RESUMEN
Peptide-mediated membrane fusion is frequently studied with in vitro bulk leaflet mixing assays based on Förster resonance energy transfer (FRET). In these, customized liposomes with fusogenic peptides are equipped with lipids which are labeled with fluorophores that form a FRET pair. Since FRET is dependent on distance and membrane fusion comes along with lipid mixing, the assays allow for conclusions on the membrane fusion process. The experimental outcome of these assays, however, greatly depends on the applied parameters. In the present study, the influence of the peptides, the size of liposomes, their lipid composition and the liposome stoichiometry on the fusogenicity of liposomes are evaluated. As fusogenic peptides, soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) protein analogues featuring artificial recognition units attached to the native SNARE transmembrane domains are used. The work shows that it is important to control these parameters in order to be able to properly investigate the fusion process and to prevent undesired effects of aggregation.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Péptidos/química , Proteínas SNARE/química , Péptidos/síntesis químicaRESUMEN
PNA-peptide conjugates are versatile tools in chemical biology, which are employed in a variety of applications. Here, we present the synthesis of PNA-peptide conjugates that serve as SNARE protein-mimicking biooligomers. They resemble the structure of native SNARE proteins but exhibit a much simpler architecture. Incorporated into liposomes, they induce lipid mixing, so that they can be used to study the SNARE-mediated membrane fusion in a simplified setting in vitro. They consist of artificial SNARE recognition units made out of PNA oligomers, which are attached to the native linker and transmembrane domains of two neuronal SNAREs. The PNA-peptide conjugates are synthesized via solid-phase peptide synthesis in a continuous fashion starting with the peptide part, followed by assembly of the PNA recognition unit. On top, we describe a strategy to synthesize PNA-peptide conjugates in a fully automated fashion by using a peptide synthesizer.
Asunto(s)
Mimetismo Biológico , Biomimética , Técnicas de Química Sintética , Ácidos Nucleicos de Péptidos/química , Péptidos/química , Proteínas SNARE/química , Biomimética/métodos , Liposomas , Fusión de Membrana , Estructura Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Técnicas de Síntesis en Fase Sólida , Relación Estructura-ActividadRESUMEN
Membrane fusion is an essential process in nature and is often accomplished by the specific interaction of SNARE proteins. SNARE model systems, in which SNARE domains are replaced by small artificial units, represent valuable tools to study membrane fusion in vitro. The synthesis and analysis is presented of SNARE model peptides that exhibit a recognition motif composed of two different types of peptide nucleic acid (PNA) sequences. This novel recognition unit is designed to mimic the SNARE zippering mechanism that initiates SNARE-mediated fusion. It contains N-(2-aminoethyl)glycine-PNA (aeg-PNA) and alanyl-PNA, which both recognize the respective complementary strand but differ in duplex topology and duplex formation kinetics. The duplex formation of PNA hybrid oligomers as well as the fusogenicity of the model peptides in lipid-mixing assays were characterized and the peptides were found to induce liposome fusion. As an unexpected discovery, peptides with a recognition unit containing only five aeg-PNA nucleo amino acids were sufficient and most efficient to induce liposome fusion.
Asunto(s)
Liposomas/química , Ácidos Nucleicos de Péptidos/química , Péptidos/química , Proteínas SNARE/química , Dicroismo Circular , Liposomas/metabolismo , Fusión de Membrana , Modelos Moleculares , Ácidos Nucleicos de Péptidos/metabolismo , Péptidos/metabolismo , Proteínas SNARE/metabolismoRESUMEN
Fusion of synaptic vesicles with the presynaptic plasma membrane is mediated by Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein Receptor proteins also known as SNAREs. The backbone of this essential process is the assembly of SNAREs from opposite membranes into tight four helix bundles forcing membranes in close proximity. With model systems resembling SNAREs with reduced complexity we aim to understand how these proteins work at the molecular level. Here, peptide nucleic acids (PNAs) are used as excellent candidates for mimicking the SNARE recognition motif by forming well-characterized duplex structures. Hybridization between complementary PNA strands anchored in liposomes through native transmembrane domains (TMDs) induces the merger of the outer leaflets of the participating vesicles but not of the inner leaflets. A series of PNA/peptide hybrids differing in the length of TMDs and charges at the C-terminal end is presented. Interestingly, mixing of both outer and inner leaflets is seen for TMDs containing an amide in place of the natural carboxylic acid at the C-terminal end. Charged side chains at the C-terminal end of the TMDs are shown to have a negative impact on the mixing of liposomes. The length of the TMDs is vital for fusion as with the use of shortened TMDs, fusion was completely prevented.
Asunto(s)
Fusión de Membrana , Modelos Biológicos , Dominios y Motivos de Interacción de Proteínas , Proteínas SNARE/metabolismo , Aminoácidos , Lípidos/química , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/metabolismo , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Proteínas SNARE/químicaRESUMEN
Neuronal exocytosis is catalysed by the SNAP receptor protein syntaxin-1A, which is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis. However, how syntaxin-1A is sequestered is unknown. Here we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Using super-resolution stimulated-emission depletion microscopy on the plasma membranes of PC12 cells, we found that PIP2 is the dominant inner-leaflet lipid in microdomains about 73 nanometres in size. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the carboxy-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein-lipid interactions can result in the formation of microdomains independently of cholesterol or lipid phases.