RESUMO
All cells use organized lipid compartments to facilitate specific biological functions. Membrane-bound organelles create defined spatial environments that favor unique chemical reactions while isolating incompatible biological processes. Despite the fundamental role of cellular organelles, there is a scarcity of methods for preparing functional artificial lipid-based compartments. Here, we demonstrate a robust bioconjugation system for sequestering proteins into zwitterionic lipid sponge phase droplets. Incorporation of benzylguanine (BG)-modified phospholipids that form stable covalent linkages with an O6 -methylguanine DNA methyltransferase (SNAP-tag) fusion protein enables programmable control of protein capture. We show that this methodology can be used to anchor hydrophilic proteins at the lipid-aqueous interface, concentrating them within an accessible but protected chemical environment. SNAP-tag technology enables the integration of proteins that regulate complex biological functions in lipid sponge phase droplets, and should facilitate the development of advanced lipid-based artificial organelles.
Assuntos
Gotículas Lipídicas , O(6)-Metilguanina-DNA Metiltransferase , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Fosfolipídeos , ProteínasRESUMO
Despite the central importance of lipid membranes in cellular organization, it is challenging to reconstitute their formation de novo from minimal chemical and biological elements. Here, we describe a chemoenzymatic route to membrane-forming noncanonical phospholipids in which cysteine-modified lysolipids undergo spontaneous coupling with fatty acyl-CoA thioesters generated enzymatically by a fatty acyl-CoA ligase. Due to the high efficiency of the reaction, we were able to optimize phospholipid formation in a cell-free transcription-translation (TX-TL) system. Combining DNA encoding the fatty acyl-CoA ligase with suitable lipid precursors enabled one-pot de novo synthesis of membrane-bound vesicles. Noncanonical sphingolipid synthesis was also possible by using a cysteine-modified lysosphingomyelin as a precursor. When the sphingomyelin-interacting protein lysenin was coexpressed alongside the acyl-CoA ligase, the in situ assembled membranes were spontaneously decorated with protein. Our strategy of coupling gene expression with membrane lipid synthesis in a one-pot fashion could facilitate the generation of proteoliposomes and brings us closer to the bottom-up generation of synthetic cells using recombinant synthetic biology platforms.
Assuntos
Sistema Livre de Células/metabolismo , Coenzima A Ligases/metabolismo , Lipídeos de Membrana/metabolismo , Sistema Livre de Células/química , Coenzima A Ligases/química , Coenzima A Ligases/genética , Humanos , Lipídeos de Membrana/químicaRESUMO
The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro. As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a noncanonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.
Assuntos
Ácido Graxo Sintase Tipo I/metabolismo , Fosfolipídeos/biossíntese , Ácido Graxo Sintase Tipo I/química , Estrutura Molecular , Fosfolipídeos/químicaRESUMO
Activating mutations in the small GTPase NRAS are responsible for driving tumor growth in several cancers. Unfortunately, the development of NRAS inhibitors has proven difficult due to the lack of hydrophobic binding pockets on the protein's surface. To overcome this limitation, we chose to target the post-translational S-palmitoyl modification of NRAS, which is required for its signaling activity. Utilizing an amphiphile-mediated depalmitoylation (AMD) strategy, we demonstrate the ability to directly cleave S-palmitoyl groups from NRAS and inhibit its function. C8 alkyl cysteine causes a dose-dependent decrease in NRAS palmitoylation and inhibits downstream signaling in melanoma cells with an activating mutation in NRAS. This compound reduces cell growth in NRAS-driven versus non-NRAS-driven melanoma lines and inhibits tumor progression in an NRAS-mutated melanoma xenograft mouse model. Our work demonstrates that AMD can effectively suppress NRAS activity and could represent a promising new avenue for discovering lead compounds for treatment of NRAS-driven cancers.
