Structural and Molecular Basis for Multifunctional Roles of Aminopeptidase N

Abstract

Aminopeptidase N (APN, also known as CD13 or alanine aminopeptidase) is a zinc-dependent metallopeptidase widely expressed on the cell surfaces of many different tissues, including kidney, intestine, endothelium and central nervous system. Aside from regulating peptide metabolism in diverse physiological pathways, APN also plays pivotal roles in tumor cell motility, tumor-homing therapy, and coronavirus infection, all of which are seemingly unrelated to its aminopeptidase activity. How APN functions in these processes is largely a mystery. Therefore, in my thesis research, I used a combination of structural and functional studies to delineate the underlying mechanisms of APN-mediated tumor cell motility, tumor-homing therapy and coronavirus infection. APN is an important target for tumor-homing therapy because it serves as a receptor for tumor-homing peptide (peptides that bring anti-cancer drugs to tumor cells) Asn-Gly-Arg (NGR). To provide mechanistic insights into this interaction, I determined the crystal structure of APN in complex with a tumor-homing peptide containing a representative NGR motif. The tumor-homing peptide binds to the APN’s enzymatic active site, but it resists APN degradation due to a distorted scissile peptide bond. APN mediates tumor cell motility via its interactions with extracellular matrix (ECM) proteins. Using a variety of biochemical assays. I found that APN binds to, but does not degrade, NGR motifs in ECM proteins that share similar conformations with the NGR motif in the APN-bound tumor-homing peptide. Therefore, APN-based tumor cell motility and tumor-homing therapy rely on a unified mechanism in which both functions are driven by the specific and stable interactions between APN and the NGR motifs in ECM proteins and tumor-homing peptides. This study not only elucidates the molecular basis for APN-based tumor cell motility and tumor-homing therapy, but also facilitates the development of APN-targeting cancer therapies. Besides its above functions, APN is also the receptor for several coronaviruses such as human respiratory coronavirus 229E (HCoV-229E), porcine transmissible gastroenteritis virus (TGEV), feline enteric coronavirus (FCoV). Previous studies have revealed the coronavirus-binding sites on APN and they are not located in APN’s active site. In 2013, a TGEV-related coronavirus, porcine epidemic diarrhea coronavirus (PEDV), emerged in the United States and has significantly damaged America’s pork industry. I investigated the receptor usage and cell entry of PEDV, and found that PEDV used APN from both pig and human and a sugar coreceptor N-acetylneuraminic acid (Neu5Ac) for cell entry. Moreover, PEDV was able to infect cells from pig, human, monkey, and bat. These results provide insights in the transmission pathway of PEDV, implicate PEDV as a potential threat to other species, and suggest antiviral strategies to control its spread. Coronavirus spike proteins mediate viral entry into cells, a process that requires the spike proteins to be proteolytically activated. It has been a conundrum what proteases activate PEDV entry. To further characterize the cell entry process of PEDV, I systematically examined the roles of different host cell proteases in PEDV entry using pseudovirus entry and biochemical assays. I discovered that PEDV spike is activated by lysosomal cysteine proteases, but not proprotein convertases or cell-surface serine proteases. Extracellular trypsin activates PEDV entry when lysosomal cysteine proteases are inhibited. I further pinpointed cathepsin L and cathepsin B as the lysosomal cysteine proteases that activate PEDV spike. These results advance our understanding of the molecular mechanism for PEDV entry and identify potential antiviral targets for curbing the spread of PEDV. Collectively, my thesis work provides critical structural and molecular insights in better understanding the diverse functions of APN involved in pathological conditions of tumor progression and coronavirus infection. In addition, this work also lays foundation for therapeutic development to treat these APN-related health-threatening diseases.