HspB8 prevents aberrant phase transitions of FUS by chaperoning its folded RNA binding domain
- Max Planck Institute of Molecular Cell Biology and Genetics, Germany
- University of Konstanz, Germany
- Max Planck Institute for the Physics of Complex Systems, Germany
- Biotec, TU Dresden, Germany
- University of Modena and Reggio Emilia, Italy
Abstract
Aberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif (RRM) of FUS as a key driver of condensate ageing. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its α-crystallin domain (αCD). These αCD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions.
Data availability
All data generated or analysed during this study are included in this published article (and its supplementary information files). The MS data (raw files, xQuest, xTract and in-house quantitation output files) have been deposited to the ProteomeXchange Consortium via the PRIDE (60) partner repository with the dataset identifier PXD021114 (Username: reviewer33076@ebi.ac.uk; Password: 5atfkbP8) and PXD021115 (Username: reviewer54149@ebi.ac.uk; Password: UZW7Gnr5).
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Funding
Deutsche Forschungsgemeinschaft (STE 2517/1-1)
- Florian Stengel
Konstanz Research School Chemical Biology (Chemicals and small Equipment Purchase)
- Florian Stengel
Deutsche Forschungsgemeinschaft (Cluster of Excellence Physics of Life"")
- Dr. Simon Alberti
Deutsche Forschungsgemeinschaft (Cluster of Excellence Physics of Life"")
- Anthony A Hyman
EU Joint Programme – Neurodegenerative Disease Research (Neurodegenerative Disease Research (JPND))
- Serena Carra
EU Joint Programme – Neurodegenerative Disease Research (Neurodegenerative Disease Research (JPND))
- Dr. Simon Alberti
AriSLA Foundation (Granulopathy and MLOpathy)
- Serena Carra
MAECI (Dissolve_ALS)
- Serena Carra
MIUR (E91I18001480001)
- Serena Carra
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Boczek et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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- Biochemistry and Chemical Biology
The protein ligase Connectase can be used to fuse proteins to small molecules, solid carriers, or other proteins. Compared to other protein ligases, it offers greater substrate specificity, higher catalytic efficiency, and catalyzes no side reactions. However, its reaction is reversible, resulting in only 50% fusion product from two equally abundant educts. Here, we present a simple method to reliably obtain 100% fusion product in 1:1 conjugation reactions. This method is efficient for protein-protein or protein-peptide fusions at the N- or C-termini. It enables the generation of defined and completely labeled antibody conjugates with one fusion partner on each chain. The reaction requires short incubation times with small amounts of enzyme and is effective even at low substrate concentrations and at low temperatures. With these characteristics, it presents a valuable new tool for bioengineering.
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- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
