Interaction Between Supercharged Guanidinium-Rich Peptides and the Lipid Bis(monoacylglycero)phosphate Enables Cytosolic Penetration into Live Human Cells: Structure-Activity Relationships and Mechanisms
Abstract
The effective delivery of hydrophilic, bioactive molecules, such as enzymes and nucleic acids, to the cytosol of cells has proven to be a multibillion-dollar problem for cell biologists and the pharmaceutical industry. In recent studies, unprecedented cytosolic delivery of such cargos was achieved by the utilization of cell-penetrating agents. These densely charged, polycationic molecules, whether of biological or synthetic origin, have shown the ability to penetrate human cells and, concomitantly, carry macromolecular cargos into the intracellular milieu, albeit with variable efficiencies. Although this method of cell delivery seems promising, the molecular underpinnings involved in such transport remain unclear and, by proxy, limit advancements in this technology. In this study, I determine the effect of charge density (net charge / kDa) on cell penetration and gain insight into the proposed mechanism of transport by using the prototypical cell-penetrating peptide TAT. Cell penetrating peptide variants were synthesized containing one, two, or three copies of the TAT peptide on a synthetic scaffold to generate branched cell-permeable prototypes with increasing charge density. I establish that increasing TAT copies dramatically increases the cell penetration efficiency of the peptides while simultaneously enabling the efficient cytosolic delivery of macromolecular cargos. In previous studies, it has been shown that TAT-mediated cellular entry involves the leaky fusion of late endosomal membranes enriched with the anionic lipid BMP. We found that the derivatives with two and three TAT branches, 2TAT and 3TAT, induce the leakage of lipid bilayers specifically containing BMP. Furthermore, these compounds lead to liposomal flocculation, fusion and an increase in lamellarity. In contrast, while the monomeric counterpart 1TAT binds to the same extent and causes liposomal flocculation, 1TAT does not induce fusion or a significant increase in lamellarity. Overall, these results indicate that an increase in the peptide density of these branched structures leads to the emergence of membrane disruption and cell penetration activities. Moreover, I sought to identify additional properties of BMP-containing membranes that play a role in endosomolysis. In this study, I found that late endosomal membranes are substantially more fluid or disordered than many other biological membranes. The source of this membrane fluidity stems from BMP itself. By utilizing phospholipid components with variable extents of unsaturation, I generated a series of late endosomal liposome mimics that increased in membrane fluidity as a function of unsaturation. As the liposomes increased in fluidity, 3TAT-induced membrane leakage increased as well. Conversely, liposomes containing saturated fatty acids leaked to a lesser extent than their unsaturated fatty acid counterparts. Taken together these results suggest that while the guanidinium-BMP interaction is necessary to cause endosomolysis, the intrinsic fluidity of BMP-enriched membranes directly impacts the extent of endosomolysis imparted by supercharged CPPs.
Subject
bis(monoacylglycero)phosphatecell-penetrating peptides
cellular delivery
endosomal escape
membrane leakage
supercharged molecules
Citation
Brock, Dakota James (2019). Interaction Between Supercharged Guanidinium-Rich Peptides and the Lipid Bis(monoacylglycero)phosphate Enables Cytosolic Penetration into Live Human Cells: Structure-Activity Relationships and Mechanisms. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /186552.