RESUMO
Subcellular compartmentalization is a defining feature of all cells. In prokaryotes, compartmentalization is generally achieved via protein-based strategies. The two main classes of microbial protein compartments are bacterial microcompartments and encapsulin nanocompartments. Encapsulins self-assemble into proteinaceous shells with diameters between 24 and 42 nm and are defined by the viral HK97-fold of their shell protein. Encapsulins have the ability to encapsulate dedicated cargo proteins, including ferritin-like proteins, peroxidases, and desulfurases. Encapsulation is mediated by targeting sequences present in all cargo proteins. Encapsulins are found in many bacterial and archaeal phyla and have been suggested to play roles in iron storage, stress resistance, sulfur metabolism, and natural product biosynthesis. Phylogenetic analyses indicate that they share a common ancestor with viral capsid proteins. Many pathogens encode encapsulins, and recent evidence suggests that they may contribute toward pathogenicity. The existing information on encapsulin structure, biochemistry, biological function, and biomedical relevance is reviewed here.
Assuntos
Bactérias , Proteínas de Bactérias , Archaea/genética , Archaea/metabolismo , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Ferro/metabolismo , FilogeniaRESUMO
The centrosome is a main orchestrator of the animal cellular microtubule cytoskeleton. Dissecting its structure and assembly mechanisms has been a goal of cell biologists for over a century. In the last two decades, a good understanding of the molecular constituents of centrosomes has been achieved. Moreover, recent breakthroughs in electron and light microscopy techniques have enabled the inspection of the centrosome and the mapping of its components with unprecedented detail. However, we now need a profound and dynamic understanding of how these constituents interact in space and time. Here, we review the latest findings on the structural and molecular architecture of the centrosome and how its biogenesis is regulated, highlighting how biophysical techniques and principles as well as quantitative modeling are changing our understanding of this enigmatic cellular organelle.
Assuntos
Centrossomo , Organelas , AnimaisRESUMO
Respiratory syncytial virus (RSV) is a worldwide public health concern for which no vaccine is available. Elucidation of the prefusion structure of the RSV F glycoprotein and its identification as the main target of neutralizing antibodies have provided new opportunities for development of an effective vaccine. Here, we describe the structure-based design of a self-assembling protein nanoparticle presenting a prefusion-stabilized variant of the F glycoprotein trimer (DS-Cav1) in a repetitive array on the nanoparticle exterior. The two-component nature of the nanoparticle scaffold enabled the production of highly ordered, monodisperse immunogens that display DS-Cav1 at controllable density. In mice and nonhuman primates, the full-valency nanoparticle immunogen displaying 20 DS-Cav1 trimers induced neutralizing antibody responses â¼10-fold higher than trimeric DS-Cav1. These results motivate continued development of this promising nanoparticle RSV vaccine candidate and establish computationally designed two-component nanoparticles as a robust and customizable platform for structure-based vaccine design.
Assuntos
Anticorpos Neutralizantes/imunologia , Vírus Sinciciais Respiratórios/imunologia , Vacinação/métodos , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Caveolina 1 , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/uso terapêutico , Cultura Primária de Células , Vírus Sinciciais Respiratórios/patogenicidade , Vacinas/imunologia , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/fisiologiaRESUMO
The ornately geometric walls of pollen grains have inspired scientists for decades. We show that the evolved diversity of these patterns is entirely recapitulated by a biophysical model in which an initially uniform polysaccharide layer in the extracellular space, mechanically coupled to the cell membrane, phase separates to a spatially modulated state. Experiments reveal this process occurring in living cells. We observe that in â¼10% of extant species, this phase separation reaches equilibrium during development such that individual pollen grains are identical and perfectly reproducible. About 90% of species undergo an arrest of this process prior to equilibrium such that individual grains are similar but inexact copies. Equilibrium patterns have appeared multiple times during the evolution of seed plants, but selection does not favor these states. This framework for pattern development provides a route to rationalizing the surface textures of other secreted structures, such as cell walls and insect cuticle.
Assuntos
Parede Celular/metabolismo , Parede Celular/fisiologia , Pólen/metabolismo , Fenômenos Biofísicos/fisiologia , Membrana Celular/metabolismo , Simulação por Computador , Regulação da Expressão Gênica de Plantas/genética , Microscopia Eletrônica de Transmissão/métodos , Morfogênese/fisiologia , Passiflora/metabolismo , FilogeniaRESUMO
The formation of ordered nanostructures by molecular self-assembly of proteins and peptides represents one of the principal directions in nanotechnology. Indeed, polyamides provide superior features as materials with diverse physical properties. A reductionist approach allowed the identification of extremely short peptide sequences, as short as dipeptides, which could form well-ordered amyloid-like ß-sheet-rich assemblies comparable to supramolecular structures made of much larger proteins. Some of the peptide assemblies show remarkable mechanical, optical, and electrical characteristics. Another direction of reductionism utilized a natural noncoded amino acid, α-aminoisobutryic acid, to form short superhelical assemblies. The use of this exceptional helix inducer motif allowed the fabrication of single heptad repeats used in various biointerfaces, including their use as surfactants and DNA-binding agents. Two additional directions of the reductionist approach include the use of peptide nucleic acids (PNAs) and coassembly techniques. The diversified accomplishments of the reductionist approach, as well as the exciting future advances it bears, are discussed.
