Fig 1: PPDPF deficiency inhibits NAD+ synthesis through NMNATs.(A) Representative images of double staining of PPDPF and mitochondrial markers ATP5A1, HSP60, or TOMM20 in HK2 cells. Scale bar, 10 μm. (B) Western blots of subcellular fractions of HK2 cells stained with PPDPF, the mitochondrial antibody TOMM20, and the nuclear antibody LAMIN B1. GAPDH was used as internal control. (C) Schematic diagram of PPDPF engages in the synthesis of NAD+ from NMN. (D) NAD+ content of sham and sgRNA-targeted HK2 cells following PBS or NMN treatment. (E) NAD+ content of primary cells isolated from WT and Ppdpf KO kidney following NMN treatment. (F) NAD/NADH ratio of primary cells isolated from WT and Ppdpf KO mice, treated with NMN for 4 hours and measured with SoNar biosensor. (G) NAD/NADH ratio of primary cells isolated from WT and Ppdpf KO mice, treated with NMN for 12 hours and measured with SoNar biosensor. (H) NMNAT activity of primary cells isolated from WT and Ppdpf KO kidney. A450, absorbance at 450 nm. (I) Representative double staining of PPDPF with NMNAT1, NMNAT2, or NMNAT3 in HK2 cells. (J) Representative images of PLA (red). (K) Quantifications of PLA. Scale bar, 10 μm. P value was calculated by two-tailed t test and one-way analysis of variance (ANOVA). P < 0.05 is statistically significant. Data are represented as mean ± SEM. (D) n = 4 for each group. (E) n = 4, WT; n = 4, KO. (F) n = 230, WT; n = 209, KO. (G) n = 87, WT; n = 119, KO. (H) n = 3, WT; n = 3, KO. (J) n = 4 for each group.
Fig 2: PPDPF functions as a thiol-disulfide oxidoreductase.(A) Schematic representation of the domain organization of TGR, TR1, TR3, GR, LDH, and PPDPF. These six proteins have active disulfide center (the CxxxxC motif; two cysteines separated by four other residues) of the pyridine nucleotide disulfide oxidoreductase family. TGR, thioredoxin (Trx) and GSSG reductase; TrxR1, cytosolic Trx reductase; TrxR2, mitochondrial Trx reductase; GR, GSSG reductase; LDH, lactate dehydrogenase. (B) Determination of the redox potential of recombinant human PPDPF by subjecting it to increasing dithiothreitol (DTT) concentrations. The band intensity increases as the redox environment becomes more reducing. This indicates that the disulfide bond is gradually being reduced and resulting in an increase in free cysteine thiols for MPB labeling. (C) Optical density readout of an insulin turbidity assay. (D) Readout of a di-eosin-GSSG assay using purified proteins. RFU, relative fluorescence unit. (E) Readout of an PPDPF RNase disulfide isomerase assay. (F) Left: Representation workflow of MPB labeling. PPDPF reduces disulfide bonds, exposing free thiol groups within NMNATs. The free thiols are labeled with MPB, and the proteins are separated by SDS–polyacrylamide gel electrophoresis. Right: Western blot of MPB-labeled NMNATs in the presence of PPDPF. (G) Schematic representation of the transfection of PPDPF WT, C30S, and C35S constructs into HEK293T followed by quantifications of NAD+ levels and NMNAT activity. (H) Western blots of PPDPF-WT, PPDPF-C30S, and PPDPF-C35S overexpression in HEK293T cells. (I) Quantifications of NMNAT activity (top) and NAD+ content (bottom) in cells transfected with indicated constructs. P value was calculated by one-way ANOVA. P < 0.05 is statistically significant. Data are represented as mean ± SEM. (C to E) n = 2 for each group. (H) n = 2 for each group. (I) For NMNAT activity, n = 4 for each group. For NAD+ measurement, n = 4 for each group. OD650, optical density at 650 nm.
Fig 3: Nmnat1-LKO abrogates NMN supplementation–alleviated ALD.(A to J) Nmnat1-Ctrl and Nmnat1-LKO mice were supplemented with/without NMN (500 mg/kg per day) in the AF group. (A) Liver nuclear NAD+ content (n = 8). (B) Liver nuclear NADH content (n = 8). (C) Liver nuclear NAD+/NADH ratio (n = 8). (D) Plasma ALT levels (n = 8). (E) Plasma AST levels (n = 8). (F) Liver TG content (n = 8). (G) Liver H&E and Oil red O staining (n = 4). (H) Liver taurine content (n = 8). (I) mRNA expressions of Csad in the mouse liver (n = 8). (J) CSAD protein expression in the mouse liver (n = 4). (K to P) Liver immunohistochemistry of acetylated-lysine staining (K), SIRT1 activity (L), acetyl-FOXO1 protein expression (M), acetyl-HNF4α protein expression (N), and mRNA expressions of Cyp7a1 (O) and G6pc (P) was detected in AF Nmnat1-Ctrl and Nmnat1-LKO mouse livers (n = 3 to 8). (Q) Acetyl-FOXO1 and acetyl-HNF4α protein expressions were measured in the liver of NMN-administered Nmnat1-LKO ALD mice (n = 4). The protein band intensity was quantified by ImageJ. Data are presented as the means ± SD. *P < 0.05 represents statistical difference. MOD, mean optical density.
Fig 4: Schematic mechanistic illustration of hepatic NMNAT1–regulated ALD.
Fig 5: CSAD-regulated taurine metabolism is potentially involved in NMNAT1-regulated ALD.(A and B) Nontargeted metabolomic KEGG analysis of the differential metabolite enrichment pathway (A) and differential metabolites in amino acid metabolic pathways (B) in AF Nmnat1-Ctrl and Nmnat1-LKO mouse livers (n = 4). (C and D) Transcriptome KEGG enrichment signal pathway analysis (C) and differential genes expression in taurine and hypotaurine metabolism signaling pathways (D) in AF Nmnat1-Ctrl and Nmnat1-LKO mouse livers (n = 4). (E) mRNA expressions of Csad, Fmo2, and Fmo3 in the mouse liver (n = 8). (F) CSAD protein expression in the mouse liver (n = 3). (G) Liver taurine content (n = 8). (H to L) Liver-specific CSAD overexpression mice were generated by caudal vein injection with an AAV8-constructed vector containing the Csad sequence. Mice injected with a null vector serve as the control. Taurine (1 g/kg per day) was supplemented in the diet of the AF + taurine group. (H) mRNA expressions of Csad in the mouse liver (n = 8). (I) Liver taurine content (n = 8). (J) Plasma ALT levels (n = 8). (K) Liver TG content (n = 8). (L) Liver H&E and Oil red O staining (n = 4). The protein band intensity was quantified by ImageJ. Data are presented as the means ± SD. *P < 0.05 represents statistical difference.
Supplier Page from Abcam for NMNAT1 Activity Assay Kit (Colorimetric)