Cytoplasmic short linear motifs in ACE2 and integrin β ₃ link SARS-CoV-2 host cell receptors to mediators of endocytosis and autophagy

Abstract

The spike protein of SARS-CoV-2 binds the angiotensin-converting enzyme 2 (ACE2) on the host cell surface and subsequently enters host cells through receptor-mediated endocytosis. Additional cell receptors may be directly or indirectly involved, including integrins. The cytoplasmic tails of ACE2 and integrins contain several predicted short linear motifs (SLiMs) that may facilitate internalization of the virus as well as its subsequent propagation through processes such as autophagy. Here, we measured the binding affinity of predicted interactions between SLiMs in the cytoplasmic tails of ACE2 and integrin β3 with proteins that mediate endocytic trafficking and autophagy. We validated that a class I PDZ-binding motif mediated binding of ACE2 to the scaffolding proteins SNX27, NHERF3, and SHANK, and that a binding site for the clathrin adaptor AP2 μ2 in ACE2 overlaps with a phospho-dependent binding site for the SH2 domains of Src family tyrosine kinases. Furthermore, we validated that an LC3-interacting region (LIR) in integrin β3 bound to the ATG8 domains of the autophagy receptors MAP1LC3 and GABARAP in a manner enhanced by LIR-adjacent phosphorylation. Our results provide molecular links between cell receptors and mediators of endocytosis and autophagy that may facilitate viral entry and propagation.

ACE2 interaction with cytoplasmic PDZ protein enhances SARS-CoV-2 invasion

“…Similar PDZ interactions manifested by ACE2-NHERF1 have also been observed between ACE2 and NHERF3, a putative ACE2-interacting partner (Kliche et al, 2021). As shown in Figure S2, full-length myc-ACE2 overexpressed in HEK293 GnTI À cells coimmunoprecipitates with NHERF3-FLAG as observed for NHERF1.…”

“…An early report showed that deletion of the ACE2 C-terminal tail causes significant change in SARS-CoV incorporation (Inoue et al, 2007), implying an important role for the carboxy-terminus region and its potential interaction with unidentified intracellular proteins mediating viral entry. While the present study was in progress, a report postulated that the interacting partners associated with the cytoplasmic tail of ACE2 are likely to be PDZ-containing (Singh et al, 2009) (PDZ is an acronym for the first three identified proteins sharing a highly similar structural domain: Postsynaptic density 95/Disc large/Zonula occludens-1) proteins SNX27, NHERF3, or SHANK based on bioinformatics analysis and fluorescence polarization assay using FITC-labeled C-terminal peptides of ACE2 and individual truncated PDZ domains from the three mentioned proteins (Kliche et al, 2021). Functional evidence for that proposition was not provided.…”

Integrin activation is an essential component of SARS-CoV-2 infection

“…This study provides mechanistic evidence for the functionality of extracellular ligand-binding domains of integrin b1 and cytoplasmic tails of integrins in general, 24,28 which offer possible molecular links between ACE2 and integrins. We show that Mn 2+ , which induces integrin extension and high affinity ligand binding, enhances the cell entry of SARS-CoV-2 R18 .…”

In-vivo Protection from SARS-CoV-2 infection by ATN-161 in k18-hACE2 transgenic mice

“…This suggests that the concomitant elevation of these integrins and the upregulation of ACE2 in an organ may render it more susceptible to preferentially binds to and inhibits activated forms of α 5β1 in areas of inflammation, hypoxia and angiogenesis [30]. ATN-161 also binds to integrin α vβ3 [30], an additional integrin that has been implicated in SARS-CoV-2 pathogenesis [33,32], and is safe, well-tolerated in human clinical trials (cancer) with no dose limiting toxicity [28,66], and can be administered i.v., i.p., and intranasally [31,67]; The latter may support a more readily accessible means of COVID-19 treatment as well as afford a prophylactic approach which is currently under investigation in our laboratory.…”

“…We further demonstrated that the small pentapeptide α 5β1 integrin inhibitor ATN-161 could inhibit this binding and also decrease SARS-CoV-2 infection and cytopathy in cultured vero-E6 cells [26]. ATN-161 has several properties that make it potentially attractive as a novel SARS-CoV-2 therapy; it is safe and welltolerated with no dose-limiting toxicity in phase I cancer clinical trials [28], has demonstrated invivo efficacy in mice against a different beta coronavirus, porcine hemagglutinating encephalomyelitis virus (PHEV) [29] as well as other disease/injury models [30,31], and can also bind to and inhibit integrin α vβ3 which is similar to α 5β1 integrin that has also been implicated in SARS-CoV-2 infection [32,33]. In the present study we examined the therapeutic potential of ATN-161 in a human ACE2 receptor-expressing K18 mouse model of SARS-CoV-2 infection.…”