Comprehensive identification of proteome in human liver tissue
A total of 14307 peptides and 3337 proteins were identified by iTRAQ analysis. Mass spectrometry results showed 112 differentially expressed proteins (DEPs), of which 73 were down regulated, and 39 were upregulated in the PNALD group (Table S1).
To identify the functional classification of DEPs, this study performed gene ontology analysis according to their molecular functions (MF), biological processes (BP), and cellular components (CC) with the assistance of DAVID Bioinformatics Resources. The top 15 annotations represented in each of the three GO categories are shown in Figure 1. Majority of enriched categories included mitochondrial and mitochondria-related proteins (Figure 2-A, Table S2; total 27, 16 downregulated and 11 upregulated). Impaired oxidative phosphorylation was the predominant process in PNALD group. Mitochondria are known to be mainly responsible for the oxidative decomposition of dextrose, fat, and protein, for providing energy. Accordingly, the glycolipid metabolism (Figure 2-B, Table S3; total 32, 28 downregulated and 4 upregulated) and amino acid metabolism (Figure 2-C, Table S4; total 22, 15 downregulated and 7 upregulated) were significantly altered between the groups.
Mitochondrial oxidative phosphorylation was impaired in PNALD patients
Mitochondrial oxidative phosphorylation system is the final biochemical pathway to produce ATP and the maintenance of cell function. Mitochondrial respiratory chain NADH dehydrogenase (complex I) is the most abundant enzyme in the electron transport chain [15], and is essential for oxidative phosphorylation in mitochondria [16]. Ten subunits of NADH dehydrogenase were found to be downregulated in the PNALD group, including NDUFB11, NDUFB7, NDUFV1, NDUFA7, NDUFV2, NDUFS8, NDUFA1, NDUFS2, NDUFA12, and NDUFS1 (Table S2). Consistent with our hypothesis that mitochondrial oxidative phosphorylation in the liver of patients with PNALD might be impaired, bioinformatics analysis indicated the DEPs to be enriched in mitochondria-associated biological processes (Figure 3), including mitochondrial electron transport (NADH to ubiquinone), response to oxidative stress, oxidation-reduction process, and ATP synthesis-coupled electron transport (Table 2). And the IPA showed that the oxidative phosphorylation was significantly inhibited (Figure 4). These all results indicated the mitochondrial oxidative phosphorylationwas impaired in PNALD patients.
Hepatic glycolipid metabolism disorder in patients with PNALD
Liver plays a significant role in the control of glucose homeostasis by regulating various pathways in glucose metabolism, including glycogenesis, glycogenolysis, glycolysis, and gluconeogenesis. In this study, we observed a strong enrichment of DEPs linked to metabolic enzymes. For example, phosphorylase kinases (PHKA2, PHKB, and PHKG2), which stimulate glycogen degradation [17], were downregulated. Fructose 1,6-bisphosphate 1-phosphatases (FBP1 and FBP2), the rate-limiting enzyme in gluconeogenesis [18], were downregulated. Pyruvate carboxylase (PC), which catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, and involved in gluconeogenesis [19,20], was also found to be downregulated. Consistent with the function of DEPs, bioinformatics analysis indicated that glycogen metabolic process (PHKA2, PHKB, PHKG2, and GNMT) and gluconeogenesis (GOT1, FBP1, FBP2, and PC) might be damaged in PNALD. Besides, we also found some glycolipid metabolism-associated DEPs, including nicotinamide N-methyltransferase (NNMT, upregulated) [21], D-3-phosphoglycerate dehydrogenase (PHGDH, downregulated) [22], and serine/threonine-protein phosphatase (CPPED1, downregulated) [23].
Besides its role in glucose metabolism, the liver plays a pivotal role in lipid metabolism and is the hub of fatty acid metabolism and lipid circulation [24]. Estrogen sulfotransferase (SULT1E1), involved in the process of adipogenesis [25,26], was found to be downregulated and acetyl-CoA carboxylase 2 (ACACB), which inhibits fatty acid oxidation [27], was also downregulated. Further, lipid metabolism associated DEPs, including glycine N-methyltransferase (GNMT, downregulated) [28-32], fatty acid-binding protein (FABP5, upregulated) [33], cathepsin S (CTSS, upregulated) [34], aldose reductase (AKR1B1, upregulated) [35], and N(G), N(G)-dimethylarginine dimethylaminohydrolase 1 (DDAH1, downregulated) were also identified [36]. These results together indicated PNALD to be related to glycolipid metabolism disorder.
Oxidative stress caused by of the antioxidant factors may generate PNALD
Previous reports had shown oxidative stress injury to be one of the significant causes of PNALD. Production of ROS is increased when mitochondrial oxidative phosphorylation is impaired and under circumstances of antioxidant defense deficiency [37]. First, the impaired mitochondrial respiratory chain complex I assembly proteins damage the normal process of oxidative phosphorylation in patients with PNALD. Second, various antioxidant factors are downregulated in PNALD. For example, peroxiredoxin-6 (PRDX6, downregulated) is a peroxiredoxin that primarily functions as an antioxidant to scavenge peroxides in biological systems [38], regucalcin (RGN, downregulated) is an antioxidant [39], delta-aminolevulinic acid dehydratase (ALAD, downregulated) is an important antioxidant enzyme, whose inhibition may result in the accumulation of its substrate d-ALA, which in turn is associated with the overproduction of ROS [40], inhibition of PHGDH (downregulated) impairs the synthesis of heme, resulting in the impairment of oxidative phosphorylation and escape of electrons to molecular oxygen generating more ROS [41], inhibition of aspartate aminotransferase (GOT1, downregulated) inhibits the synthesis of the NADPH antioxidant[42], and DDAH1 (downregulated) deficiency significantly enhances cellular oxidative stress [43]. Third, we found histidine metabolism-associated proteins (AMDHD1, HNMT, FTCD, HAL, and UROC1) to be downregulated in PNALD. Previous studies had reported the histidine supplementation to inhibit oxidative stress and preserve mitochondrial membrane potential as well as dehydrogenase activity [44]. Moreover, IPA results showed that the “NRF2-mediated oxidative stress response”, “EIF2 signaling”, “PFKFB4 signaling pathway”, and “Glutathione mediated detoxification” were suppressed (Fig. S2). Previous studies had reported these pathways all participated in decreasing oxidative stress[45-48]. Combined with the above results, we can hypothesize that oxidative stress caused by ownregulation of the antioxidant factors may also participate in the development of PNALD.
Validation of differential expression proteins by western blotting
In order to verify the credibility of proteomics, we randomly selected several up- and downregulated DEPs for semi-quantitative verification by western blotting, including CYP2B6, DDAH1, NDUFA1, FABP5, and CAPG (Figure 5 4). As shown, expression of CYP2B6, DDAH1, and NDUFA1 was significantly downregulated, and that of FABP5 and CAPG was upregulated in the PNALD group, compared to the control group. Western blotting results were in agreement with iTRAQ proteomics results.