Coating and Stabilization of Liposomes by Clathrin-Inspired DNA Self-Assembly

The self-assembly of the protein clathrin on biological membranes facilitates essential processes of endocytosis and has provided a source of inspiration for materials design by the highly ordered structural appearance. By mimicking the architecture of the protein building blocks and clathrin self-assemblies to coat liposomes with biomaterials, advanced hybrid carriers can be derived. Here we present a method for fabricating DNA-coated liposomes by hydrophobically anchoring and subsequently connecting DNA-based triskelion structures on the liposome surface inspired by the assembly of the protein clathrin. Dynamic light scattering, ζ-potential, confocal microscopy and cryo-electron microscopy measurements independently demonstrate successful DNA coating. Nanomechanical measurements conducted with atomic force microscopy show that the DNA coating enhances the mechanical stability of the liposomes relative to uncoated ones. Furthermore, we provide the possibility to reverse the coating process by triggering the disassembly of the DNA coats through a toehold-mediated displacement reaction. Our results describe a straightforward, versatile, and reversible approach for coating and stabilizing lipid vesicles through the assembly of rationally designed DNA structures. This method has potential for further development towards the ordered arrangement of tailored functionalities on the surface of liposomes and for applications as hybrid nanocarrier

Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism

The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-ß (Aß) in Alzheimer’s disease and a-synuclein (aS) in Parkinson’s disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of aS, Aß and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases

Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism

10 pags., 5 figs.The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-β (Aβ) in Alzheimer’s disease and α-synuclein (αS) in Parkinson’s disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aβ and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.This work was supported by the Cambridge Centre for Misfolding Diseases (R.L., B.M., F.S.R., C.K.X., M.P., S.C., S.W.C., J.H., T.K., J.R.K., T.P.J.K., M.V., and C.M.D.), the UK Biotechnology and Biochemical Sciences Research Council (M.V. and C.M.D.), the Wellcome Trust (203249/Z/16/Z to T.P.J.K and M.V.), the Frances and Augustus Newman Foundation (T.P.J.K.), the Regione Toscana – FAS Salute, project SUPREMAL (R.C., A.B., C.C., and F.C.), the Gates Cambridge Trust and St. John’s College Cambridge (R.L.), Darwin College Cambridge (F.S.R.), the Herchel Smith Fund (C.K.X.), a Faculty Development Research Fund grant from the United States Military Academy, West Point (R.L.) and a DTRA Service Academy Research Initiative grant (HDTRA1033862 to R.L.)

A natural product inhibits the initiation of a-synuclein aggregation & suppresses its toxicity

The self-Assembly of a-synuclein is closely associated with Parkinson''s disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects a-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces a-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of a-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing a-synuclein, observing a dramatic reduction of a-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson''s disease and related conditions

Silk micrococoons for protein stabilisation and molecular encapsulation

Naturally spun silks generate fibres with unique properties, including strength, elasticity and biocompatibility. Here we describe a microfluidics-based strategy to spin liquid native silk, obtained directly from the silk gland of Bombyx mori silkworms, into micron-scale capsules with controllable geometry and variable levels of intermolecular β-sheet content in their protein shells. We demonstrate that such micrococoons can store internally the otherwise highly unstable liquid native silk for several months and without apparent effect on its functionality. We further demonstrate that these native silk micrococoons enable the effective encapsulation, storage and release of other aggregation-prone proteins, such as functional antibodies. These results show that native silk micrococoons are capable of preserving the full activity of sensitive cargo proteins that can aggregate and lose function under conditions of bulk storage, and thus represent an attractive class of materials for the storage and release of active biomolecules

In situ kinetic measurements of α-synuclein aggregation reveal large population of short-lived oligomers

Biological and Soft Matter Physic

The component polypeptide chains of bovine insulin nucleate or inhibit aggregation of the parent protein in a conformation-dependent manner

Amyloid fibrils are typically rigid, unbrariched structures with diameters of similar to 10 nm and lengths up to several micrometres, and are associated with more than 20 diseases including Alzheimer's disease and type II diabetes. Insulin is a small, predominantly alpha-helical protein consisting of 51 residues in two disulfide-linked polypeptide chains that readily assembles into amyloid fibrils under conditions of low PH and elevated temperature. We demonstrate here that both the A-chain and the B-chain of insulin are capable of forming amyloid fibrils in isolation under similar conditions, with fibrillar morphologies that differ from those composed of intact insulin. Both the A-chain and B-chain fibrils were found to be able to cross-seed the fibrillization of the parent protein, although these reactions were substantially less efficient than self-seeding with fibrils composed of full-length insulin. In both cases, the cross-seeded fibrils were morphologically distinct from the seeding, material, but shared common characteristics with typical insulin fibrils, including a very similar helical repeat. The broader distribution of heights of the cross-seeded fibrils compared to typical insulin fibrils, however, indicates that their underling protofilament hierarchy may be subtly different. In addition, and remarkably in view of this seeding behavior, the soluble forms of the A-chain and B-chain peptides were found to be capable of inhibiting insulin fibril formation. Studies using mass spectrometry suggest that this behavior might be attributable to complex formation between insulin and the A-chain and B-chain peptides. The finding that the same chemical form of a polypeptide chain in different physical states can either stimulate or inhibit the conversion of a protein into amyloid fibrils sheds new light on the mechanisms underlying fibril formation, fibril strain propagation and amyloid disease initiation and progression. (c) 2006 Elsevier Ltd. All rights reserved