Saturday, February 18, 2012
Exhibit Hall A-B1 (VCC West Building)
Background: We previously found that Dysferlin is expressed in vascular endothelium, an innermost layer of the blood vessels. Down-regulation of Dysferlin in vascular endothelial cells (VEC) causes a deficiency in cell adhesion of subconfluent VEC, but not in confluent cells. This phenomenon is due to decreased expression of platelet endothelial cell adhesion molecule 1 (PECAM-1), indicating Dysferlin has a functional role in maintaining vascular homeostasis in blood vessels that perfused skeletal muscles. However, the mechanistic pathway of Dysferlin and membrane protein trafficking remains unknown. Goal: In this study, we aim to identify Dysferlin-interacting proteins that may unravel the underlying linkage between Dysferlin and protein trafficking. Methods: To identify Dysferlin-interacting protein complexes, we generated glutathione-S-transferase (GST)-dysferlin recombinant proteins as a bait for pulldown assays. Briefly, truncated Dysferlin sequences containing the first 3 C2 domains (C2A to C2C), last 4 C2 domains (C2D to C2G) and highly conserved domains (DysF N and C domains) were amplified by polymerase chain reaction and inserted into glutathione-S-transferase (GST) vector. Resins bound with GST-Dysferlin were incubated with human umbilical vein endothelial cell (HUVEC) lysates to pull down Dysferlin-interacting protein complexes. Dysferlin-interacting proteins were identified by mass spectrometry. Results: With the use of proteomics, we identified several transport, signaling, contractile and motor proteins that interact with Dysferlin directly or indirectly. These results further suggest that Dysferlin mediates the trafficking of vesicles that contain protein cargos. Conclusion: Collectively, our study identifies Dysferlin-interacting proteins in human VEC and may initiate subsequent hypothesis-driven research to further elucidate the mechanistic pathway of Dysferlin function in human vasculature and also intact skeletal muscles.