Assuntos
Nanoestruturas/química , Peptídeos/química , Proteínas Amiloidogênicas/química , Animais , Biotecnologia , Humanos , Modelos Moleculares , Nanotecnologia/métodos , Oligopeptídeos/química , Ácidos Nucleicos Peptídicos/química , Engenharia de ProteínasRESUMO
Synthetic multicellular systems hold promise as models for understanding natural development of biofilms and higher organisms and as tools for engineering complex multi-component metabolic pathways and materials. However, such efforts require tools to adhere cells into defined morphologies and patterns, and these tools are currently lacking. Here, we report a 100% genetically encoded synthetic platform for modular cell-cell adhesion in Escherichia coli, which provides control over multicellular self-assembly. Adhesive selectivity is provided by a library of outer membrane-displayed nanobodies and antigens with orthogonal intra-library specificities, while affinity is controlled by intrinsic adhesin affinity, competitive inhibition, and inducible expression. We demonstrate the resulting capabilities for quantitative rational design of well-defined morphologies and patterns through homophilic and heterophilic interactions, lattice-like self-assembly, phase separation, differential adhesion, and sequential layering. Compatible with synthetic biology standards, this adhesion toolbox will enable construction of high-level multicellular designs and shed light on the evolutionary transition to multicellularity.
Assuntos
Adesão Celular/fisiologia , Engenharia Metabólica/métodos , Biologia Sintética/métodos , Fenômenos Fisiológicos Bacterianos , Evolução Biológica , Adesão Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Escherichia coli/genética , Biblioteca Gênica , Redes e Vias Metabólicas , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/fisiologiaRESUMO
Aging is associated with progressive phenotypic changes. Virtually all cellular phenotypes are produced by proteins, and their structural alterations can lead to age-related diseases. However, we still lack comprehensive knowledge of proteins undergoing structural-functional changes during cellular aging and their contributions to age-related phenotypes. Here, we conducted proteome-wide analysis of early age-related protein structural changes in budding yeast using limited proteolysis-mass spectrometry (LiP-MS). The results, compiled in online ProtAge catalog, unraveled age-related functional changes in regulators of translation, protein folding, and amino acid metabolism. Mechanistically, we found that folded glutamate synthase Glt1 polymerizes into supramolecular self-assemblies during aging, causing breakdown of cellular amino acid homeostasis. Inhibiting Glt1 polymerization by mutating the polymerization interface restored amino acid levels in aged cells, attenuated mitochondrial dysfunction, and led to lifespan extension. Altogether, this comprehensive map of protein structural changes enables identifying mechanisms of age-related phenotypes and offers opportunities for their reversal.
Assuntos
Senescência Celular , Longevidade , Longevidade/genética , Polimerização , AminoácidosRESUMO
The assembly of individual proteins into functional complexes is fundamental to nearly all biological processes. In recent decades, many thousands of homomeric and heteromeric protein complex structures have been determined, greatly improving our understanding of the fundamental principles that control symmetric and asymmetric quaternary structure organization. Furthermore, our conception of protein complexes has moved beyond static representations to include dynamic aspects of quaternary structure, including conformational changes upon binding, multistep ordered assembly pathways, and structural fluctuations occurring within fully assembled complexes. Finally, major advances have been made in our understanding of protein complex evolution, both in reconstructing evolutionary histories of specific complexes and in elucidating general mechanisms that explain how quaternary structure tends to evolve. The evolution of quaternary structure occurs via changes in self-assembly state or through the gain or loss of protein subunits, and these processes can be driven by both adaptive and nonadaptive influences.
Assuntos
Proteínas/química , Proteínas/metabolismo , Archaea/química , Bactérias/química , Cristalografia por Raios X , Eucariotos/química , Evolução Molecular , Humanos , Complexos Multiproteicos/química , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Estrutura Quaternária de ProteínaRESUMO
Ion channels play a central role in the regulation of nearly every cellular process. Dating back to the classic 1952 Hodgkin-Huxley model of the generation of the action potential, ion channels have always been thought of as independent agents. A myriad of recent experimental findings exploiting advances in electrophysiology, structural biology, and imaging techniques, however, have posed a serious challenge to this long-held axiom, as several classes of ion channels appear to open and close in a coordinated, cooperative manner. Ion channel cooperativity ranges from variable-sized oligomeric cooperative gating in voltage-gated, dihydropyridine-sensitive CaV1.2 and CaV1.3 channels to obligatory dimeric assembly and gating of voltage-gated NaV1.5 channels. Potassium channels, transient receptor potential channels, hyperpolarization cyclic nucleotide-activated channels, ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3Rs) have also been shown to gate cooperatively. The implications of cooperative gating of these ion channels range from fine-tuning excitation-contraction coupling in muscle cells to regulating cardiac function and vascular tone, to modulation of action potential and conduction velocity in neurons and cardiac cells, and to control of pacemaking activity in the heart. In this review, we discuss the mechanisms leading to cooperative gating of ion channels, their physiological consequences, and how alterations in cooperative gating of ion channels may induce a range of clinically significant pathologies.
Assuntos
Ativação do Canal Iônico , Canal de Liberação de Cálcio do Receptor de Rianodina , Potenciais de Ação , Humanos , Ativação do Canal Iônico/fisiologia , NeurôniosRESUMO
CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection. Here, we introduce a CRISPR-based peptide display technology to facilitate customized, high-throughput in vitro protein interaction studies. We show that bespoke peptide libraries fused to catalytically inactive Cas9 (dCas9) and barcoded with unique single guide RNA (sgRNA) molecules self-assemble from a single mixed pool to programmable positions on a DNA microarray surface for rapid, multiplexed binding assays. We develop dCas9-displayed saturation mutagenesis libraries to characterize antibody-epitope binding for a commercial anti-FLAG monoclonal antibody and human serum antibodies. We also show that our platform can be used for viral epitope mapping and exhibits promise as a multiplexed diagnostics tool. Our CRISPR-based peptide display platform and the principles of complex library self-assembly using dCas9 could be adapted for rapid interrogation of varied customized protein libraries or biological materials assembly using DNA scaffolding.