Biology subjects

Savastano, A.

Publications and source records attributed to Savastano, A..

3 recordsLinked to original sources

bioRxiv2022

Contributions of the N-terminal intrinsically disordered region of the SARS-CoV-2 nucleocapsid protein to RNA-induced phase separation

SARS-CoV-2 nucleocapsid protein is an essential structural component of mature virions, encapsulating the genomic RNA and modulating RNA transcription and replication. Several of its activities might be associated with the proteins ability to undergo liquid-liquid phase separation. NSARS-CoV-2 contains an intrinsically disordered region at its N-terminus (NTE) that can be phosphorylated and is affected by disease-relevant mutations. Here we show that NTE deletion decreases the range of RNA concentrations that can induce phase separation of NSARS-CoV-2. In addition, deletion of the prion-like NTE allows NSARS-CoV-2 droplets to retain their liquid-like nature during incubation. We further demonstrate that RNA-binding engages multiple parts of the NTE and changes NTEs structural properties. The results form the foundation to characterize the impact of N-terminal mutations and post-translational modifications on the molecular properties of the SARS-CoV-2 nucleocapsid protein. StatementThe nucleocapsid protein of SARS-CoV-2 plays an important role in both genome packaging and viral replication upon host infection. Replication has been associated with RNA-induced liquid-liquid phase separation of the nucleocapsid protein. We present insights into the role of the N-terminal part of the nucleocapsid protein in the proteins RNA-mediated liquid-liquid phase separation.

biophysics↗

bioRxiv2021

The Pathogenic R5L Mutation Disrupts Formation of Tau Complexes on the Microtubule by Altering Local N-Terminal Structure

The microtubule-associated protein (MAP) Tau is an intrinsically disordered protein (IDP) primarily expressed in axons, where it functions to regulate microtubule dynamics, modulate motor protein motility, and participate in signaling cascades. Tau misregulation and point mutations are linked to neurodegenerative diseases, including Progressive Supranuclear Palsy (PSP), Picks Disease and Alzheimers disease. Many disease-associated mutations in Tau occur in the C-terminal microtubule-binding domain of the protein. Effects of C-terminal mutations in Tau have led to the widely accepted disease-state theory that missense mutations in Tau reduce microtubule-binding affinity or increase Tau propensity to aggregate. Here, we investigate the effect of an N-terminal disease-associated mutation in Tau, R5L, on Tau-microtubule interactions using an in vitro reconstituted system. Contrary to the canonical disease-state theory, we determine the R5L mutation does not reduce Tau affinity for the microtubule using Total Internal Reflection Fluorescence (TIRF) Microscopy. Rather, the R5L mutation decreases the ability of Tau to form larger order complexes, or Tau patches, at high concentrations of Tau. Using Nuclear Magnetic Resonance (NMR), we show that the R5L mutation results in a local structural change that reduces interactions of the projection domain in the presence of microtubules. Altogether, these results challenge both the current paradigm of how mutations in Tau lead to disease and the role of the projection domain in modulating Tau behavior on the microtubule surface. Significance StatementThe microtubule-associated protein Tau is strongly linked to a number of neurological diseases. Disease onset is typically associated with weakened interaction with the microtubule, but this widely accepted model is based on hyperphosphorylation or mutations within the C-terminal microtubule-binding domain of Tau. Here, we find an N-terminal disease-associated mutation in Tau, R5L, does not reduce Tau affinity for microtubules, but instead modifies the N-terminal structure, altering Taus behavior and ability to condense on the microtubule surface. Our findings challenge the current paradigms of both how mutations in Tau lead to disease and the functional role of the N-terminal region of Tau.

biophysics↗

bioRxiv2020

Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

The etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription and is essential for viral replication. Here we show that RNA induces cooperative liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid-liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

biophysics↗