Date of Award
Doctor of Philosophy (PhD)
The hepatocyte is one of the major secretory cell types in the body. It fulfills many of the liver's essential functions in protein secretion, lipid storage and transport, and excretion. Some of these functions are carried out via polarized secretion of simple protein cargo, such as serum albumin, or large macromolecular lipid-protein complexes, the lipoproteins. The hepatocyte is also the site of infection of several hepatotropic viruses. Of these, hepatitis C virus (HCV) is peculiar due to its close structural and functional association with the hepatic lipoproteins. All these cargoes are transported from the endoplasmic reticulum (ER) to the cell surface by the vesicular secretory pathway, yet insufficient knowledge exists regarding the molecular regulation of their secretion by the hepatocyte. Furthermore, differential modalities of regulation may be involved in the shuttling of such a diverse set of cargoes as albumin, the lipoproteins and HCV. The work presented here head-starts a comprehensive examination of how the hepatocyte regulates the secretion of the following cargoes: serum albumin, the apolipoproteins E and B100 (ApoE and ApoB100, respectively, both lipoprotein components, and surrogate markers for these complex macromolecular particles), and HCV, a lipoprotein-associated virus. I propose to combine genetic, biochemical, virological and imaging approaches to identify which vesicular secretory pathways are utilized by each of these cargoes. These approaches include inactivation of specific vesicular transport pathways, accompanied by measurements of their effects on cargo secretion efficiencies, and establishment of functional fluorescent protein-tagged cargo markers to be used in live cell imaging experiments. I begin by describing a dominant negative (DN) Rab GTPase screen that I performed to identify Rab proteins involved in ApoE, ApoB100 or albumin secretion. The small Rab GTPases control individual steps of vesicular transport. I analyzed how expression of individual dominant negative Rab proteins affected cargo secretion compared to expression of their wild type (WT) counterparts. I identified several Rabs that caused significant changes in secretion, many of which had previously been described as regulators of various exocytic vesicular transport steps. I next present ongoing work that aims to define the involvement of the Rabs 11a, 11b, 8a, and 8b in hepatic cargo secretion. Their dominant negative mutants exhibited some of the largest secretion phenotypes in my dominant negative Rab screen. These Rabs have been implicated in various aspects of post-Golgi secretion in polarized and non-polarized cell types. I thus discuss the implications of their involvement in cargo secretion in the polarized hepatocyte and outline my ongoing efforts to define the parameters of this involvement. I also investigated the function of Rab1b in hepatic secretion. I show that inactivation of Rab1 function, by expression of a set of dominant negative mutants, or by expression of a bacterial effector which affects Rab1 function, led to impairment of albumin, ApoE, ApoB100 and HCV secretion. I implicate Rab1, for the first time to my knowledge, in the transport of these cargoes. I also document differences in the sensitivity of cargo secretion to the various means of Rab1 inactivation. ApoE secretion, in particular, was insensitive to several means of transport inactivation, consistent with existing models of differential regulation of hepatic cargo transport. Lastly, I functionally characterize an ApoE-green fluorescent protein fusion (ApoE-GFP). I show that while ApoE-GFP does not support infectious HCV release, a hallmark function of untagged ApoE, ApoE-GFP nevertheless reproduces several known behaviors of ApoE that have been associated with lipoprotein release. I thus conclude that ApoE-GFP may be a useful marker for live cell imaging of lipoprotein release. This work therefore identifies potential regulators of hepatic cargo transport, establishes molecular tools useful for the continued study of cargo secretion in hepatocytes and elsewhere, and advances the understanding of the involvement of Rabs 11, 8, and, in particular, Rab1, in the regulation of hepatic cargo transport. I propose that this work forms a solid foundation for extensive studies on how these biomedically relevant hepatic cargoes are secreted.
Takacs, Constantin N., "Analysis of Hepatocyte Secretion Pathways: A Case Study on Hepatic Apolipoproteins, Serum Albumin, and Hepatitis C Virus" (2016). Student Theses and Dissertations. 305.