Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium.
Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles. Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.
UBC James Hogg Research Centre, Institute for Heart + Lung Health, Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada.
Cleo
C
Leung
Soraya
S
Utokaparch
Arpeeta
A
Sharma
Carol
C
Yu
Thomas
T
Abraham
Christoph
C
Borchers
Pascal
P
Bernatchez
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D000073939:Dysferlin; D004730:Endothelium, Vascular / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism*; D021381:Protein Transport; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
C114839:DYSF protein, human; D000073939:Dysferlin; D008565:Membrane Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins