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Nitrate-Sialin2 signaling promotes mitochondrial biogenesis and functional homeostasis <t>via</t> <t>LKB1-AMPK</t> activation a, Immunoblot analysis of NT-PGC1α, TFAM, mitochondrial-encoded proteins (MT- ND5, <t>CYTB,</t> 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.
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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.

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