Proteomic Analysis of the Intestinal Resistance to Thyroid Hormone Mouse Model With Thyroid Hormone Receptor Alpha Mutations

Abstract

Thyroid hormone is critical during the development of vertebrates and affects the function of many organs and tissues, especially the intestine. Triiodothyronine (T₃) is the active form and can bind to thyroid hormone nuclear receptors (TRs) to play a vital role in the development of vertebrates. The resistance to thyroid hormone α, as seen in patients, has been mimicked by the Thra^E403X mutation. To investigate the mechanisms underlying the effect of TRα1 on intestinal development, the present study employed proteomic analysis to identify differentially expressed proteins (DEPs) in the distal ileum between homozygous Thra^E403X/E403X and wild-type Thra^+/+ mice. A total of 1,189 DEPs were identified, including 603 upregulated and 586 downregulated proteins. Proteomic analysis revealed that the DEPs were highly enriched in the metabolic process, the developmental process, the transporter of the nutrients, and the intestinal immune system-related pathway. Of these DEPs, 20 proteins were validated by parallel reaction monitoring analysis. Our intestinal proteomic results provide promising candidates for future studies, as they suggest novel mechanisms by which TRα1 may influence intestinal development, such as the transport of intestinal nutrients and the establishment of innate and adaptive immune barriers of the intestine.

Keywords: bioinformatic; intestine; parallel reaction monitoring analysis; proteomic analysis; thyroid hormone receptor.

Figure 1

Overview of the mass spectrometry results. Bar plot summarizing the detected peptides and proteins in Wt Thra ^+/+ (n = 4) and Hom Thra ^E403X/E403X (n = 5) mice.

Figure 2

Identification of differentially expressed proteins (DEPs) between Thra ^E403X/E403X and Thra ^+/+ mice. (A) The total number of upregulated and downregulated DEPs. The fold change (FC) >1.30 and p-value <0.05 were set as the significant thresholds for the upregulated DEPs. FC <0.77 and p-value <0.05 were set as the significant thresholds for the downregulated DEPs. (B) Volcano plot of the identified DEPs between Thra ^E403X/E403X (n = 5) and Thra ^+/+ (n = 4) mice. The x-axis shows the log2 fold change of each protein, and the y-axis shows the p-value of the significant difference test after log10 logarithmic conversion. The red dots denote upregulated DEPs, the blue dots denote downregulated DEPs, and the gray dots denote unchanged proteins.

Figure 3

Gene ontology (GO) analysis for the identified differentially expressed proteins (DEPs). The DEPs were annotated into three categories based on GO terms, including biological processes, cellular components, and molecular functions. (A) GO enrichment analysis of the upregulated DEPs. (B) GO enrichment analysis of the downregulated DEPs.

Figure 4

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed proteins (DEPs). Bubble diagrams displaying the top 20 KEGG pathways for which upregulated (A) and downregulated (B) DEPs were significantly enriched, respectively. The y-axis shows the IDs and the names of the enriched pathways, and the x-axis shows the converted log2 fold enrichment. The size of the bubbles indicates the number of DEPs in each pathway.

Figure 5

Hierarchical cluster analysis for the differentially expressed proteins (DEPs). (A) Fold change >1.30 or <0.77 and p <0.05 were set as the significant thresholds for the DEPs. According to the degree of fold change (FC), the DEPs were divided into four groups from Q1 to Q4. The different colors indicate the different Q categories: Q1 (FC ≤0.667, severely downregulated), Q2 (0.667 < FC ≤0.769, mildly downregulated), Q3 (1.3 < FC ≤1.5, mildly upregulated), and Q4 (FC>1.5, severely upregulated). (B) The distribution of Q categories in the Kyoto Encyclopedia of Genes and Genomes pathways. (C) The distribution of Q categories for the analysis of the protein domain. (D) The distribution of Q categories for the analysis of the biological process. (E) The distribution of Q categories for the analysis of the cellular component. (F) The distribution of Q categories for the analysis of the molecular function. The red color indicates stronger enrichment. The blue color indicates weaker enrichment.

Figure 6

Validation by parallel reaction monitoring analysis. The absolute quantification levels of 20 differentially expressed proteins were evaluated in Thra ^+/+ (n = 4) and Thra ^E403X/E403X mice (n = 5). Data are presented as mean ± SEM. Statistical analysis was performed by Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001.