ABSTRACT
Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular ß-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.
Subject(s)
Arginine/chemistry , Molecular Chaperones/chemistry , RNA-Binding Protein FUS/chemistry , Amyotrophic Lateral Sclerosis/metabolism , Animals , Cations , DNA Methylation , Frontotemporal Dementia/metabolism , Frontotemporal Lobar Degeneration/metabolism , Humans , Microscopy, Atomic Force , Microscopy, Fluorescence , Protein Binding , Protein Domains , Protein Processing, Post-Translational , Protein Structure, Secondary , RNA-Binding Protein FUS/metabolism , Tyrosine/chemistry , Xenopus laevisABSTRACT
Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species, which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies, but its role remains controversial. Using high-throughput screening, we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.
Subject(s)
Electron Transport Complex III/metabolism , Free Radical Scavengers/pharmacology , Mitochondria/drug effects , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Superoxides/antagonists & inhibitors , Adenosine Triphosphate/biosynthesis , Animals , Antimycin A/analogs & derivatives , Antimycin A/antagonists & inhibitors , Antimycin A/pharmacology , Dose-Response Relationship, Drug , Female , HEK293 Cells , High-Throughput Screening Assays , Humans , Hydrogen Peroxide/antagonists & inhibitors , Hydrogen Peroxide/metabolism , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Male , Mitochondria/metabolism , Oxidative Phosphorylation/drug effects , Oxidative Stress , Rats , Rats, Sprague-Dawley , Rats, Wistar , Signal Transduction , Superoxides/metabolismABSTRACT
Using high-throughput screening we identified small molecules that suppress superoxide and/or H2O2 production during reverse electron transport through mitochondrial respiratory complex I (site IQ) without affecting oxidative phosphorylation (suppressors of site IQ electron leak, "S1QELs"). S1QELs diminished endogenous oxidative damage in primary astrocytes cultured at ambient or low oxygen tension, showing that site IQ is a normal contributor to mitochondrial superoxide-H2O2 production in cells. They diminished stem cell hyperplasia in Drosophila intestine in vivo and caspase activation in a cardiomyocyte cell model driven by endoplasmic reticulum stress, showing that superoxide-H2O2 production by site IQ is involved in cellular stress signaling. They protected against ischemia-reperfusion injury in perfused mouse heart, showing directly that superoxide-H2O2 production by site IQ is a major contributor to this pathology. S1QELs are tools for assessing the contribution of site IQ to cell physiology and pathology and have great potential as therapeutic leads.