Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Microbiology and Immunology

Supervisor

Dikeakos, Jimmy D.

Abstract

Membrane trafficking events are required to direct proteins to their precise subcellular locations. The cellular Phosphofurin Acidic Cluster Sorting protein – 1 (PACS-1) has emerged as a protein of interest in controlling the localization of a multitude of cellular and viral proteins. Specifically, PACS-1 is hijacked by type-1 Human Immunodeficiency Virus (HIV-1) to contribute to immune evasion in addition to regulating neuroendocrine hormone storage and release. To accomplish this, PACS-1 connects the cytoplasmic tail of cellular receptors to the heterotetrameric adaptor proteins (APs) to form a functional trafficking unit. Throughout this dissertation, I explored the role of PACS-1 and AP-1 to drive the localization of unique cargo proteins; the type-1 major histocompatibility complex (MHC-I) and the adrenocorticotropic hormone (ACTH). I have utilized the intracellular protein-protein interaction reporter assay: bimolecular fluorescence complementation (BiFC), in combination with super-resolution microscopy to uncover the membrane trafficking route undertaken by the HIV-viral accessory protein Nef and the cellular receptor MHC-I. Additionally, I have generated a tool termed “viral BiFC” to study virus:host interactions in cells. These studies revealed a mechanism by which Nef re-routes MHC-I from the cell surface, toward the trans-Golgi Network (TGN) by hijacking both early and recycling endosomes. Interestingly, Nef requires PACS-1 to permit the transport of MHC-I from the endosomes to the TGN, a process which is also dependent on AP-1 recruitment. Moreover, the role of PACS-1 extends beyond its implication in HIV-1 infection. The regulation of neuroendocrine cell hormone storage and secretion is a tightly regulated process requiring coordinated trafficking of multiple proteins. Thus, I hypothesized that PACS-1 would promote proper storage of ACTH within specialized storage granules. These studies identified a key role for PACS-1 and AP-1 in directing ACTH to the storage granule. Undoubtedly, the function of PACS-1 is important in not only the immune evasive capabilities of HIV-1 Nef, but also in the regulation of hormone secretion. By understanding how cargo molecules are targeted throughout the cell by PACS-1, we can begin to unravel the molecular details of viral pathogenesis and cellular homeostasis.

Available for download on Monday, June 01, 2020

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