiv

Article Title: Sialin2 Functions as a Mammalian Nitrate Sensor to Sustain Mitochondrial Homeostasis

doi: 10.1101/2025.05.04.652104

Figure Lengend Snippet: Nitrate-Sialin2 signaling promotes mitochondrial biogenesis and functional homeostasis via LKB1-AMPK activation a, Immunoblot analysis of NT-PGC1α, TFAM, mitochondrial-encoded proteins (MT- ND5, CYTB, MT-CO2, and ATP8), and Sialin2 in control and sg SLC17A5 HEK293T cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. See full quantitation in Extended Data Fig. 12a–d. b, c, IF staining images (left) and quantification (right) of Cox IV (mitochondria; b) and JC-1 (mitochondrial membrane potential; c) in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. d, e, ATP production (d) and mitochondrial DNA (mtDNA) copy number (e) in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). N = 3. f, Mitochondrial ROS (mtROS) production in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Production normalized to control. N = 3 from three independent experiments, each in triplicate. g, Proliferation (%EdU positive cells) in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See EdU images in Extended Data Fig. 12m. h, Immunoblot analysis of NT-PGC1α, TFAM, mitochondrial-encoded proteins (MT- ND5, CYTB, MT-CO2, and ATP8), Flag, and Sialin2 in control and sg SLC17A5 HEK293T cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1- M (D194A). Representative images of n = 3 independent experiments were shown. See full quantitation in Extended Data Fig. 13a–d. i, j, IF staining images (left) and quantification (right) of Cox IV (i) and JC-1 (j) in control and sg SLC17A5 NRK cells reconstituted with vector, Flag-LKB1, Flag-LKB1- M, or Flag-LKB1-M (D194A). N = 30 cells from representative experiments of three repeats. k, l, ATP production (k) and mtDNA copy number (l) in control and sg SLC17A5 NRK cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). N = 3. m, mtROS production in control and sg SLC17A5 NRK cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). Production normalized to control. N = 3 from three independent experiments, each in triplicate. n, Proliferation (%EdU positive cells) in control and sg SLC17A5 NRK cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). N = 30 cells from representative experiments of three repeats. o, Schematic illustration of nitrate triggers CTSB-mediated cleavage of Sialin at residues K256/R257/I258 to generate Sialin2, which translocates to mitochondria, acts as the nitrate sensor, recruits LKB1 to activate mitochondrial AMPK phosphorylation, and drives mitochondrial biogenesis to sustain mitochondrial function and cellular homeostasis. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Monoclonal antibodies against CTSB (Santa Cruz Biotechnology, clone H-5, #sc- 365558), Tomm20 (Cell Signaling, clone D8T4N, #42406), LAMP1 (Invitrogen, clone LY1C6, #MA1-164), pAMPK T172 (Cell Signaling, clone 40H9, #2535), AMPK (Cell Signaling, clone D5A2, #5831), LKB1 (Santa Cruz Biotechnology, clone G-12, #sc- 374300), CAMKK2 (Cell Signaling, clone 6G9, #50049), Cox IV (Cell Signaling, clone 3E11, #4850), PGC1α (Santa Cruz Biotechnology, clone D-5, #sc-518025), HA- tag (Cell Signaling, clone C29F4, #3724; clone 6E2, #2367), Flag-tag (Cell Signaling, clone D6W5B, #14793; clone 9A3, #8146), GAPDH (Cell Signaling, clone 14C10, #2118), ACTB (Proteintech, clone 2D4H5, #66009), HSP90 (Cell Signaling, clone C45G5, #4877), and polyclonal antibodies against Sialin (Invitrogen, #PA5-30517), pAMPK T172 (Invitrogen, #PA5-37821), LKB1 (Proteintech, #10746), MT-ND5 (Proteintech, #55410), CYTB (Proteintech, #55090), MT-CO2 (Proteintech, #55070), ATP8 (Proteintech, #26723), TFAM (Proteintech, #22586), GFP-tag (Abcam, #ab290), and mCherry (Abcam, #167453) were used in this study.

Techniques: Functional Assay, Activation Assay, Western Blot, Control, Quantitation Assay, Staining, Membrane, Plasmid Preparation, Phospho-proteomics

Journal: bioRxiv

Article Title: Sialin2 Functions as a Mammalian Nitrate Sensor to Sustain Mitochondrial Homeostasis

