Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Microbiology and Immunology

Supervisor

Mymryk, Joe S.

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.

Summary for Lay Audience

A human cell can be broken down into several different compartments. The two largest compartments are represented by the nucleus and the cytoplasm, which are physically separate from each other by the nuclear envelope (NE). This separation ultimately means that all human genes are expressed within the nucleus and translated into proteins within the cytoplasm. Proteins are generally regarded as the functional molecules of a cell and are responsible for carrying out cellular processes in every compartment, including the nucleus. For proteins to enter the nucleus, they must contain a nuclear localization sequence (NLS). Importantly, many viral proteins have NLSs as well, allowing these proteins to enter the nucleus and promote viral replication. Here, I show that nuclear import can be targeted by the drug ivermectin to block the replication of human adenovirus, a clinically important virus which currently lacks specific antiviral treatments. Additionally, I demonstrated that many nuclear proteins in humans do not have an NLS. Looking specifically at these proteins without an NLS, I provided evidence that proteins can “piggyback” into the nucleus. This means that a protein with an NLS can physically interact with a non-NLS protein and carry it into the nucleus. In particular, I provide evidence that an important cellular protein complex, Mediator, is likely to use piggybacking as a strategy. Since many nuclear proteins do not have an NLS, strategies for finding how they are transported into the nucleus are needed. To address this, I developed a genetic selection in yeast. This approach used an engineered protein that is too large to enter the nucleus unless it has an NLS. Expressing this protein in yeast with random protein sequences allowed me to select for those that could mediate nuclear import, since only these yeast were able to survive. Using this selection, I identified several protein sequences that look nothing like current NLSs. Interestingly, two of these NLSs were also able to function in human cells. Together, my findings demonstrate that additional strategies to gain access to the nucleus exist and that the nuclear import pathway can be targeted to inhibit viral replication.

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