Small molecule modulation of a redox-sensitive stress granule protein dissolves stress granules with beneficial outcomes for familial amyotrophic lateral sclerosis models

Wheeler, Richard J.; Lee, Hyun O.; Poser, Ina; Pal, Arun; Doeleman, T; Kishigami, Satoshi; Kour, Sukhleen; Anderson, Eric N.; Marrone, Lara; Murthy, AC; Jahnel, Marcus; Zhang, X; Boczek, Edgar E.; Fritsch, Anatol; Fawzi, NL; Sterneckert, Jared; Pandey, Udai Bhan; David, Della; Davis, Benjamin G.; Baldwin, Andrew J.; Hermann, Andreas; Bickle, Marc; Alberti, Simon; Hyman, Anthony

doi:10.1101/721001

Cited by 71 publications

(72 citation statements)

References 141 publications

(252 reference statements)

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“…Cells with higher levels of transfection were more likely to form spherical droplets in the cytoplasm ( Figure 4A We reasoned that small molecules that increased or decreased N-protein LLPS or altered RNA recruitment could slow viral infection. We examined 1,6-hexanediol (19), lipoic acid (20), the aminoglycoside kanamycin (21) and cyclosporin (22), each of which potentially alters LLPS by a representative and distinctive mechanism. As a simple positive test, we examined 1,6-Hexanediol which disrupts LLPS (19).…”

Section: N-protein Phase Separates In Mammalian Cells and Can Be Disrmentioning

confidence: 99%

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Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate

Iserman

Roden

Boerneke

et al. 2020

Preprint

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A mechanistic understanding of the SARS-CoV-2 viral replication cycle is essential to develop new therapies for the COVID-19 global health crisis. In this study, we show that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome, and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5'-End) under physiological conditions and is enhanced at human upper airway temperatures. N-protein condensates exclude non-packaged RNA sequences. We comprehensively map sites bound by N-protein in the 5'-End and find preferences for single-stranded RNA flanked by stable structured elements. Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2. IntroductionThe outbreak of COVID-19, caused by the severe acute respiratory syndrome-related coronavirus SARS-CoV-2, is a global public health crisis. Coronaviruses, including SARS-CoV-2, are RNA viruses with ~30 kb genomes that are replicated and packaged in host cells. Packaging is thought to be highly specific for the complete viral genome (gRNA), and excludes host RNA and abundant virus-produced subgenomic RNAs (1). Viral replication and gRNA packaging depends on the nucleocapsid protein (N-protein) (2, 3). The N-protein has two RNA-binding domains, forms multimers (4) and is predicted to contain intrinsically disordered regions ( Figure 1A). N-protein thus has hallmarks of proteins that undergo liquid-liquid phase separation (LLPS), a process which may provide selectivity and efficiency to viral replication and packaging. N-protein phase separates with viral RNA in a length, sequence and concentration dependent mannerWe reconstituted purified N-protein under physiological buffer conditions with viral RNA segments and observed that N-protein produced in mammalian cells (post-translationally modified) or bacteria (unmodified) phase separated with viral RNA segments. However, unmodified protein yielded larger and more abundant droplets ( Figure S1A). Since N-protein in SARS-CoV1 virions is hypophoshorylated (5) and packaging (initiated by binding of N-protein to gRNA) first occurs in the cytoplasm of coronaviruses (6,7), where N-protein is thought to be in its unphosphorylated state (8), we used unmodified protein for subsequent experiments.Pure N-protein demixed into droplets on its own and phase separation was enhanced by fulllength genomic SARS-CoV-2 RNA ( Figure 1B). To determine if certain segments of SARS-CoV-2 genome had preferential ability to drive phase separation, we identified regions of the gRNA under synonymous codon constraints. We hypothesized that LLPS occurs specifically with gRNA carrying a viral packaging signal(s), whose ex...