doi: 10.1101/2025.05.04.652104

Figure Lengend Snippet: Nitrate-Sialin2 signaling promotes LKB1-mitochondrial recruitment to activate AMPK phosphorylation a, Schematic workflow of phosphoproteomic analysis. b, Top enriched KEGG pathways in HEK293T cells treated with 4 mM nitrate or control vehicle for 4 h listed by the rank of P value based on DAVID analysis. Data represent three biological replicates per condition. c, IF staining images (left) and quantification (right) of pAMPK T172 in control and SLC17A5 knockout (sg SLC17A5 ) NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. d, Quantification of pAMPK T172 in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA), Sialin, and Sialin2. N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 8e. e, Schematic diagram of subcellular compartment-specific biosensor osABKAR. f, Quantification of FRET/CFP ratio of Mito-ABKAR in control and sg SLC17A5 NRK cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 9d. g, h, IF staining images (upper, representative YFP images, lower, representative pseudocolor images of FRET/CFP ratio show the FRET response; g) and quantification (h) of FRET/CFP ratio of Mito-ABKAR in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA), Sialin, and Sialin2. N = 30 cells from representative experiments of three repeats. i, Quantification of FRET/CFP ratio of Lyso-ExRai-ABKAR in control and sg SLC17A5 NRK cells treated with metformin (Met, 10 mM) for 4 h. N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 9h. j, k, PLA of Sialin/Sialin2-LKB1 in NRK (j) or HEK293T (k) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images of HEK293T cells in Extended Data Fig. 10c. l, PLA of Sialin/Sialin2-CaMKK2 in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 10d. m, Quantification of FRET/CFP ratio of Mito-ABKAR in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See images in Extended Data Fig. 10e. n, o, IF staining images of LKB1 and MitoTracker in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA), Sialin, and Sialin2 (n). Colocalization quantified by Pearson’s correlation coefficient (o). N = 30 cells from representative experiments of three repeats. p, Immunoblot analysis of pAMPK T172 , AMPK, Flag, and Sialin2 in control and sg SLC17A5 HEK293T cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). Representative images of n = 3 independent experiments were shown. q, r, IF staining images (upper, representative YFP images, lower, representative pseudocolor images of FRET/CFP ratio show the FRET response; q) and quantification (r) of FRET/CFP ratio of Mito-ABKAR in control and sg SLC17A5 HEK293T cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). N = 30 cells from representative experiments of three repeats. s, Schematic illustration of nitrate enhances Sialin2-LKB1 interaction, promotes the recruitment of LKB1 to mitochondria, which in turn promotes the phosphorylation and activation of AMPK. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: HRP-conjugated LKB1 (Santa Cruz Biotechnology, #sc-374334HRP) and Flag (Proteintech, #HRP-66008) antibodies were purchased.

Techniques: Phospho-proteomics, Control, Staining, Knock-Out, Western Blot, Plasmid Preparation, Activation Assay

Journal: bioRxiv

Article Title: Sialin2 Functions as a Mammalian Nitrate Sensor to Sustain Mitochondrial Homeostasis

doi: 10.1101/2025.05.04.652104

Figure Lengend Snippet: Nitrate-Sialin2 signaling promotes mitochondrial biogenesis and functional homeostasis via LKB1-AMPK activation a, Immunoblot analysis of NT-PGC1α, TFAM, mitochondrial-encoded proteins (MT- ND5, CYTB, MT-CO2, and ATP8), and Sialin2 in control and sg SLC17A5 HEK293T cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Representative images of n = 3 independent experiments were shown. See full quantitation in Extended Data Fig. 12a–d. b, c, IF staining images (left) and quantification (right) of Cox IV (mitochondria; b) and JC-1 (mitochondrial membrane potential; c) in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. d, e, ATP production (d) and mitochondrial DNA (mtDNA) copy number (e) in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). N = 3. f, Mitochondrial ROS (mtROS) production in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). Production normalized to control. N = 3 from three independent experiments, each in triplicate. g, Proliferation (%EdU positive cells) in control and sg SLC17A5 NRK cells reconstituted with Sialin (KRI/AAA) or Sialin2 and treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. See EdU images in Extended Data Fig. 12m. h, Immunoblot analysis of NT-PGC1α, TFAM, mitochondrial-encoded proteins (MT- ND5, CYTB, MT-CO2, and ATP8), Flag, and Sialin2 in control and sg SLC17A5 HEK293T cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1- M (D194A). Representative images of n = 3 independent experiments were shown. See full quantitation in Extended Data Fig. 13a–d. i, j, IF staining images (left) and quantification (right) of Cox IV (i) and JC-1 (j) in control and sg SLC17A5 NRK cells reconstituted with vector, Flag-LKB1, Flag-LKB1- M, or Flag-LKB1-M (D194A). N = 30 cells from representative experiments of three repeats. k, l, ATP production (k) and mtDNA copy number (l) in control and sg SLC17A5 NRK cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). N = 3. m, mtROS production in control and sg SLC17A5 NRK cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). Production normalized to control. N = 3 from three independent experiments, each in triplicate. n, Proliferation (%EdU positive cells) in control and sg SLC17A5 NRK cells reconstituted with vector, Flag-LKB1, Flag-LKB1-M, or Flag-LKB1-M (D194A). N = 30 cells from representative experiments of three repeats. o, Schematic illustration of nitrate triggers CTSB-mediated cleavage of Sialin at residues K256/R257/I258 to generate Sialin2, which translocates to mitochondria, acts as the nitrate sensor, recruits LKB1 to activate mitochondrial AMPK phosphorylation, and drives mitochondrial biogenesis to sustain mitochondrial function and cellular homeostasis. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: HRP-conjugated LKB1 (Santa Cruz Biotechnology, #sc-374334HRP) and Flag (Proteintech, #HRP-66008) antibodies were purchased.

Techniques: Functional Assay, Activation Assay, Western Blot, Control, Quantitation Assay, Staining, Membrane, Plasmid Preparation, Phospho-proteomics