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1.
J Cell Physiol ; 228(11): 2222-31, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23625794

ABSTRACT

FUsed in Sarcoma/Translocated in LipoSarcoma (FUS/TLS or FUS) has been linked to several biological processes involving DNA and RNA processing, and has been associated with multiple diseases, including myxoid liposarcoma and amyotrophic lateral sclerosis (ALS). ALS-associated mutations cause FUS to associate with stalled translational complexes called stress granules under conditions of stress. However, little is known regarding the normal role of endogenous (non-disease linked) FUS in cellular stress response. Here, we demonstrate that endogenous FUS exerts a robust response to hyperosmolar stress induced by sorbitol. Hyperosmolar stress causes an immediate re-distribution of nuclear FUS to the cytoplasm, where it incorporates into stress granules. The redistribution of FUS to the cytoplasm is modulated by methyltransferase activity, whereas the inhibition of methyltransferase activity does not affect the incorporation of FUS into stress granules. The response to hyperosmolar stress is specific, since endogenous FUS does not redistribute to the cytoplasm in response to sodium arsenite, hydrogen peroxide, thapsigargin, or heat shock, all of which induce stress granule assembly. Intriguingly, cells with reduced expression of FUS exhibit a loss of cell viability in response to sorbitol, indicating a prosurvival role for endogenous FUS in the cellular response to hyperosmolar stress.


Subject(s)
Cytoplasmic Granules/metabolism , Hypertonic Solutions/pharmacology , RNA-Binding Protein FUS/metabolism , Stress, Physiological/drug effects , Animals , Cell Death/drug effects , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Survival/drug effects , Cytoplasmic Granules/drug effects , HEK293 Cells , HeLa Cells , Humans , Methylation/drug effects , Mice , Sorbitol/pharmacology , Sorbitol/toxicity
2.
Nat Neurosci ; 13(11): 1396-403, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20953194

ABSTRACT

Many mutations confer one or more toxic function(s) on copper/zinc superoxide dismutase 1 (SOD1) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we found that oxidized wild-type SOD1 and mutant SOD1 share a conformational epitope that is not present in normal wild-type SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord were markedly C4F6 immunoreactive, indicating that an aberrant wild-type SOD1 species was present. Recombinant, oxidized wild-type SOD1 and wild-type SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to that of FALS-linked mutant SOD1. Our findings suggest that wild-type SOD1 can be pathogenic in SALS and identify an SOD1-dependent pathogenic mechanism common to FALS and SALS.


Subject(s)
Amyotrophic Lateral Sclerosis , Mutation/genetics , Proteostasis Deficiencies/complications , Superoxide Dismutase/genetics , Adult , Aged , Amyotrophic Lateral Sclerosis/etiology , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Antibodies, Anti-Idiotypic , Crystallography, X-Ray/methods , Epitope Mapping/methods , Female , Humans , Male , Mass Spectrometry/methods , Middle Aged , Models, Molecular , Mutation/immunology , Oxidation-Reduction , Protein Folding , Proteostasis Deficiencies/genetics , Proteostasis Deficiencies/pathology , Superoxide Dismutase/chemistry , Superoxide Dismutase/immunology , Superoxide Dismutase-1
3.
Hum Mol Genet ; 19(21): 4160-75, 2010 Nov 01.
Article in English | MEDLINE | ID: mdl-20699327

ABSTRACT

Mutations in the RNA-binding protein FUS (fused in sarcoma) are linked to amyotrophic lateral sclerosis (ALS), but the mechanism by which these mutants cause motor neuron degeneration is not known. We report a novel ALS truncation mutant (R495X) that leads to a relatively severe ALS clinical phenotype compared with FUS missense mutations. Expression of R495X FUS, which abrogates a putative nuclear localization signal at the C-terminus of FUS, in HEK-293 cells and in the zebrafish spinal cord caused a striking cytoplasmic accumulation of the protein to a greater extent than that observed for recessive (H517Q) and dominant (R521G) missense mutants. Furthermore, in response to oxidative stress or heat shock conditions in cultures and in vivo, the ALS-linked FUS mutants, but not wild-type FUS, assembled into perinuclear stress granules in proportion to their cytoplasmic expression levels. These findings demonstrate a potential link between FUS mutations and cellular pathways involved in stress responses that may be relevant to altered motor neuron homeostasis in ALS.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , RNA-Binding Protein FUS/physiology , Adult , Animals , Cell Line , Cytoplasm/metabolism , Female , Green Fluorescent Proteins/genetics , Humans , Male , Middle Aged , Mutation, Missense , Oxidative Stress , RNA-Binding Protein FUS/genetics , RNA-Binding Protein FUS/metabolism , Zebrafish
4.
Chem Phys ; 348(1-3): 152-160, 2008 Jun 02.
Article in English | MEDLINE | ID: mdl-19079566

ABSTRACT

Green Fluorescent Proteins (GFP) and GFP-like proteins all undergo an autocatalytic post-translational modification to form a centrally located chromophore. Structural analyses of all the GFP and GFP-like proteins in the protein databank were undertaken to determine the role of the tight-turn, broken hydrogen bonding, Gly67, Glu222 and Arg96 in the biosynthesis of the imidazolone group from 65SYG67. The analysis was supplemented by computational generation of the conformation adopted by uncyclized wild-type GFP. The data analysis suggests that Arg96 interacts with the Tyr66 carbonyl, stabilizing the reduced enolate intermediate that is required for cyclization; the carboxylate of Glu 222 acts as a base facilitating, through a network of two waters, the abstraction of a hydrogen from the alpha-carbon of Tyr66; a tight-turn conformation is required for autocatalytic cyclization. This conformation is responsible for a partial reduction in the hydrogen bonding network around the chromophore-forming region of the immature protein.

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