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

Baxter SS, Dibble CF, Byrd WC, Carlson J, Mack CR, Saldarriaga I, Bencharit S - Mol Biosyst (2014)

Bottom Line: Loss of expression of each CCM gene results in loss of in vitro endothelial tube formation.Principal component analysis and cluster analysis show the effects of individual knockdown.While all CCM mutations result in similar pathology, different CCM mutations have their own distinct pathogenesis in cell signaling.

View Article: PubMed Central - PubMed

Affiliation: David H. Murdock Research Institute, North Carolina Research Campus, Kannapolis, NC 28081, USA.

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.

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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.
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fig3: 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.

Mentions: Our proteomic analysis shows that eight proteins differentially expressed among five cell lines are known proteins that interact with the CCM protein complex.24 Cytoskeletal proteins are among the most common (Table 1 and Fig. 3A–D). Both wild type (WT) and mock knockdown were used as a control. However, we compared the proteomic profiling of each knockdown with the mock knockdown to minimize confounding effects from the knockdown process. Gunel et al.32 showed that Krit1, CCM1 protein, interacts with beta-tubulin. Béraud-Dufour et al.33 further showed that Krit1-microtubule interaction is mediated with Rap1/Icap1. Rap1 and tubulin interact with Krit1's FERM domain, while Icap1 interacts through the N-terminal NPXY motif. The proteomic analysis shows a remarkable alteration of several tubulin isoforms. Our group previously showed that OSM, CCM2 protein, interacts with several isoforms of tubulin as well as elongation factor 1 alpha 1 (EF1A1). EF1A1 is one of the major regulators for actin cytoskeleton.24 This proteomic study shows the changes in EF1A1 and one of its partners-EF1G-in addition to direct interaction with cytoskeletal proteins, Krit1, OSM, and PDCD10 (CCM3 protein), may also regulate actin cytoskeleton through their interaction with phosphatidylinositol phosphates.24,32,33


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

Baxter SS, Dibble CF, Byrd WC, Carlson J, Mack CR, Saldarriaga I, Bencharit S - Mol Biosyst (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043921&req=5

fig3: 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.
Mentions: Our proteomic analysis shows that eight proteins differentially expressed among five cell lines are known proteins that interact with the CCM protein complex.24 Cytoskeletal proteins are among the most common (Table 1 and Fig. 3A–D). Both wild type (WT) and mock knockdown were used as a control. However, we compared the proteomic profiling of each knockdown with the mock knockdown to minimize confounding effects from the knockdown process. Gunel et al.32 showed that Krit1, CCM1 protein, interacts with beta-tubulin. Béraud-Dufour et al.33 further showed that Krit1-microtubule interaction is mediated with Rap1/Icap1. Rap1 and tubulin interact with Krit1's FERM domain, while Icap1 interacts through the N-terminal NPXY motif. The proteomic analysis shows a remarkable alteration of several tubulin isoforms. Our group previously showed that OSM, CCM2 protein, interacts with several isoforms of tubulin as well as elongation factor 1 alpha 1 (EF1A1). EF1A1 is one of the major regulators for actin cytoskeleton.24 This proteomic study shows the changes in EF1A1 and one of its partners-EF1G-in addition to direct interaction with cytoskeletal proteins, Krit1, OSM, and PDCD10 (CCM3 protein), may also regulate actin cytoskeleton through their interaction with phosphatidylinositol phosphates.24,32,33

Bottom Line: Loss of expression of each CCM gene results in loss of in vitro endothelial tube formation.Principal component analysis and cluster analysis show the effects of individual knockdown.While all CCM mutations result in similar pathology, different CCM mutations have their own distinct pathogenesis in cell signaling.

View Article: PubMed Central - PubMed

Affiliation: David H. Murdock Research Institute, North Carolina Research Campus, Kannapolis, NC 28081, USA.

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.

Show MeSH
Related in: MedlinePlus