Characterization and Discovery of Short Linear Motifs Mediating Protein Nuclear Import
Abstract
Protein-protein interactions (PPI) mediated through short linear motifs (SLiMs) are ubiquitous throughout the human proteome and are involved in many essential cellular processes. One such type of SLiM is the classical nuclear localization sequence (cNLS), which facilitates nuclear import by binding importin-α (Imp-α). This pathway is indispensable to many cellular processes and is extensively used by viral proteins that function within the nucleus of infected cells. Based on this, I demonstrated that the classical nuclear import pathway inhibitor, ivermectin, can inhibit replication of human adenovirus. Treatment with ivermectin blocks nuclear localization of the E1A protein, an essential viral nuclear protein that functions early during infection. I also demonstrate, for the first time, that ivermectin inhibits the Imp-α/cNLS interaction. Interestingly though, despite the classical nuclear import pathway being extensively studied, up to 50% of Imp-α cargo in yeast do not have a cNLS, as one would expect. However, whether this is true with humans remained unclear. To address this, I used currently available databases and datasets for human Imp-α PPIs and computationally searched for cNLSs. Using my approach, I found that 20–50% of Imp-α interactors do not have predictable cNLS. Furthermore, I found that the majority of proteins in the Mediator complex associate with Imp-α without having a predictable cNLS. Based on these findings I hypothesized that components of Mediator are likely to be using a “piggybacking” mechanism. These findings also demonstrated a need for identifying piggybacking mechanisms and/or novel NLSs. To explore these questions, I developed a yeast-based genetic selection to identify peptides conferring nuclear import. This system uses a large recombinant protein to express randomly generated peptides that can be subsequently selected for based on their ability to facilitate nuclear import in yeast. Peptides that I identified in this selection were also able to localize EGFP to the nucleus and interact with Imp-α in human cells. This approach also represents a novel strategy to identify SLiMs in a high throughput fashion, an area of SLiM discovery that currently lacks high throughput experimental methods.