Branched amphiphilic peptide capsules (BAPCs) are peptide nanospheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble to form bi-layer delimited capsules. In two recent publications we described the lipid analogous characteristics of our BAPCs, examined their initial assembly, mode of fusion, solute encapsulation, and resizing and delineated their capability to be maintained at a specific size by storing them at 4 °C. In this report we describe the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs in vivo. The ability of our constructs to retain alpha particle emitting radionuclides without any apparent leakage and their persistence in the peri-nuclear region of the cell for extended periods of time, coupled with their ease of preparation and potential tune-ability, makes them attractive as biocompatible carriers for targeted cancer therapy using particle emitting radioisotopes. This article is part of a Special Issue entitled: Interfacially active peptides and proteins.
In a recent article (Gudlur et al., (2012) PLOS ONE, 7 (9) e45374) we described the special properties of a mixed branched peptide assembly in which equimolar bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK self-associate to form bilayer delimited capsules capable of trapping solutes. These poly-cationic vesicle-like capsules are readily taken up by epithelial cells in culture, escape or evade the endocytic pathway, and accumulate in the peri-nuclear region where they persist without any apparent degradation. In this report we examine the lipid-like properties of this system including initial assembly; solute encapsulation and washing; fusion and resizing by membrane extrusion through polycarbonate filters with defined pore sizes. The resized peptide capsules have uniform diameters in nm size ranges. Once resized, the capsules can be maintained at the new size by storing them at 4° C. Having the ability to prepare stable uniform nano-scale capsules of desired sizes makes them potentially attractive as biocompatible delivery vehicles for various solutes/drugs.
Branched amphiphilic peptide capsules (BAPCs) are biocompatible, bilayer delimited polycationic nanospheres that spontaneously form at room temperature through the coassembly of two amphiphilic branched peptides: bis(FLIVI)-K-K4 and bis(FLIVIGSII)-K-K4. BAPCs are readily taken up by cells in culture, where they escape and/or evade the endocytic pathway and accumulate in the perinuclear region, persisting there without apparent degradation or extravasation. Drugs, small proteins, and solutes as well as α particle emitting radionuclides are stably encapsulated for extended periods of time. BAPC formation at room temperature proceeds via a fusogenic process and after 48 h a range of BAPCs sizes are observed, from 50 nm to a few microns in diameter. It was previously reported that cooling BAPCs from 25 to 4 °C and then back to 25 °C eliminated their fusogenic property. In this report, biophysical techniques reveal that BAPCs undergo thermosensitive conformational transitions as a function of both time and temperature and that the properties of BAPCs vary based on the temperature of assembly. The solvent dissociation properties of BAPCs were studied as well as the contributions of specific amino acid residues to the observed conformations. The roles of the potential stabilizing forces present within the bilayer that bestow the unusal stability of the BAPCs are discussed. Ultimately this study presents revised assembly protocols for preparing BAPCs with discrete sizes and solvent-induced extravasation properties.
Various strategies are being developed to improve delivery and increase the biological half-lives of pharmacological agents. To address these issues, drug delivery technologies rely on different nano-sized molecules including: lipid vesicles, viral capsids and nano-particles. Peptides are a constituent of many of these nanomaterials and overcome some limitations associated with lipid-based or viral delivery systems, such as tune-ability, stability, specificity, inflammation, and antigenicity. This review focuses on the evolution of bio-based drug delivery nanomaterials that self-assemble forming vesicles/capsules. While lipid vesicles are preeminent among the structures; peptide-based constructs are emerging, in particular peptide bilayer delimited capsules. The novel biomaterial— Branched Amphiphilic Peptide Capsules (BAPCs) display many desirable properties. These nano-spheres are comprised of two branched peptides— bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK, designed to mimic diacyl-phosphoglycerides in molecular architecture. They undergo supramolecular self-assembly and form solvent-filled, bilayer delineated capsules with sizes ranging from 20 nm to 2 microns depending on annealing temperatures and time. They are able to encapsulate different fluorescent dyes, therapeutic drugs, radionuclides and even small proteins. While sharing many properties with lipid vesicles, the BAPCs are much more robust. They have been analyzed for stability, size, cellular uptake and localization, intra-cellular retention and, bio-distribution both in culture and in vivo.
C-terminus of Hsc/p70-Interacting Protein (CHIP) is a homodimeric E3 ubiquitin ligase. Each CHIP monomer consists of a tetratricopeptide-repeat (TPR), helix-turn-helix (HH), and U-box domain. In contrast to nearly all homodimeric proteins, CHIP is asymmetric. To uncover the origins of asymmetry, we performed molecular dynamics simulations of dimer assembly. We determined that a CHIP monomer is most stable when the HH domain has an extended helix that supports intra-monomer TPR-U-box interaction, blocking the E2-binding surface of the U-box. We also discovered that monomers first dimerize symmetrically through their HH domains, which then triggers U-box dimerization. This brings the extended helices into close proximity, including a repulsive stretch of positively charged residues. Unable to smoothly unwind, this conflict bends the helices until the helix of one protomer breaks to relieve the repulsion. The abrupt snapping of the helix forces the C-terminal residues of the other protomer to disrupt that protomer's TPR-U-box tight binding interface, swiftly exposing and activating one of the E2 binding sites. Mutagenesis and biochemical experiments confirm that C-terminal residues are necessary both to maintain CHIP stability and function. This novel mechanism indicates how a ubiquitin ligase maintains an inactive monomeric form that rapidly activates only after asymmetric assembly.Generally, the lowest energy state of protein assembly is symmetrical, whereas asymmetry is associated with energy frustration and structural instability 1 . However, symmetry is not evolutionarily constrained, and many oligomeric enzymes are known to be asymmetric with only half-of-sites active 2-5 . In the majority of such oligomers, asymmetry results from conformational changes triggered by ligand binding 2,6,7 . Another mechanism to fold and activate asymmetric dimers requires that one of the ligand-binding sites is deformed 3,6,7 . However, very few of the vast number of known homo-multimeric proteins assemble into asymmetric structures 6 . Among the exceptions are the C-terminus of Hsc/p70-Interacting Protein (CHIP) 8 , an E3 ubiquitin ligase that associates with cyoplasmic Hsp70 and Hsp90 chaperones, and Hikeshi 9 , a nuclear import protein that also binds Hsp70. Unveiling the mechanism of symmetry breaking in homo-oligomers will shed light on new principles of folding and assembly for this important class of proteins.The CHIP homodimer consists of three domains: tetratricopeptide repeat (TPR), helix-turn-helix (HH), and U-box 8 (Fig. 1). CHIP targets misfolded, chaperone-bound substrates for proteasomal degradation by transferring ubiquitin from a compatible E2 ubiquitin conjugating-enzyme, which associates with the U-box domain, to a lysine residue on the target protein 10, 11 . The crystal structure of murine CHIP (CHIP, PDB: 2C2L), which differs from human CHIP by one residue at the N-terminus, showcases an asymmetric homodimer in which only one U-box domain has an accessible E2-binding surface. The E2 binding site/U-box in t...
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