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-induced CTSB-dependent cleavage of Sialin at K256/R257/I258 generates Sialin2, which localizes to mitochondria a, Immunoblot analysis of Sialin and Sialin2 protein normalized to HSP90 in cerebral cortex from Alzheimer’s disease (AD) mice, salivary glands from irradiation (IR)- damaged mice. Representative data of n = 6 independent experiments were shown. See full immunoblot images and quantitation in Extended Data Fig. 1a. b, Endogenous Sialin2 was immunoprecipitated (IP’ed) from HEK293T cells, with IgG as negative control. Representative data of n = 3 independent experiments were shown. c, Immunoblot analysis of Sialin and Sialin2 in HEK293T cells pre-treated for 6 h with DMSO (vehicle), 200 nM Baf-A1/40 μM CQ (lysosomal inhibitors), or 10 μM MG132 (proteasome inhibitor), followed by 4 h stimulation with 4 mM nitrate. Representative images of n = 3 independent experiments were shown. d, Protein-protein interaction network (GeneMANIA) predicted cathepsin B (CTSB) as a top candidate protease interacting with Sialin. e, Co-IP of Sialin and CTSB in HEK293T cells treated with 4 mM nitrate for 4 h. Representative data of n = 3 independent experiments were shown. f, g, Proximity ligation assay (PLA) of Sialin/Sialin2-CTSB in HeLa (f) or NRK (g) cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. h, Immunoblot analysis of Sialin and Sialin2 in HEK293T (left panel), HeLa (middle panel) or NRK (right panel) cells pre-treated for 6 h with a vehicle control (DMSO) and 20 μM CTSB inhibitor CA-074, followed by 4 h stimulation with 4 mM nitrate. Representative images of n = 3 independent experiments were shown. i, j, HEK293T (left panel) or HeLa cells (right panel) introduced with HA/Flag-tagged full length (FL) Sialin or mutant full-length Sialin (KRI/AAA) were treated with nitrate (4 mM, 4 h), followed by analysis of HA, Flag, Sialin, and Sialin2. Representative images of n = 3 independent experiments were shown. I, Ile; K, Lys; R, Arg. k, Top enriched cellular component GO terms for unique Sialin2-related proteomes in HEK293T cells listed by the rank of P values based on DAVID analysis. l, PLA of Tomm20-LAMP1 in HeLa cells treated with nitrate (4 mM, 4 h). N = 30 cells from representative experiments of three repeats. m, High-sensitivity structured illumination microscopy (HiS-SIM) live-cell images of Sialin or Sialin2 colocalized with MitoTracker-labeled mitochondria (PK Mito) in HeLa cells stably expressed GFP-Sialin or GFP-Sialin2. See quantification in Extended Data Fig. 2f. n, Immunoelectron microscopy images of Sialin or Sialin2 localized to mitochondria in NRK cells stably expressed GFP-Sialin and mCherry-Sialin2. N = 30 cells from representative experiments of three repeats. o, Schematic illustration of nitrate-activated CTSB cleaving Sialin at K256/R257/I258 to generate Sialin2, which is preferentially localized to mitochondria. For all panels, data are represented as mean ± SD, P value denotes t -test. Scale bar: 10 μm.

Article Snippet: Mito-ABKAR (organelle-targeting sequence: MAIQLRSLFP LALPGMLALLGWWWFFSRKKA, N-terminus; Addgene: #61509) and Lyso-ExRai- AMPKAR (organelle-targeting sequence: full length of lysosome-associated membrane protein 1 (LAMP1), N-terminus; Addgene: # 192449) were created by Jin Zhang lab.

Techniques: Western Blot, Irradiation, Quantitation Assay, Immunoprecipitation, Negative Control, Co-Immunoprecipitation Assay, Proximity Ligation Assay, Control, Mutagenesis, Microscopy, Labeling, Stable Transfection, Immuno-Electron Microscopy