a b s t r a c tArginine-rich cell-penetrating peptides are short cationic peptides capable of traversing the plasma membranes of eukaryotic cells. While successful intracellular delivery of many biologically active macromolecules has been accomplished using these peptides, their mechanisms of cell entry are still under investigation. Recent dialogue has centered on a debate over the roles that direct translocation and endocytotic pathways play in internalization of cell-penetrating peptides. In this paper, we review the evidence for the broad range of proposed mechanisms, and show that each distinct process requires negative Gaussian membrane curvature as a necessary condition. Generation of negative Gaussian curvature by cell-penetrating peptides is directly related to their arginine content. We illustrate these concepts using HIV TAT as an example.
Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using "nunchuck" CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay ("negative Gaussian") membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.protein transduction domain | polyarginine | peptide-lipid interactions | pore-forming peptide | antimicrobial peptide C ell-penetrating peptides (CPPs) are effective intracellular delivery systems (1-5). These peptides are usually short (<20 amino acids) and cationic. Examples include the TAT peptide from HIV, antennapedia (ANTP) from Drosophila, and even simple polyarginines. Although unique molecular architectures incorporating CPPs have been designed for drug delivery (3, 6-8), the molecular mechanisms of cellular entry, and the relations between them, are not well understood. Different uptake mechanisms have been proposed for CPPs (9). Cell-based assays have indicated that multiple endocytotic pathways are involved (10-15). In addition to these, CPPs are also capable of direct entry mechanisms* (17-20). In general, cell-penetrating activity of CPPs has proven to be difficult to eliminate completely using a specific set of conditions (3,12,21), suggesting the existence of multiple mechanisms. A unified understanding of CPPs, which is currently lacking, must engage why the same sequence can readily activate the qualitatively distinct outcomes.How do relatively simple molecules like HIV TAT peptide facilitate mechanisms as different as direct translocation, and multiple endocytotic processes? Rather than debate priority between mechanisms, we focus on the physical chemistry of what these different mechanisms and CPPs have in common. Here, we show how the TAT peptide can multiplex different interactions with the same sequence, thus interacting with the membrane, the actin cytoskeleton, and specific receptors to produce multiple pathways of translocation under different condition...
Infection of mice with sporozoites of Plasmodium berghei or Plasmodium yoelii has been used extensively to evaluate liver-stage protection by candidate preerythrocytic malaria vaccines. Unfortunately, repeated success of such vaccines in mice has not translated readily to effective malaria vaccines in humans. Thus, mice may be used better as models to dissect basic parameters required for immunity to Plasmodium-infection than as preclinical vaccine models. In turn, this basic information may aid in the rational design of malaria vaccines. Here, we describe a model of circumsporozoite-specific memory CD8 T cell generation that protects mice against multiple P. berghei sporozoite challenges for at least 19 months. Using this model we defined a threshold frequency of memory CD8 T cells in the blood that predicts long-term sterilizing immunity against liver-stage infection. Importantly, the number of Plasmodium-specific memory CD8 T cells required for immunity greatly exceeds the number required for resistance to other pathogens. In addition, this model allowed us to identify readily individual immunized mice that exceed or fall below the protective threshold before infection, information that should greatly facilitate studies to dissect basic mechanisms of protective CD8 T cell memory against liver-stage Plasmodium infection. Furthermore, the extremely large threshold in memory CD8 T cell frequencies required for long-term protection in mice may have important implications for development of effective malaria vaccines.
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