RESUMO
Most antiviral proteins recognize specific features of viruses. In contrast, the recently described antiviral factor retroCHMP3 interferes with the "host endosomal complexes required for transport" (ESCRT) pathway to inhibit the budding of enveloped viruses. RetroCHMP3 arose independently on multiple occasions via duplication and truncation of the gene encoding the ESCRT-III factor CHMP3. However, since the ESCRT pathway is essential for cellular membrane fission reactions, ESCRT inhibition is potentially cytotoxic. This raises fundamental questions about how hosts can repurpose core cellular functions into antiviral functions without incurring a fitness cost due to excess cellular toxicity. We reveal the evolutionary process of detoxification for retroCHMP3 in New World monkeys using a combination of ancestral reconstructions, cytotoxicity, and virus release assays. A duplicated, full-length copy of retroCHMP3 in the ancestors of New World monkeys provides modest inhibition of virus budding while exhibiting subtle cytotoxicity. Ancient retroCHMP3 then accumulated mutations that reduced cytotoxicity but preserved virus inhibition before a truncating stop codon arose in the more recent ancestors of squirrel monkeys, resulting in potent inhibition. In species where full-length copies of retroCHMP3 still exist, their artificial truncation generated potent virus-budding inhibitors with little cytotoxicity, revealing the potential for future antiviral defenses in modern species. In addition, we discovered that retroCHMP3 restricts LINE-1 retrotransposition, revealing how different challenges to genome integrity might explain multiple independent origins of retroCHMP3 in different species to converge on new immune functions.
Assuntos
Liberação de Vírus , Vírus , Animais , Antivirais , Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Primatas/genéticaRESUMO
Complexes of specifically interacting molecules, such as transcription factor proteins (TFs) and the DNA response elements (REs) they recognize, control most biological processes, but little is known concerning the functional and evolutionary effects of epistatic interactions across molecular interfaces. We experimentally characterized all combinations of genotypes in the joint protein-DNA sequence space defined by an historical transition in TF-RE specificity that occurred some 500 million years ago in the DNA-binding domain of an ancient steroid hormone receptor. We found that rampant epistasis within and between the two molecules was essential to specific TF-RE recognition and to the evolution of a novel TF-RE complex with unique derived specificity. Permissive and restrictive epistatic mutations across the TF-RE interface opened and closed potential evolutionary paths accessible by the other, making the evolution of each molecule contingent on its partner's history and allowing a molecular complex with novel specificity to evolve.
Assuntos
DNA/genética , DNA/metabolismo , Epistasia Genética , Evolução Molecular , Elementos de Resposta , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sítios de Ligação , Biologia ComputacionalRESUMO
Complex gene regulatory networks require transcription factors (TFs) to bind distinct DNA sequences. To understand how novel TF specificity evolves, we combined phylogenetic, biochemical, and biophysical approaches to interrogate how DNA recognition diversified in the steroid hormone receptor (SR) family. After duplication of the ancestral SR, three mutations in one copy radically weakened binding to the ancestral estrogen response element (ERE) and improved binding to a new set of DNA sequences (steroid response elements, SREs). They did so by establishing unfavorable interactions with ERE and abolishing unfavorable interactions with SRE; also required were numerous permissive substitutions, which nonspecifically improved cooperativity and affinity of DNA binding. Our findings indicate that negative determinants of binding play key roles in TFs' DNA selectivity and-with our prior work on the evolution of SR ligand specificity during the same interval-show how a specific new gene regulatory module evolved without interfering with the integrity of the ancestral module.
Assuntos
Evolução Molecular , Redes Reguladoras de Genes , Receptores de Esteroides/química , Receptores de Esteroides/genética , Elementos de Resposta , Animais , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Receptores de Esteroides/metabolismoRESUMO
We previously proposed three hypotheses relating the mechanism of antimicrobial and cytolytic peptides in model membranes to the Gibbs free energies of binding and insertion into the membrane [Almeida, P. F., and Pokorny, A. (2009) Biochemistry 48, 8083-8093]. Two sets of peptides were designed to test those hypotheses, by mutating of the sequences of δ-lysin, cecropin A, and magainin 2. Peptide binding and activity were measured on phosphatidylcholine membranes. In the first set, the peptide charge was changed by mutating basic to acidic residues or vice versa, but the amino acid sequence was not altered much otherwise. The type of dye release changed from graded to all-or-none according to prediction. However, location of charged residues in the sequence with the correct spacing to form salt bridges failed to improve binding. In the second set, the charged and other key residues were kept in the same positions, whereas most of the sequence was significantly but conservatively simplified, maintaining the same hydrophobicity and amphipathicity. This set behaved completely different from predicted. The type of release, which was expected to be maintained, changed dramatically from all-or-none to graded in the mutants of cecropin and magainin. Finally, contrary to the hypotheses, the results indicate that the Gibbs energy of binding to the membrane, not the Gibbs energy of insertion, is the primary determinant of peptide activity.
Assuntos
Anti-Infecciosos/metabolismo , Membranas Artificiais , Peptídeos/metabolismo , Fosfatidilcolinas/metabolismo , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Galanina/química , Galanina/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Magaininas/química , Magaininas/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Fosfatidilcolinas/química , Fosfatidilcolinas/genética , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Eletricidade Estática , Termodinâmica , Venenos de Vespas/química , Venenos de Vespas/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
We report a first test of the hypothesis that the mechanism of antimicrobial, cytolytic, and amphipathic cell-penetrating peptides in model membranes is determined by the thermodynamics of insertion of the peptide into the lipid bilayer from the surface-associated state. Three peptides were designed with minimal mutations relative to the sequence of TP10W, the Y3W variant of transportan 10, which is a helical, amphipathic cell-penetrating peptide previously studied. Binding to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes and release of dye from those vesicles were assessed by stopped-flow fluorescence, and the secondary structure of the peptides on the membrane was determined by circular dichroism. The Gibbs energy of binding determined experimentally was in excellent agreement with that calculated using the Wimley-White interfacial hydrophobicity scale, taking into account the helical content of the membrane-associated peptide. Release of dye from POPC vesicles remained graded, as predicted by the hypothesis. More significantly, as the Gibbs energy of insertion into the bilayer became more unfavorable, which was estimated using the Wimley-White octanol hydrophobicity scale, dye release became slower, in quantitative agreement with the prediction.
Assuntos
Membrana Celular/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Sequência de Aminoácidos , Peptídeos Penetradores de Células/genética , Cinética , Bicamadas Lipídicas/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , TermodinâmicaRESUMO
The sculpting of membranes into dynamic, curved shapes is central to intracellular cargo trafficking. Though the generation of membrane curvature during trafficking necessarily involves both lipids and membrane-associated proteins, current mechanistic views focus primarily on the formation of rigid cages and curved scaffolds by protein assemblies. Here we report on a different mechanism for the control of membrane deformation, unrelated to the imposition of predefined curvature, involving modulation of membrane material properties: Sar1, a GTPase that regulates vesicle trafficking from the endoplasmic reticulum, lowers the rigidity of the lipid bilayer membrane to which it binds. In vitro assays in which optically trapped microspheres create controlled membrane deformations revealed a monotonic decline in bending modulus as a function of Sar1 concentration, down to nearly zero rigidity, indicating a dramatic lowering of the energetic cost of curvature generation. This is the first demonstration that a vesicle trafficking protein lowers the rigidity of its target membrane, leading to a new conceptual framework for vesicle biogenesis.