RESUMEN
The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.
Asunto(s)
Proteínas de la Nucleocápside de Coronavirus , SARS-CoV-2 , COVID-19 , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Humanos , Microscopía Electrónica , Simulación de Dinámica Molecular , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , ARN Viral/genética , SARS-CoV-2/química , SARS-CoV-2/genética , SARS-CoV-2/metabolismoRESUMEN
The gut microbiota influence neurodevelopment, modulate behavior, and contribute to neurodegenerative disorders. Several studies have consistently reported a greater abundance of Akkermansia muciniphila in Parkinson disease (PD) fecal samples. Therefore, we investigated whether A.muciniphila-conditioned medium (CM) could initiate α-synuclein (αSyn) misfolding in enteroendocrine cells (EEC) - a component of the gut epithelium featuring neuron-like properties. We found that A. muciniphila CM composition is influenced by the ability of the strain to degrade mucin. Our in vitro experiments showed that the protein-enriched fraction of mucin-free CM induces RyR-mediated Ca2+ release and increased mitochondrial Ca2+ uptake leading to ROS generation and αSyn aggregation. Oral administration of A. muciniphila cultivated in the absence of mucin to mice led to αSyn aggregation in cholecystokinin (CCK)-positive EECs but no motor deficits were observed. Noteworthy, buffering mitochondrial Ca2+ reverted the damaging effects observed. These molecular insights offer evidence that bacterial proteins can induce αSyn aggregation in EECs.