Phosphate-Based Amphiphile and Lipidated Lysine Assemble into Superior Protocellular Membranes over Carboxylate and Sulfate-Based Systems: A Potential Missing Link between Prebiotic and the Modern Era?

Amphiphiles are among the most extensively studied building blocks that self-assemble into cell-like compartments. Most literature suggested that the building blocks/amphiphiles of early Earth (fatty acid-based membrane) were much simpler than today's phospholipids. To establish the bridge between the prebiotic fatty acid era and the modern phospholipid era, the investigation and characterization of alternate building blocks that form protocellular membranes are necessary. Herein, we report the potential prebiotic synthesis of alkyl phosphate, alkyl carboxylate, and alkyl sulfate amphiphiles (anionic) using dry-down reactions and demonstrate a more general role of cationic amino acid-based amphiphiles to recruit the anionic amphiphiles via ion-pair, hydrogen bonding, and hydrophobic interactions. The formation and self-assembly of the catanionic (mixed) amphiphilic system to vesicular morphology were characterized by turbidimetric, dynamic light scattering, transmission electron microscopy, fluorescence lifetime imaging microscopy, and glucose encapsulation experiments. Further experiments suggest that the phosphate-based vesicles were more stable than the alkyl sulfate and alkyl carboxylate-based systems. Moreover, the alkyl phosphate system can form vesicles at prebiotically relevant acidic pH (5.0), while alkyl carboxylate mainly forms cluster-type aggregates. An extended supramolecular polymer-type network formation via H-bonding and ion-pair interactions might order the membrane interface and stabilize the phosphate-based vesicles. The results suggest that phosphate-based amphiphiles might be a superior successor to fatty acids as early compartment building blocks. The work highlights the importance of previously unexplored building blocks that participate in protocellular membrane formation to encapsulate important precursors required for the functions of early life.

C-Terminal Lipidation of SARS-CoV-2 Fusion Peptide Reinstates Superior Membrane Fusion Catalytic Ability.

The spike (S) protein of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) mediates a critical stage in infection, the fusion between viral and host membranes. The protein is categorized as a class I viral fusion protein and has two distinct cleavage sites that can be activated by proteases. The activation deploys the fusion peptide (FP) for insertion into the target cell membranes. Recent studies including our experiments showed that the FP was unable to modulate the kinetics of fusion at a low peptide-to-lipid ratio akin to the spike density at the viral surface. Therefore, we modified the C terminus of FP and attached a myristoyl chain (C-myr-FP) to restrict the C terminus near to the interface, bridge both membranes, and increase the effective local concentration. The lipidated FP (C-myr-FP) of SARS-CoV-2 greatly accelerates membrane fusion at a low peptide-to-lipid ratio as compared to the FP with no lipidation. Biophysical experiments suggest that C-myr-FP adopts a helical structure, perturbs the membrane interface, and increases water penetration to catalyze fusion. Scrambled peptide (C-myr-sFP) and truncated peptide (C-myr-8FP) could not significantly catalyze the fusion, thus suggesting the important role of myristoylation and the N terminus. C-myr-FP enhances murine coronavirus infection by promoting syncytia formation in L2 cells. The C-terminal lipidation of the FP might be a useful strategy to induce artificial fusion in biomedical applications.

Lipid and Lipidation in Membrane Fusion.

Membrane fusion plays a lead role in the transport of vesicles, neurotransmission, mitochondrial dynamics, and viral infection. There are fusion proteins that catalyze and regulate the fusion. Interestingly, various types of fusion proteins are present in nature and they possess diverse mechanisms of action. We have highlighted the importance of the functional domains of intracellular heterotypic fusion, homotypic endoplasmic reticulum (ER), homotypic mitochondrial, and type-I viral fusion. During intracellular heterotypic fusion, the SNAREs and four-helix bundle formation are prevalent. Type-I viral fusion is controlled by the membrane destabilizing properties of fusion peptide and six-helix bundle formation. The ER/mitochondrial homotypic fusion is controlled by GTPase activity and the membrane destabilization properties of the amphipathic helix(s). Although the mechanism of action of these fusion proteins is diverse, they have some similarities. In all cases, the lipid composition of the membrane greatly affects membrane fusion. Next, examples of lipidation of the fusion proteins were discussed. We suggest that the fatty acyl hydrophobic tail not only acts as an anchor but may also modulate the energetics of membrane fusion intermediates. Lipidation is also important to design more effective peptide-based fusion inhibitors. Together, we have shown that membrane lipid composition and lipidation are important to modulate membrane fusion.