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Section: N-protein Phase Separates In Mammalian Cells and Can Be Disrmentioning

confidence: 99%

“…Indeed, 1,6-Hexanediol prevented condensate formation ( Figure 4G/H). Lipoic acid dissolves cellular stress granules (20), which are impacted in SARS-CoV (23).…”

Section: N-protein Phase Separates In Mammalian Cells and Can Be Disrmentioning

confidence: 99%

Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate

Iserman

Roden

Boerneke

et al. 2020

Preprint

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“…For example, Burke et al (24) mapped the nonspecific interactions between the low-complexity domain of FUS and RNA polymerase II C-terminal heptad tail in the dispersed phase. Finally, interactions between LLPS-prone proteins and small molecules can be monitored using chemical shifts (20,39). For example, the interaction of the low-complexity domain of FUS and potential therapeutic small molecules was quantified-the chemotherapeutic mitoxantrone was found to interact with tyrosine residues present in the low-complexity domain of FUS in the dispersed phase (39).…”

Section: Chemical Shiftsmentioning

confidence: 99%

The (un)structural biology of biomolecular liquid-liquid phase separation using NMR spectroscopy

Murthy

Fawzi

2020

Journal of Biological Chemistry

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Liquid-liquid phase separation (LLPS) of proteins and nucleic acids is a phenomenon that underlies membraneless compartmentalization of the cell. The underlying molecular interactions that underpin biomolecular LLPS have been of increased interest due to the importance of membraneless organelles in facilitating various biological processes and the disease association of several of the proteins that mediate LLPS. Proteins that are able to undergo LLPS often contain intrinsically disordered regions and remain dynamic in solution. Solution-state NMR spectroscopy has emerged as a leading structural technique to characterize protein LLPS due to the variety and specificity of information that can be obtained about intrinsically disordered sequences. This review discusses practical aspects of studying LLPS by NMR, summarizes recent work on the molecular aspects of LLPS of various protein systems, and discusses future opportunities for characterizing the molecular details of LLPS to modulate phase separation.

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“…In addition, aberrant phase separation of FUS, TDP-43, and Annexin A11, the proteins we study here, has been associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis, frontotemporal dementia, and Alzheimer's disease (7). The discovery of salt-mediated reentrant phase separation for these systems suggests a crucial feature of protein LLPS behavior that may be important when developing therapies for phase-separation implicated diseases (77).…”

Section: Resultsmentioning

confidence: 76%

Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions

Krainer

Welsh

Joseph

et al. 2020

Preprint

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Many cellular proteins have the ability to demix spontaneously from solution to form liquid condensates. These phase-separated structures form membraneless compartments in living cells and have wide-ranging roles in health and disease. Elucidating the molecular driving forces underlying liquid-liquid phase separation (LLPS) of proteins has thus become a key objective for understanding biological function and malfunction. Here we show that proteins implicated in cellular phase separation, such as FUS, TDP-43, and Annexin A11, which form condensates at low salt concentrations via homotypic multivalent interactions, also have the ability to undergo LLPS at high salt concentrations by reentering into a phase-separated regime. Through a combination of experiments and simulations, we demonstrate that phase separation in the high-salt regime is mainly driven by hydrophobic and non-ionic interactions. As such, it is mechanistically distinct from the low-salt regime, where condensates are stabilized by a broad mix of electrostatic, hydrophobic, and non-ionic forces. Our work thus expands the molecular grammar of interactions governing LLPS of cellular proteins and provides a new view on hydrophobicity and non-ionic interactions as non-specific driving forces for the condensation process, with important implications for the aberrant function, druggability, and material properties of biomolecular condensates. One Sentence SummaryProteins implicated in cellular phase separation can undergo a salt-mediated reentrant liquid-liquid phase transition.

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Small molecule modulation of a redox-sensitive stress granule protein dissolves stress granules with beneficial outcomes for familial amyotrophic lateral sclerosis models

Cited by 71 publications

References 141 publications

Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate

Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate

The (un)structural biology of biomolecular liquid-liquid phase separation using NMR spectroscopy

Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions

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