Role of cytoskeletal proteins in cerebral cavernous malformation signaling pathways: a proteomic analysis

Abstract

Three genetic mutations were found to cause cerebral cavernous malformation (CCM), a vascular anomaly predisposing affected individuals to hemorrhagic stroke. These CCM proteins function together as a protein complex in the cell. Loss of expression of each CCM gene results in loss of in vitro endothelial tube formation. Label-free differential protein expression analysis using multidimensional liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) was applied to explore the proteomic profile for loss of each CCM gene expression in mouse endothelial stem cells (MEES) compared to mock shRNA and no shRNA control cell-lines. Differentially expressed proteins were identified (p < 0.05). 120 proteins were differentially expressed among the cell-lines. Principal component analysis and cluster analysis show the effects of individual knockdown. In all knockdown cell-lines, altered expression of cytoskeletal proteins is the most common. While all CCM mutations result in similar pathology, different CCM mutations have their own distinct pathogenesis in cell signaling.

Fig. 1. In vitro tube formation assays in MEES cell lines; (A) knockdown CCM1 (cell line 1), (B) knockdown CCM2 (cell line 2), (C) knockdown CCM2 (cell line 3), (D) wild-type (cell line 4), and (E) mock knockdown (cell line 5).

Fig. 2. Mass Spectrometry Analysis (A–B). Aligned Mass Data. An example of aligned peptide signals for the data set is presented. Sample labels: figures are color coded according to cell line. Selection (red) references an option to manually select a single sample to be highlighted. This option was not selected. (A) Extracted ion chromatogram for m/z 633.0408–633.7484 from retention time 548 391–556 203 demonstrates retention time alignment across all samples for this signal. (B) Aligned masses in the mass range of 633.0408–633.7484 for all samples in the study set. (C). PCA plot for the study set. An ANOVA test was performed using all mass signals detected to compare the expression results from the five cell lines. Results demonstrate an unsupervised separation of the five cell lines based on the mass signal patterns. (D). PCA for cell line replicates. An ANOVA test was performed at the protein level to compare the protein expression results from the five cell lines. Candidate differentially expressed proteins were determined and are presented in the ESI, Table S1. Results demonstrate separation of the five cell lines based on the protein patterns determined based on an ANOVA test. (E). Cell line cluster based on differentially expressed proteins determined by ANOVA.

Fig. 3. Differential expression of selected cytoskeletal proteins. Expression of cytoskeleton-related proteins based on relative fold expression (A) Myosin 9, (B) Tubulin β4, (C) Myosin B binding protein, and (D) α actin 4.

Fig. 4. Summary model for signaling pathway for CCM development.