There are about 100 single point mutations of copper, zinc superoxide dismutase 1 (SOD1) which are reported (http://alsod.iop.kcl.ac.uk/Als/index.aspx) to be related to the familial form (fALS) of amyotrophic lateral sclerosis (ALS). These mutations are spread all over the protein. It is well documented that fALS produces protein aggregates in the motor neurons of fALS patients, which have been found to be associated to mitochondria. We selected eleven SOD1 mutants, most of them reported as pathological, and characterized them investigating their propensity to aggregation using different techniques, from circular dichroism spectra to ThT-binding fluorescence, size-exclusion chromatography and light scattering spectroscopy. We show here that these eleven SOD1 mutants, only when they are in the metal-free form, undergo the same general mechanism of oligomerization as found for the WT metal-free protein. The rates of oligomerization are different but eventually they give rise to the same type of soluble oligomeric species. These oligomers are formed through oxidation of the two free cysteines of SOD1 (6 and 111) and stabilized by hydrogen bonds, between beta strands, thus forming amyloid-like structures. SOD1 enters the mitochondria as demetallated and mitochondria are loci where oxidative stress may easily occur. The soluble oligomeric species, formed by the apo form of both WT SOD1 and its mutants through an oxidative process, might represent the precursor toxic species, whose existence would also suggest a common mechanism for ALS and fALS. The mechanism here proposed for SOD1 mutant oligomerization is absolutely general and it provides a common unique picture for the behaviors of the many SOD1 mutants, of different nature and distributed all over the protein.
The structural and dynamical properties of the metal-free form of WT human superoxide dismutase 1 (SOD1) and its familial amyotrophic lateral sclerosis (fALS)-related mutants, T54R and I113T, were characterized both in solution, through NMR, and in the crystal, through X-ray diffraction. We found that all 3 X-ray structures show significant structural disorder in 2 loop regions that are, at variance, well defined in the fully-metalated structures. Interestingly, the apo state crystallizes only at low temperatures, whereas all 3 proteins in the metalated form crystallize at any temperature, suggesting that crystallization selects one of the most stable conformations among the manifold adopted by the apo form in solution. Indeed, NMR experiments show that the protein in solution is highly disordered, sampling a large range of conformations. The large conformational variability of the apo state allows the free reduced cysteine Cys-6 to become highly solvent accessible in solution, whereas it is essentially buried in the metalated state and the crystal structures. Such solvent accessibility, together with that of Cys-111, accounts for the tendency to oligomerization of the metal-free state. The present results suggest that the investigation of the solution state coupled with that of the crystal state can provide major insights into SOD1 pathway toward oligomerization in relation to fALS.amyotrophic lateral sclerosis ͉ NMR ͉ X-ray ͉ mobility ͉ H2O/D2O exchange M ore than 100 different variants of human copper-zinc superoxide dismutase (Cu 2 Zn 2 SOD) have been identified and linked to the neurodegenerative disease familial amyotrophic lateral sclerosis (fALS) by a gain-of-function mechanism (1, 2). Although the mechanism of the toxicity is unknown, aberrant SOD1 protein oligomerization has been strongly implicated in disease causation (3,4). Several recent publications (5, 6) have presented compelling evidence that in vivo abnormal disulfide cross-linking of ALS mutant SOD1 plays a role in this oligomerization, and disulfide-linked SOD1 multimers have been detected mainly in mitochondria of neuronal tissues of SOD1-linked fALS patients and transgenic mice (7-9).WT human SOD1 is an exceptionally stable, homodimeric 32-kDa protein, located mainly in the cytoplasm, but it is also present in the peroxisomes, the mitochondrial intermembrane space, and the nucleus of eukaryotic cells (10, 11). Each subunit of the dimer binds 1 copper and 1 zinc ion and folds as an 8-stranded Greek-key -barrel that is stabilized by an intrasubunit disulfide bond (Cys-57, Cys-146) near the active site (12). In vivo, in the highly reducing cytoplasm environment, the existence of this intrasubunit disulfide bond points to its very low reduction potential.In addition to the 2 cysteines involved in the formation of the intramolecular disulfide bond, 2 reduced cysteines, Cys-6 and Cys-111, are located on -strand 1 and loop VI of WT human SOD1, respectively. Among the loops connecting the 8 -strands, 2 have structural and functional roles. The ...
Liquid–liquid phase separation enables compartmentalization of biomolecules in cells, notably RNA and associated proteins in the nucleus. Besides having critical functions in RNA processing, there is a major interest in deciphering the molecular mechanisms of compartmentalization orchestrated by RNA-binding proteins such as TDP-43 (also known as TARDBP) and FUS because of their link to neuron diseases. However, tools for probing compartmentalization in cells are lacking. Here, we developed a method to analyze the mixing and demixing of two different phases in a cellular context. The principle is the following: RNA-binding proteins are confined on microtubules and quantitative parameters defining their spatial segregation are measured along the microtubule network. Through this approach, we found that four mRNA-binding proteins, HuR (also known as ELAVL1), G3BP1, TDP-43 and FUS form mRNA-rich liquid-like compartments on microtubules. TDP-43 is partly miscible with FUS but immiscible with either HuR or G3BP1. We also demonstrate that mRNA is essential to capture the mixing and demixing behavior of mRNA-binding proteins in cells. Taken together, we show that microtubules can be used as platforms to understand the mechanisms underlying liquid–liquid phase separation and their deregulation in human diseases.
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