Proteomic Analysis of Marinesco-Sjogren Syndrome Fibroblasts Indicates Pro-Survival Metabolic Adaptation to SIL1 Loss

Abstract

Marinesco-Sjogren syndrome (MSS) is a rare multisystem pediatric disorder, caused by loss-of-function mutations in the gene encoding the endoplasmic reticulum cochaperone SIL1. SIL1 acts as a nucleotide exchange factor for BiP, which plays a central role in secretory protein folding. SIL1 mutant cells have reduced BiP-assisted protein folding, cannot fulfil their protein needs, and experience chronic activation of the unfolded protein response (UPR). Maladaptive UPR may explain the cerebellar and skeletal muscle degeneration responsible for the ataxia and muscle weakness typical of MSS. However, the cause of other more variable, clinical manifestations, such as mild to severe mental retardation, hypogonadism, short stature, and skeletal deformities, is less clear. To gain insights into the pathogenic mechanisms and/or adaptive responses to SIL1 loss, we carried out cell biological and proteomic investigations in skin fibroblasts derived from a young patient carrying the SIL1 R111X mutation. Despite fibroblasts not being overtly affected in MSS, we found morphological and biochemical changes indicative of UPR activation and altered cell metabolism. All the cell machineries involved in RNA splicing and translation were strongly downregulated, while protein degradation via lysosome-based structures was boosted, consistent with an attempt of the cell to reduce the workload of the endoplasmic reticulum and dispose of misfolded proteins. Cell metabolism was extensively affected as we observed a reduction in lipid synthesis, an increase in beta oxidation, and an enhancement of the tricarboxylic acid cycle, with upregulation of eight of its enzymes. Finally, the catabolic pathways of various amino acids, including valine, leucine, isoleucine, tryptophan, lysine, aspartate, and phenylalanine, were enhanced, while the biosynthetic pathways of arginine, serine, glycine, and cysteine were reduced. These results indicate that, in addition to UPR activation and increased protein degradation, MSS fibroblasts have profound metabolic alterations, which may help them cope with the absence of SIL1.

Keywords: BiP; autophagosome; fibroblast; neurodegenerative disease; pathway analysis; protein folding; unfolded protein response.

Figure 1

UPR activation in MSS patient fibroblasts. (A,C) HF-CT (CT) and HF-MSS (MSS) cultured under standard growth conditions were analyzed by Western blotting to assess the expression of SIL1, ATF4, ATF6, and the phosphorylated form of EIF2α (A), BiP, ORP150, and PDI (C) as indicated. Immunoblots are representative of at least two independent experiments. Ponceau S staining of the blots is shown to control for equal protein loading. (B) XBP1 splicing was assessed by PCR. HF-CT (first lane) treated with 1 μM thapsigargin (Tg) for 6 h was used as a positive control. The higher and lower bands represent the unspliced (U) and spliced (S) forms of XBP1.

Figure 2

LAMP1-positive structures increase in HF-MSS cells. (A) HF-CT (CT) and HF-MSS (MSS) were plated on glass coverslips, fixed in PFA 4%, and processed for immunofluorescence. Confocal images of cells immunostained for LAMP1 (green) and reacted with Hoechst 33342 (blue) to stain the nuclei. The merge of green and blue channels is represented in the third panel. A magnification of the squared area is also shown. White arrows indicate ring-like structures. Scale bar: 20 µm. (B) Quantification of total LAMP1 staining shown in A. * p < 0.05.

Figure 3

Electron microscopy (EM) and immunoblot analysis of autophagic vacuoles. (A) Ultrastructural alterations in HF-MSS. Representative EM micrographs of HF-CT (a) and HF-MSS (c) cells. Details at higher magnification of the cytoplasm of HF-CT (b) and HF-MSS (d) cells. HF-MSS cells accumulate vacuoles containing large clusters of membranes (arrowheads) and multilamellar bodies (asterisks). Scale bars: a and c: 2 μm; b and d: 1 μm. (B) Upper graph, percentage of HF-CT and HF-MSS cells exhibiting vacuoles/multilamellar bodies (n = 100 cells/group); lower graph, average number of vacuoles/multilamellar bodies per cell calculated by considering only the cells showing such structures. Statistical significance Chi square test (lower) and t-test (upper) * p < 0.05. (C) HF-CT (CT) and HF-MSS (MSS) cultured under standard growth conditions were analyzed by Western blotting for the expression of p62/SQSTM1 (p62), LC3, and LAMP1. Immunoblots are representative of at least two independent experiments. Ponceau S staining of the blots is shown to control for equal protein loading.

Figure 4

Proteomic analysis graphical networks. (A) Volcano Plot: Statistical analysis of differentially expressed proteins between HF-CT and HF-MSS cells, run by Perseus. LQF intensity of HF-MSS minus that of HF-CT was plotted against the negative logarithm of the p-values. (B) STRING network of proteins downregulated (left) and upregulated (right) in HF-MSS, confidence level set at 0.9. Squares indicate examples of clusters of interacting proteins involved in the indicated functions.

Figure 5

Fluorescence microscopy of representative DE proteins. (A) HF-CT (CT) and HF-MSS (MSS) were plated on glass coverslips, fixed in PFA 4%, and processed for immunofluorescence. Confocal images of cells stained for CNX (red) and NCL (green) are shown. The merge of green and red channels is shown in the third panel. Scale bar: 20 µm. (B) Quantification of total CNX staining and the number of NCL-positive nucleoli/cell of the experiment shown in A. Blue and red bars indicate HF-CT and HF-MSS, respectively. ** p < 0.01.

Figure 6

Fluorescence microscopy validation of proteomic analysis. (A) HF-CT (CT) and HF-MSS (MSS) were plated on glass coverslips, fixed in PFA 4%, and processed for immunofluorescence. Confocal images of cells stained for PDI (green) and NPM (red) are shown. The merge of green and red signal is also shown. Scale bar: 20 µm. (B) Quantification of total PDI staining and the number of NPM-positive nucleoli/cell of the experiment shown in A. Blue and red bars indicate HF-CT and HF-MSS, respectively. **** p < 0.0001.

Figure 7

Western blot validation of proteomic analysis. (A,B) HF-CT (CT) and HF-MSS (MSS) cultured under standard growth conditions were analyzed by Western blotting to assess the expression of NPM, NCL, NES, CAV-1, ANXA-5, and CTHRC1 as indicated. The proteins downregulated or upregulated in HF-MSS are shown in A and B, respectively. Immunoblots are representative of at least two independent experiments. Ponceau S staining of the blots is shown to control for equal protein loading.