1. Conformational studies of a bombolitin III-derived peptide mimicking the four-helix bundle structural motif of proteins
Elisabetta Schievano, Stefano Mammi, Luca Monticelli, Marta Ciardella, Evaristo Peggion J Am Chem Soc. 2003 Dec 17;125(50):15314-23. doi: 10.1021/ja0300970.
Bombolitins are five structurally related heptadecapeptides originally isolated from the venom of a bumblebee. In aqueous solution, bombolitins at sufficiently high concentration form oligomeric aggregates with consequent conformational transition from a random coil to the alpha-helical structure. Previous studies suggested that oligomeric aggregates could mimic the four-helix bundle structural motif of proteins. In the present work, we synthesized the following peptide sequence formed by two bombolitin III sequences linked head-to-tail by the tetrapeptide bridge -Gly-Pro-Val-Asp-: I(1)-K(2)-I(3)-M(4)-D(5)-I(6)-L(7)-A(8)-K(9)-L(10)-G(11)-K(12)-V(13)-L(14)-A(15)-H(16)-V(17)-G(18)-P(19)-V(20)-D(21)-I(22)-K(23)-I(24)-M(25)-D(26)-I(27)-L(28)-A(29)-K(30)-L(31)-G(32)-K(33)-V(3)(4)-L(35)-A(36)-H(37)-V(38)-NH(2). The tetrapeptide GPVD connecting the two helical peptide sequences was chosen to facilitate the formation of the helix-loop-helix structural motif. The conformational properties of the peptide were studied by CD, NMR, and molecular dynamics calculations. The results indicate the presence of a helix-loop-helix conformation at 10(-)(5) M concentration. At higher concentrations, NOESY connectivities were detected which are compatible with the presence of dimers or higher aggregates of peptide molecules in the helix-loop-helix structure packed in an antiparallel fashion. Molecular dynamics simulation were run either with NOE distance restraints or without restraints in explicit solvent for extended time. The results of these simulations support the dimerization of the molecules in the helix-loop-helix structure with formation of the four-helix bundle motif.
2. Novel endosomolytic peptides for enhancing gene delivery in nanoparticles
Aqeel Ahmad, Sanjeev Ranjan, Weikai Zhang, Jing Zou, Ilmari Pyykkö, Paavo K J Kinnunen Biochim Biophys Acta. 2015 Feb;1848(2):544-53. doi: 10.1016/j.bbamem.2014.11.008. Epub 2014 Nov 15.
Trapping in the endosomes is currently believed to represent the main barrier for transfection. Peptides, which allow endosomal escape have been demonstrated to overcome this barrier, similarly to the entry of viruses. However, the design principles of such endosomolytic peptides remain unclear. We characterized three analogs derived from membrane disrupting antimicrobial peptides (AMP), viz. LL-37, melittin, and bombolitin V, with glutamic acid substituting for all basic residues. These analogs are pH-sensitive and cause negligible membrane permeabilization and insignificant cytotoxicity at pH7.4. However, at pH5.0, prevailing in endosomes, membrane binding and hemolysis of human erythrocytes become evident. We first condensed the emerald green fluorescent protein (emGFP) containing plasmid by protamine, yielding 115 nm diameter soluble nanoplexes. For coating of the nanoplex surface with a lipid bilayer we introduced a hydrophobic tether, stearyl-octa-arginine (SR8). The indicated peptides were dissolved in methanol and combined with lipid mixtures in chloroform, followed by drying at RT under a nitrogen flow. The dry residues were hydrated with nanoplexes in Hepes, pH7.4 yielding after a 30 min incubation at RT,rather monodisperse nanoparticles having an average diameter of 150-300 nm, measured by DLS and cryo-TEM. Studies with cell cultures showed the above peptides to yield expression levels comparable to those obtained using Lipofectamine 2000. However, unlike the polydisperse aggregates formed upon mixing Lipofectamine 2000 and plasmid, the procedure described yields soluble, and reasonably monodisperse nanoparticles, which can be expected to be suitable for gene delivery in vivo, using intravenous injection.
3. Bombolitins, a new class of mast cell degranulating peptides from the venom of the bumblebee Megabombus pennsylvanicus
A Argiolas, J J Pisano J Biol Chem. 1985 Feb 10;260(3):1437-44.
Five structurally related heptadecapeptides rich in hydrophobic amino acids have been discovered in the venom of the bumblebee Megabombus pennsylvanicus. We have named them bombolitin I (Ile-Lys-Ile-Thr-Thr-Met-Leu-Ala-Lys-Leu-Gly-Lys-Val-Leu-Ala-His-Val-NH2 ), bombolitin II (Ser-Lys-Ile-Thr-Asp-Ile-Leu-Ala-Lys-Leu-Gly-Lys-Val-Leu-Ala-His-Val-NH2 ), bombolitin III (Ile-Lys-Ile-Met-Asp-Ile-Leu-Ala-Lys-Leu-Gly-Lys-Val-Leu-Ala-His-Val-NH2 ), bombolitin IV (Ile-Asn-Ile-Lys-Asp-Ile-Leu-Ala-Lys-Leu-Val-Lys-Val-Leu-Gly-His-Val-NH2 ), and bombolitin V (Ile-Asn-Val-Leu-Gly-Ile-Leu-Gly-Leu-Leu-Gly-Lys-Ala-Leu-Ser-His-Leu-NH2 ). Bombolitins are structurally and functionally very similar. They lyse erythrocytes and liposomes, release histamine from rat peritoneal mast cells, and stimulate phospholipase A2 from different sources. The threshold dose is 0.5-2.5 micrograms/ml depending on the peptide and the bioassay. Bombolitin V is as potent as the well-known melittin in lysing guinea pig erythrocytes (ED50 = 0.7 microgram/ml = 4 X 10(-7) M) and is 5 times more potent than mastoparan in causing mast cell degranulation, making it one of the most potent degranulating peptides discovered so far (ED50 = 2 micrograms/ml = 1.2 X 10(-6) M). The bombolitins represent a unique structural class of peptides but they have the same biological properties as melittin (from honeybees), mastoparan (wasps, hornets, and yellow jackets), and crabrolin (European hornets). This unusual circumstance (peptides with different amino acid sequences having the same biological properties) may be a manifestion of their amphiphilic nature, a property these peptides have in common.
