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 up-regulated in the PNALD group (Table S1).
To identify the functional classification of DEPs, this study performed gene ontology analysis with the assistance of DAVID Bioinformatics Resources, according to their molecular functions (MF), biological processes (BP), and cellular components (CC). The top 15 annotations represented in each of the three GO categories are shown in Fig. 1. Majority of enriched categories included mitochondrial and mitochondria-related proteins (Fig. 2-A, Table S2; total 27, 16 down-regulated and 11 up-regulated); 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, glycolipid metabolism (Fig. 2-B, Table S3; total 32, 28 down-regulated and 4 up-regulated) and amino acid metabolism (Fig. 2-C, Table S4; total 22, 15 down-regulated and 7 up-regulated) were significantly changed between the groups.
Mitochondrial oxidative phosphorylation was impaired in PNALD patients
The mitochondrial oxidative phosphorylation system is the final biochemical pathway for the production of ATP and maintenance of cell function. Mitochondrial respiratory chain NADH dehydrogenase (complex I) is the most abundant enzyme of the electron transport chain [15], and essential for oxidative phosphorylation in mitochondria [16]. Ten subunits of NADH dehydrogenase were found to be down-regulated 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 (Fig. 3), including mitochondrial electron transport (NADH to ubiquinone), response to oxidative stress, oxidation-reduction process, and ATP synthesis-coupled electron transport (Table 2).
Hepatic glycolipid metabolism disorder in patients with PNALD
The liver plays a significant role in the control of glucose homeostasis by regulating various pathways of 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 down-regulated; fructose 1,6-bisphosphate 1-phosphatases (FBP1 and FBP2), the rate-limiting enzyme in gluconeogenesis [18], were down-regulated; pyruvate carboxylase (PC), which catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate, and is involved in gluconeogenesis [19, 20], was down-regulated. 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, up-regulated) [21], D-3-phosphoglycerate dehydrogenase (PHGDH, down-regulated) [22], and serine/threonine-protein phosphatase (CPPED1, down-regulated) [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 down-regulated while acetyl-CoA carboxylase 2 (ACACB), which inhibits fatty acid oxidation [27], was also down-regulated. Further, lipid metabolism-associated DEPs, including glycine N-methyltransferase (GNMT, down-regulated) [28–32], fatty acid-binding protein (FABP5, up-regulated) [33], cathepsin S (CTSS, up-regulated) [34], aldose reductase (AKR1B1, up-regulated) [35], and N(G), N(G)-dimethylarginine dimethylaminohydrolase 1 (DDAH1, down-regulated) were also identified [36]. These results together indicated PNALD to be related to glycolipid metabolism disorder.
Oxidative stress caused by increased production of reactive oxygen species (ROS) 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. Next, various antioxidant factors are down-regulated in PNALD. For example, peroxiredoxin-6 (PRDX6, down-regulated) is a peroxiredoxin that primarily functions as an antioxidant to scavenge peroxides in biological systems [38]; regucalcin (RGN, down-regulated) is an antioxidant [39]; delta-aminolevulinic acid dehydratase (ALAD, down-regulated) 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 (down-regulated) 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 (down-regulated) deficiency significantly enhances cellular oxidative stress [43]. Moreover, we found histidine metabolism-associated proteins (AMDHD1, HNMT, FTCD, HAL, and UROC1) to be down-regulated in PNALD, and previous studies had reported histidine supplementation to inhibit oxidative stress and preserve mitochondrial membrane potential as well as dehydrogenase activity [44]. Combined with the above results, we can hypothesize that oxidative stress caused by increased production of ROS 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 down-regulated DEPs for semi-quantitative verification by western blotting, including CYP2B6, DDAH1, NDUFA1, FABP5, and CAPG (Fig. 4). As shown, expression of CYP2B6, DDAH1, and NDUFA1 was significantly down-regulated, and that of FABP5 and CAPG was up-regulated in the PNALD group, compared to the control group. Western blotting results were in agreement with iTRAQ proteomics results.