Graduation Date

Spring 5-8-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pathology & Microbiology

First Advisor

Hamid Band

Abstract

STUDY 1: Role of endocytic regulator EHD1 and its binding partner RUSC2 in EGFR traffic

Abstract

Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors. Cell surface display of EGFR is essential for cellular responses to its ligands. While post activation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/pre-activation surface display of EGFR. Here, we identify a novel role of the endocytic regulator EHD1 and a potential EHD1 partner, RUSC2, in cell surface display of EGFR. EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular structures and at the Golgi compartment. Inducible EHD1 knockdown reduced the cell surface EGFR expression with accumulation at the Golgi compartment, a phenotype rescued by exogenous EHD1. RUSC2 knockdown phenocopied the EHD1 depletion effects. EHD1 or RUSC2 depletion impaired the EGF-induced cell proliferation, demonstrating that the novel, EHD1- and RUSC2-dependent transport of unstimulated EGFR from the Golgi compartment to the cell surface that we describe is functionally important, with implications for physiologic and oncogenic roles of EGFR and targeted cancer therapies.


STUDY 2: ROLE OF CLATHRIN-MEDIATED ENDOCYTOSIS REGULATOR AAK1 IN SARS-CoV-2 ENTRY INTO HOST CELLS

Abstract

The immune system of human populations is naïve towards the newly-emerged Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Combined with the high transmission rate and a huge burden of morbidity and mortality among patients, Coronavirus disease (COVID-19) has led to enormous healthcare and economic emergency worldwide. SARS-CoV-2 uses its spike (S) protein to bind to the cell surface receptor Angiotensin-Converting Enzyme 2 (ACE2). The first step in subsequent viral infection is the entry of the virus-receptor complex into the target cell through endocytosis. Most viruses use the endocytic pathways to deliver viral contents into the host cell. Among them, the most used pathway is clathrin-mediated endocytosis. It has been shown that SARS-CoV, a close relative of SARS-CoV-2, uses the clathrin-mediated endocytosis for entry into acidic endosomes where viral envelope to cell membrane fusion takes place. Thus, we posited that SARS-CoV-2 also utilizes the same mechanism to enter the host cells. Studies of non-coronavirus systems have shown that AP2 adaptor-associated kinase AAK1 functions as a key positive regulator of clathrin-mediated viral endocytosis by phosphorylating the mu subunit of Adaptor protein 2 complex (AP2M1). Here, we modeled the SARS-CoV-2 entry into host cells using the receptor-binding domain of the viral Spike protein (S-RBD) and used a combination of genetic and pharmacological approaches to test a hypothesis that AAK1 is required for the endocytosis of SARS-CoV-2. We found that genetic knockdown of AAK1 with siRNA reduced the internalization of S-RBD, suggesting a role for AAK1 in the entry of SARS-CoV-2 into host cells. Towards a chemical inhibitor approach to determine the role of AAK1 in SARS-CoV-2 entry into host cells, we employed AAK1 specific inhibitors and clinically-used drugs that have emerged to have an AAK1 inhibitory activity. In all analyses of chemical inhibitors, we used their ability to reduce the AP2M1 phosphorylation as a readout of AAK1 inhibitory activity to build dose responses. The dose-response analyses showed that while all the inhibitors tested had AAK1 inhibitory activity, only the specific AAK1 inhibitors LP-935509 and LP-922761 and the anticancer agent Sunitinib among the clinically-used drugs displayed AAK1 inhibitory activity at low concentrations. Accordingly, Sunitinib, and the AAK1 specific inhibitors LP-935509 and LP-922761 exhibited a dose-dependent inhibition of the SARS-CoV-2 S-RBD internalization into host cells. These genetic and pharmacological approaches suggest the requirement of AAK1 in the entry of SARS-CoV-2 and provide an insight into the mechanism of viral entry by regulating the clathrin-mediated endocytic machinery. These studies support the potential of AAK1 inhibition as an approach to thwart SARS-CoV-2 infection.

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