Proximity-Labeling of Near Neighbors of Lamin A and Lamin A-Δ50 (PROGERIN).

• Main Author: Sabri, Mohammad
• Format: Others
• Published: Digital Commons @ East Tennessee State University 2013
• Subjects: sabri302; Life Sciences; Microbiology
• Online Access: https://dc.etsu.edu/honors/100; https://dc.etsu.edu/cgi/viewcontent.cgi?article=1104&context=honors

In an attempt to isolate and identify proteins that differentially interact with or locate near lamin A and progerin, we used a previously described method named BioID (proximity-dependent biotin identification). This method is based on fusion of a promiscuous E. coli biotin-protein ligase (BL) to a targeting protein (in this study, lamin A-GFP and progerin-GFP). The biotin ligase biotinylates amino residues in proteins that are near-neighbors of the fusion protein. To create the fusion proteins, BL was sub-cloned from a pcDNA3.1 MCS-BirA(R118G)-HA plasmid donated by Kyle Roux from University of South Dakota. The BL fragment was ligated into a pNEBR-X1-lamin A-GFP and pNEBR-X1-progerin-GFP which inducibly express the fusion proteins in mammalian cells, under control of pNEBR-R1 Rheoswitch regulator plasmid. Two stable cell lines expressing the GFP-BL-lamin A and GFP-BL-progerin chimeras were created. The expression of the chimeras was induced by incubation with 500nM of GenoStat for 24 hours in the presence of 50mM Biotin. Biotinylated proteins were isolated from cell lysates by incubation with streptavidin magnetic beads. Proteins were separated by SDS-PAGE and sent for identification by mass spectrometry. In conclusion, we isolated multiple proteins that differentially associate with and/or are proximate to lamin A and progerin in vivo. The identification of such proteins may shed light into the mechanism by which progerin causes its deleterious effects. The purpose of this research is to attempt to identify the neighboring proteins that differentially interact with or locate near lamin A and progerin.