RESUMEN
It is now well established in humans that there is a bidirectional pathway of communication between the central and enteric nervous systems in which members of the microbiome participate. This microbiota-gut-brain axis (MGBA) is crucial for normal development and physiology, and its dysregulation has been implicated in a range of neurological and intestinal disorders. Investigations into the mechanistic underpinnings of the MGBA have identified serotonin as a molecule of particular interest. In this review, we highlight recent advances toward understanding the role of endogenous serotonin in microbial communities, how microbial communities bidirectionally interact with host serotonin, and potential future engineering opportunities to leverage these novel mechanisms for biomedical applications.
Asunto(s)
Microbioma Gastrointestinal , Microbiota , Humanos , Serotonina/metabolismo , Eje Cerebro-Intestino , Encéfalo/metabolismoRESUMEN
Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.
Asunto(s)
ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Desoxirribonucleasa I/metabolismo , Conformación de Ácido Nucleico , Conformación Proteica , Ingeniería de Proteínas/métodos , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Transactivadores/metabolismo , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Circoviridae/enzimología , Secuencia Conservada , Cristalografía por Rayos X , ADN Helicasas/química , ADN de Cadena Simple/química , Desoxirribonucleasa I/química , Biblioteca de Genes , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Virus de Plantas/enzimología , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Origen de Réplica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/química , Especificidad por Sustrato , Transactivadores/química , Proteínas Virales/químicaRESUMEN
The Rep domain of Wheat dwarf virus (WDV Rep) is an HUH endonuclease involved in rolling-circle replication. HUH endonucleases coordinate a metal ion to enable the nicking of a specific ssDNA sequence and the subsequent formation of an intermediate phosphotyrosine bond. This covalent protein-ssDNA adduct makes HUH endonucleases attractive fusion tags (HUH-tags) in a diverse number of biotechnological applications. Solving the structure of an HUH endonuclease in complex with ssDNA will provide critical information about ssDNA recognition and sequence specificity, thus enabling rationally engineered protein-DNA interactions that are programmable. The structure of the WDV Rep domain reported here was solved in the apo state from a crystal diffracting to 1.24â Å resolution and represents an initial step in the direction of solving the structure of a protein-ssDNA complex.