Assuntos
GTP Fosfo-Hidrolases/antagonistas & inibidores , Lipoilação , Melanoma/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Melanoma/patologia , Proteínas de Membrana/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Neoplasias Cutâneas/patologiaRESUMO
Biology utilizes multiple strategies, including sequestration in lipid vesicles, to raise the rate and specificity of chemical reactions through increases in effective molarity of reactants. We show that micelle-assisted reaction can facilitate native chemical ligations (NCLs) between a peptide-thioester - in which the thioester leaving group contains a lipid-like alkyl chain - and a Cys-peptide modified by a lipid-like moiety. Hydrophobic lipid modification of each peptide segment promotes the formation of mixed micelles, bringing the reacting peptides into close proximity and increasing the reaction rate. The approach enables the rapid synthesis of polypeptides using low concentrations of reactants without the need for thiol catalysts. After NCL, the lipid moiety is removed to yield an unmodified ligation product. This micelle-based methodology facilitates the generation of natural peptides, like Magainin 2, and the derivatization of the protein Ubiquitin. Formation of mixed micelles from lipid-modified reactants shows promise for accelerating chemical reactions in a traceless manner.
Assuntos
Lipídeos/química , Micelas , Peptídeos/química , Tensoativos/química , Sequência de Aminoácidos , Cinética , Luz , Magaininas/síntese química , Magaininas/química , Peptídeos/síntese química , Ubiquitina/metabolismoRESUMO
Amphiphilic molecules self-assemble into supramolecular structures of various sizes and morphologies depending on their molecular packing and external factors. Transformations between various self-assembled morphologies are a matter of great fundamental interest. Recently, we reported the discovery of a novel class of single-chain galactopyranosylamide amphiphiles that self-assemble to form vesicles in water. Here, we describe how the vesicles composed of the amphiphile N-oleoyl ß-d-galactopyranosylamine (GOA) undergo a morphological transition to fibers consisting of mainly flat sheet-like structures. Moreover, we show that this transformation is reversible in a temperature-dependent manner. We used several optical microscopy and electron microscopy techniques, circular dichroism spectroscopy, small-angle X-ray scattering, and differential scanning calorimetry, to fully investigate and characterize the morphological transformations of GOA and provide a structural basis for such phenomena. These studies provide significant molecular insight into the structural polymorphism of sugar-based amphiphiles and foresee future applications in rational design of self-assembled materials.
Assuntos
Água , Varredura Diferencial de Calorimetria , TemperaturaRESUMO
Post-translational S-palmitoylation plays a central role in protein localization, trafficking, stability, aggregation, and cell signaling. Dysregulation of palmitoylation pathways in cells can alter protein function and is the cause of several diseases. Considering the biological and clinical importance of S-palmitoylation, tools for direct, in vivo modulation of this lipid modification would be extremely valuable. Here, we describe a method for the cleavage of native S-palmitoyl groups from proteins in living cells. Using a cell permeable, cysteine-functionalized amphiphile, we demonstrate the direct depalmitoylation of cellular proteins. We show that amphiphile-mediated depalmitoylation (AMD) can effectively cleave S-palmitoyl groups from the native GTPase HRas and successfully depalmitoylate mislocalized proteins in an infantile neuronal ceroid lipofuscinosis (INCL) disease model. AMD enables direct and facile depalmitoylation of proteins in live cells and has potential therapeutic applications for diseases such as INCL, where native protein thioesterase activity is deficient.
Assuntos
Lipoilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cisteína/análogos & derivados , Cisteína/farmacologia , Proteína GAP-43/química , Proteína GAP-43/metabolismo , Humanos , Lipopeptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/químicaRESUMO
Autocatalytic chemical reactions, whereby a molecule is able to catalyze its own formation from a set of precursors, mimic nature's ability to generate identical copies of relevant biomolecules, and are thought to have been crucial for the origin of life. While several molecular autocatalysts have been previously reported, coupling autocatalytic behavior to macromolecular self-assembly has been challenging. Here, we report a non-enzymatic and chemoselective methodology capable of autocatalytically producing triskelion peptides that self-associate into spherical bioinspired nanostructures. Serial transfer experiments demonstrate that oligotriazole autocatalysis successfully leads to continual self-assembly of three-dimensional nanospheres. Triskelion-based spherical architectures offer an opportunity to organize biomolecules and chemical reactions in unique, nanoscale compartments. The use of peptide-based autocatalysts that are capable of self-assembly represents a promising method for the development of self-synthesizing biomaterials, and may shed light on understanding life's chemical origins.Molecules that act as both autocatalysts and material precursors offer exciting prospects for self-synthesizing materials. Here, the authors design a triazole peptide that self-replicates and then self-assembles into nanostructures, coupling autocatalytic and assembly pathways to realize a reproducing supramolecular system.
