Global Proteomics for Identifying the Alteration Pathway of Niemann-Pick Disease Type C Using Hepatic Cell Models

Abstract

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder with progressive neurodegeneration. Although the causative genes were previously identified, NPC has unclear pathophysiological aspects, and patients with NPC present various symptoms and onset ages. However, various novel biomarkers and metabolic alterations have been investigated; at present, few comprehensive proteomic alterations have been reported in relation to NPC. In this study, we aimed to elucidate proteomic alterations in NPC and perform a global proteomics analysis for NPC model cells. First, we developed two NPC cell models by knocking out NPC1 using CRISPR/Cas9 (KO1 and KO2). Second, we performed a label-free (LF) global proteomics analysis. Using the LF approach, more than 300 proteins, defined as differentially expressed proteins (DEPs), changed in the KO1 and/or KO2 cells, while the two models shared 35 DEPs. As a bioinformatics analysis, the construction of a protein-protein interaction (PPI) network and an enrichment analysis showed that common characteristic pathways such as ferroptosis and mitophagy were identified in the two model cells. There are few reports of the involvement of NPC in ferroptosis, and this study presents ferroptosis as an altered pathway in NPC. On the other hand, many other pathways and DEPs were previously suggested to be associated with NPC, supporting the link between the proteome analyzed here and NPC. Therapeutic research based on these results is expected in the future.

Keywords: Niemann–Pick disease type C; enrichment pathway analysis; global proteomics; knock out; liquid chromatography–electrospray ionization tandem mass spectrometry; model cell.

Figure 1

Accumulated cholesterol quantitation in wild-type cells and two types of NPC1 knockout cells. Data represent the means ± SDs, n = 9, ** p < 0.01 (two-tailed Welch’s t-test). WT—Hep G2 cells; KO1—Sites A and C mutant NPC1 model cells; KO2—Sites B and D mutant NPC1 model cells.

Figure 2

Venn diagram showing protein numbers identified from the results of the label-free global proteomics analysis. WT—Hep G2 cells; KO1—Sites A and C mutant NPC1 model cells; KO2—Sites B and D mutant NPC1 model cells.

Figure 3

The principal component analysis of the LF method. (a) A PCA plot using 3331 proteins identified in the KO1, KO2, and WT cells. (b) A loading plot using 3331 proteins identified in the KO1, KO2, and WT cells. WT—Hep G2 cells; KO1—Sites A and C mutant NPC1 model cells; KO2—Sites B and D mutant NPC1 model cells.

Figure 4

Volcano plots from the results of the label-free global proteomics analysis. (a) KO1 versus WT using 3390 proteins identified in KO1 and WT cells. (b) KO2 versus WT using 3434 proteins identified in KO2 and WT cells. Red-colored plots—greater than 2-fold upregulated and p < 0.05 (adjusted using the Benjamini–Hochberg correction) proteins in NPC1 model cells; green-colored plots—greater than 0.5-fold downregulated and p < 0.05 (adjusted using the Benjamini–Hochberg correction) proteins in NPC1 model cells; grey-colored plots—non-differentially changed proteins. WT—Hep G2 cells; KO1—Sites A and C mutant NPC1 model cells; KO2—Sites B and D mutant NPC1 model cells.

Figure 5

Enrichment analysis in the KEGG pathway from the results of the label-free global proteomics analysis. (a) KO1 versus WT using 136 DEPs in KO1. (b) KO2 versus WT using 249 DEPs in KO2. Proteins with p < 0.05 (Fisher’s exact test) are shown in these figures. WT—Hep G2 cells; KO1—Sites A and C mutant NPC1 model cells; KO2—Sites B and D mutant NPC1 model cells.