Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Master of Science

Program

Microbiology and Immunology

Supervisor

Dikeakos, Jimmy

Abstract

Phosphofurin acidic cluster sorting (PACS) proteins 1 and 2 are essential components of the cytoplasmic protein trafficking machinery. Recent reports have described novel roles for PACS-1 in the nucleus. Here, we defined the mechanism of PACS-1 nucleocytoplasmic shuttling by identifying the interactions of PACS-1 with nuclear transport receptors and mapping its nuclear localization/export signals. Furthermore, we described the molecular basis and the subcellular location of the interactions between PACS-1 and RNA-binding proteins, including the cellular polypyrimidine tract-binding protein 1 and the HIV-1 Rev protein. In addition, we characterized the poorly understood biochemical properties of a PACS-2 E209K mutation involved in neurodevelopmental epilepsy. Specifically, we elucidated that PACS-2 E209K had a longer half-life and enhanced interaction with the 14-3-3ε protein. The PACS-2 E209K mutation also increased susceptibility to staurosporine-induced apoptosis. Overall, this thesis extends our understanding of the PACS proteins and expands their roles in RNA metabolism and cellular stress responses.

Summary for Lay Audience

The biological cell is compartmentalized into discrete functional regions. Specialized transport proteins navigate within the subcellular metropolis and deliver biomolecules to their destinations. For example, the phosphofurin acidic cluster sorting (PACS) proteins 1 and 2 act as cellular mail carriers responsible for targeting proteins towards various subcellular locations: the endoplasmic reticulum, the Golgi apparatus, the trans-Golgi network (TGN), and the plasma membrane. While past studies have determined that PACS-1 ensures the proper localization of proteins within the TGN, it is unclear if PACS-1 has additional functions within other cellular regions.

Recently, PACS-1 was also observed to traffic into the nucleus of the cell. Nuclear PACS-1 interacts with additional nuclear proteins to increase DNA replication efficiency and ensure rapid cellular division. In this thesis, we explored the mechanism responsible for the ability of PACS-1 to move in and out of the nucleus. Using subcellular fractionation and microscopy, we identified the specific amino acid sequences required for PACS-1 to interact with the well-known nuclear receptor proteins, importin α5 and exportin 1.

We also observed that PACS-1 interacts with RNA transport molecules including the cellular polypyrimidine tract-binding protein 1 and HIV-1 Rev protein. RNA transport is an essential process for cells to produce proteins, but also a key step for viruses to establish successful infection. Thus, our results demonstrate a novel contribution of PACS-1 in RNA-related physiology, and this involvement of PACS-1 in RNA in biology is further hijacked by viruses, such as HIV-1, to enhance viral replication.

Lastly, both PACS proteins have mutations associated with developmental epileptic syndromes. My thesis explored the mechanisms of disease pathogenesis by specifically examining the PACS-2 E209K mutation at the molecular level. Here, we elucidated that PACS-2 E209K shifts its protein-protein interactions to decrease the susceptibility threshold for cells to undergo stress-induced death. These results hint at how the molecular properties of PACS-2 E209K pathologically disrupt cellular pathways, ultimately leading to neurological symptoms present in patients.

Overall, this work expands our understanding of the PACS proteins as multi-functional modulators, bridging biochemical processes across various facets of the subcellular landscape.

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