Charge and membrane curvature induced transitions in biomolecular systems: from actin bundles to cell-penetrating peptides to Bcl-2 cell death regulation in the mitochondria

Actin bundling;Cell-penetrating peptides;transactivator of transcription protein transduction domain from HIV-1 (TAT);negative Gaussian membrane curvature;Membrane-mediated interactions;Bcl-2 apoptotic proteins;bcl-2-like protein 4 (Bax, Bcl-xL)

Lai, Ghee Hwee

关键词: Actin bundling;    Cell-penetrating peptides;    transactivator of transcription protein transduction domain from HIV-1 (TAT);    negative Gaussian membrane curvature;    Membrane-mediated interactions;    Bcl-2 apoptotic proteins;    bcl-2-like protein 4 (Bax, Bcl-xL);   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/29442/Lai_GheeHwee.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

This thesis reports several research directions that all converge on the common goal of understanding the structural transitions induced by interactions among important biological polyelectrolytes and biological membranes. Essential biopolymers F-actin and DNA have high negative charge densities, and yet can condense onto themselves as close-packed structures. Correlations between multivalent counterions condensed on the biopolymers are thought to mediate the attraction, but predict macroscopic phase separation inconsistent with the experimental observation of finite bundles. Equilibrium or kinetic constraints may limit the bundle size. Here, we examined the evolution of growth modes of F-actin condensed by Mg2+ and proposed that F-actin bundling is unlikely to be constrained by kinetics. Next, we examined DNA/F-actin mixtures as a model system of like-charged rigid rods and flexible chains. We found elongated nematic F-actin domains reticulating via defect-free vertices into a network that was embedded in a random mesh of DNA. Synchrotron scattering and microscopy showed a drastic transition as the system evolved from a counterion-controlled regime to a depletion-controlled regime with small changes in monovalent salt level.Extending the field of interest from 1D polyelectrolyte to 2D membrane systems, we examined the fundamental deformation modes cell-penetrating peptides, such as TAT, induce on bacterial-like membranes.TAT belongs to a class of arginine-rich cationic peptides capable of direct translocation as well as endocytotic pathways in eukaryotic cells, and we have recently shown that TAT is capable of generating negative Gaussian membrane curvature topologically necessary for pore formation. By additionally applying earlier studied concepts from polyelectrolyte condensation, we demonstrated and discussed how the arginine-rich and highly cationic sequence of these cell-penetrating peptides can multiplex interactions with the membrane, the actin cytoskeleton and cell-surface receptors to facilitate different cellular entry pathways. Generalizing to other pore-forming systems, the final piece of work details the experimental and theoretical observations on the Bcl-2 family of proteins which dictate cell death by permeabilizing the outer membrane of the mitochondria during apoptosis and are important therapeutic targets for fighting cancer. Direct binding between BH3 domains common within the Bcl-2 protein family are thought to regulate apoptotic activity: BH3-only proteins bind and activate the pore former Bax, whereas Bcl-xL bind to Bax or BH3-only activators to prevent permeabilization. While the physiologic roles of different Bcl-2 proteins are well understood, details of regulatory mechanisms remain contentious, and there is no unified picture that integrates the non-passive role of lipid membranes during apoptosis. Correlating membrane deformational modes inferred from synchrotron x-ray scattering with confocal microscopy of giant vesicle dye leakage, we report that Bax induces the same class of curvature (negative Gaussian curvature) as cell-penetrating peptides and antimicrobial peptides. More importantly, Bcl-xL can suppress not only Bax-induced pores, but also membrane remodeling by entirely unrelated peptides from the cell-penetrating, antimicrobial or fusion peptide families. We formulate a theoretical understanding, and experimentally verify a membrane curvature-mediated model in which Bax and Bcl-xL induce antagonistic Gaussian membrane curvatures to regulate pore formation. We propose that the universal nature of curvature-mediated interactions affords synergy with direct protein-protein binding pathways, enables Bcl-xL to suppress negative Gaussian curvature induced by entirely unrelated peptides, bacterial toxins, and even high salt screening in pure lipid-water systems without other proteins or peptides, and suggests a new general strategy for engineering apoptosis.