Identification Of Proteins That Interact With NUP155, A Key Nuclear Pore Complex Protein

Background and objective: Atrial fibrillation (AF) is the most common form ofsustained clinical arrhythmias. Genetic factors play an important role in the pathogenesisand development of AF. The mutation in the NUP155gene can cause AF, whichprovides a non-ion channel basis for AF and a new mechanism for the pathogenesis of AF.NUP155, which encodes a member of the nucleoporins, the components of the nuclearpore complex (NPC). The molecular mechanism by which NUP155mutation causes AFis not completely clear. One hypothesis is that the NUP155protein acts as an upstreamkey protein, and can interact with other proteins to affect the expression of downstreamgenes. The defects of NUP155function or its interaction with other proteins due to AFmutation may affect the expression of downstream genes by altering transport of mRNAsand proteins, causing AF. To test this hypothesis, I carried out the studies onprotein-protein interactions of NUP155, which may be highly significant to theunderstanding of the function of NUP155and its role in the development of AF.Methods: In order to investigate the important function of NUP155in AF, I created aseries of plasmids, pET-28a-NUP155(1~10) and pET-28a-GST-TEV-NUP155(1~10)for overexpression and purification of his-tagged or GST-fused NUP155protein fragments.The plasmids were transformed into E. coli BL21(DE3)Rosetta. The bacteria wereinduced to make the fusion proteins. Bacterial strains with successful expressionof fusion proteins were selected for follow-up studies. His-tagged or GST-fusedNUP155proteins were then purified using His affinity chromatography and GSTaffinity chromatography. The purified proteins were incubated with crude cellprotein extracts form HeLa cells, and potential NUP155-interacting proteins werepulled down. The proteins pulled down from HeLa extracts were separated, anddetected by SDS-PAGE, isolated from the gels, and subjected to MALDI-TOF massspectrometry to identify the pulled-down proteins.Results: I constructed20bacterial expression plasmids for expression andpurification of NUP155protein fragments, including10pET-28a-NUP155plasmids(#1~10) and10pET-28a-GST-TEV-NUP155plasmids (#1~10). Two of the20 plasmids, pET-28a-GST-TEV-NUP155(1) and pET-28a-GST-TEV-NUP155(9), werefound to lead to high expression of soluble fusion proteins in BL21(DE3)Rosettaand thus used for purification of the two GST-NUP155fragment fusion proteins.GST pull-down studies yielded several protein bands that represent potentialNUP155-interacting proteins. Mass spectrometry identified one protein, BRE, as astrong candidate protein that may interact with NUP155.Conclusions: GST pull-down is an effective strategy for identify potential proteinsthat may interact with NUP155. My study identified BRE as one potential protein thatmay interact with NUP155. Future GST pull-down and co-immunoprecipitation studiesmay confirm the interaction between BRE and NUP155, and functional studies may revealwhether BRE affects the function of NUP155involved in cardiac physiology anddevelopment of AF.