Assuntos
Nanosferas , Peptídeos , Multimerização Proteica , Triazóis , Catálise , Cobre , NanoestruturasRESUMO
Cell transmembrane receptors play a key role in the detection of environmental stimuli and control of intracellular communication. G protein-coupled receptors constitute the largest transmembrane protein family involved in cell signaling. However, current methods for their functional reconstitution in biomimetic membranes remain both challenging and limited in scope. Herein, we describe the spontaneous reconstitution of adenosine A2A receptor (A2AR) during the de novo formation of synthetic liposomes via native chemical ligation. The approach takes advantage of a nonenzymatic and chemoselective method to rapidly generate A2AR embedded phospholiposomes from receptor solubilized in n-dodecyl-ß-d-maltoside analogs. In situ lipid synthesis for protein reconstitution technology proceeds in the absence of dialysis and/or detergent absorbents, and A2AR assimilation into synthetic liposomes can be visualized by microscopy and probed by radio-ligand binding.
Assuntos
Lipossomos/metabolismo , Receptor A2A de Adenosina/metabolismo , Humanos , Lipossomos/síntese química , Lipossomos/química , Modelos Moleculares , Estrutura Molecular , Receptor A2A de Adenosina/químicaRESUMO
Transmembrane proteins are critical for signaling, transport, and metabolism, yet their reconstitution in synthetic membranes is often challenging. Non-enzymatic and chemoselective methods to generate phospholipid membranes inâ situ would be powerful tools for the incorporation of membrane proteins. Herein, the spontaneous reconstitution of functional integral membrane proteins during the deâ novo synthesis of biomimetic phospholipid bilayers is described. The approach takes advantage of bioorthogonal coupling reactions to generate proteoliposomes from micelle-solubilized proteins. This method was successfully used to reconstitute three different transmembrane proteins into synthetic membranes. This is the first example of the use of non-enzymatic chemical synthesis of phospholipids to prepare proteoliposomes.
Assuntos
Bicamadas Lipídicas/química , Proteínas de Membrana/química , Fosfolipídeos/química , Proteolipídeos/química , Animais , Bovinos , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Micelas , Fosfolipídeos/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Proteolipídeos/metabolismoRESUMO
In recent years, considerable effort has been devoted to the preparation of artificial nanotubular materials. One of the most successful approaches for the construction of noncovalently bonded nanotube entities is the self-assembly of cyclic polypeptides in stacks that are stabilized by hydrogen bonds. This tutorial review covers the history and current situation for synthetic organic nanostructures obtained from self-assembling cyclic peptides. In particular, we describe the evolution to cyclic peptides that not only allow the modification of the outer surface but also the inner cavity by paying special attention to peptide rings that contain cyclic gamma-amino acids. In this respect, we describe the synthesis, properties and application of a new class of homo- and heterodimeric supramolecular assemblies that are precursors of cyclic alpha,gamma-peptide nanotubes.
Assuntos
Nanotubos de Peptídeos/química , Peptídeos Cíclicos/química , Anti-Infecciosos/química , Materiais Biocompatíveis/química , Técnicas Biossensoriais , Dimerização , Eletrônica , Estrutura Secundária de ProteínaRESUMO
Bio-inspired cyclopeptidic heterodimers built on beta-sheet-like hydrogen-bonding networks and bearing photoactive and electroactive chromophores on the outer surface have been prepared. Different cross-strand pairwise relationships between the side chains of the cyclic alpha,gamma-peptides afford the heterodimers as three nonequivalent dimeric species. Steady-state and time-resolved spectroscopies clearly show an electron transfer process from pi-extended tetrathiafulvalene, covalently attached to one of the cyclopeptides, to photoexcited [60]fullerene, located on the complementary cyclopeptide. The charge-separated state was stabilized for up to 1 micros before recombining and repopulating the ground state. Our current example shows that cyclopeptidic templates can be successfully used to form light-harvesting/light-converting hybrid ensembles with a distinctive organization of donor and acceptor units able to act as efficient artificial photosystems.