Investigating the role of lipid monolayer properties in ACE2 and TMPRSS2 incorporation

The entry of SARS-CoV-2 into host cells primarily involves binding of the viral spike (S) protein to the angiotensin-converting enzyme 2 (ACE2) receptor and subsequent fusion of the viral envelope with the host membrane, a process facilitated by host proteases such as transmembrane serine protease 2 (TMPRSS2). Lipid raft domains are believed to influence this internalization pathway, although the precise localization and functional roles of ACE2 and TMPRSS2 within these domains remain unclear. In this study, we employed mixed Langmuir monolayers-representing the plasma membrane (PM) and two lipid raft models (LR and Chol/SM)-to investigate the interfacial behavior of ACE2 and TMPRSS2 fragments. Using tensiometric, microscopic, and spectroscopic techniques, we found that both proteins were more readily incorporated into fluid and loosely packed monolayers (PM and LR), leading to increased molecular area and disruption of lipid organization. In contrast, the tightly packed Chol/SM monolayer exhibited minimal changes, indicating limited protein insertion. These results demonstrate that monolayer composition and packing significantly influence protein incorporation and arrangement, which may in turn affect their accessibility to viral components. Although lipid rafts are proposed sites of ACE2 and TMPRSS2 enrichment, our findings suggest that their structural organization within such domains may be modulated by the physicochemical properties of the surrounding lipid environment, with potential implications for SARS-CoV-2 infection mechanisms.