Nuclear transport impairment of amyotrophic lateral sclerosis‐linked mutations in FUS/TLS

Ito, Daisuke; Seki, Morinobu; Tsunoda, Yoshiko; Uchiyama, Hidemi; Suzuki, Noriyuki

doi:10.1002/ana.22246

Cited by 154 publications

(158 citation statements)

References 43 publications

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“…Furthermore, we also demonstrated previously that mutant FUS, which is missing the nuclear traffic activity of the C terminus, is mislocated to the cytoplasm and assembled into similar SG-like mRNP granules (6). Therefore, excessive cytoplasmic RNA binding proteins such as TDP-43 and FUS induce a conjoint pathological cascade of neurodegeneration that leads to ALS/FTLD-U (8).…”

supporting

confidence: 55%

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Roles of Ataxin-2 in Pathological Cascades Mediated by TAR DNA-binding Protein 43 (TDP-43) and Fused in Sarcoma (FUS)

Nihei

Ito

Suzuki

2012

Journal of Biological Chemistry

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Background:The pathological mechanism of the potent modifier of TDP-43 toxicity, ataxin-2, is unknown. Result: Ataxin-2 modified the subcellular distributions of truncated TDP-43 and mutant FUS. Conclusion: Increased ataxin-2 leads to a mislocation of TDP-43 and FUS, leading the RNA dysregulation. Significance: An aberrant distribution of TDP-43 and FUS mediated by ataxin-2 may be a key therapeutic target against ALS.

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supporting

confidence: 55%

“…Cell Culture and Reagents-HeLa human carcinoma cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum, as described previously (6,7,15,16). Human neuroblastoma SK-N-SH cells were cultured in ␣MEM (Invitrogen) supplemented with 10% fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

Roles of Ataxin-2 in Pathological Cascades Mediated by TAR DNA-binding Protein 43 (TDP-43) and Fused in Sarcoma (FUS)

Nihei

Ito

Suzuki

2012

Journal of Biological Chemistry

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“…4 and Table 1). Loss of Kapβ2 binding in these mutants correlates with the degree of cytoplasmic mislocalization in cells (2,(6)(7)(8)(9). Fig.…”

Section: Resultsmentioning

confidence: 86%

“…More recently, the signal was shown to direct Kapβ2-mediated nuclear import in cells (2) and to be heavily mutated in ∼5% of familial amyotrophic lateral sclerosis (ALS) (3)(4)(5), a progressive and fatal neurodegenerative disorder. ALS mutations in the PY-NLS disrupted nuclear import of FUS, causing its mislocalization and aggregation in the cytoplasm, as evidenced by cytoplasmic FUS inclusions in motor neurons of ALS patients (2,(6)(7)(8)(9). The PY-NLS of FUS is also mutated in another neurodegenerative disease, frontotemporal lobar dementia (FTLD), which also is characterized by cytoplasmic mislocalization and aggregation of FUS (10,11).…”

mentioning

confidence: 99%

Structural and energetic basis of ALS-causing mutations in the atypical proline–tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS)

Zhang

Chook²

2012

Proc. Natl. Acad. Sci. U.S.A.

133

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Mutations in the proline/tyrosine-nuclear localization signal (PY-NLS) of the Fused in Sarcoma protein (FUS) cause amyotrophic lateral sclerosis (ALS). Here we report the crystal structure of the FUS PY-NLS bound to its nuclear import receptor Karyopherinβ2 (Kapβ2; also known as Transportin). The FUS PY-NLS occupies the structurally invariant C-terminal arch of Kapβ2, tracing a path similar to that of other characterized PY-NLSs. Unlike other PY-NLSs, which generally bind Kapβ2 in fully extended conformations, the FUS peptide is atypical as its central portion forms a 2.5-turn α-helix. The Kapβ2-binding epitopes of the FUS PY-NLS consist of an N-terminal PGKM hydrophobic motif, a central arginine-rich α-helix, and a C-terminal PY motif. ALS mutations are found almost exclusively within these epitopes. Each ALS mutation site makes multiple contacts with Kapβ2 and mutations of these residues decrease binding affinities for Kapβ2 (K D for wild-type FUS PY-NLS is 9.5 nM) up to ninefold. Thermodynamic analyses of ALS mutations in the FUS PY-NLS show that the weakening of FUSKapβ2 binding affinity, the degree of cytoplasmic mislocalization, and ALS disease severity are correlated.T he proline/tyrosine-nuclear localization signal (PY-NLS) of RNA-binding protein Fused in Sarcoma (FUS) was first identified in a structure-based bioinformatics approach and shown to bind the import-karyopherin, karyopherinβ2 (Kapβ2) in a Ran-sensitive manner (1). More recently, the signal was shown to direct Kapβ2-mediated nuclear import in cells (2) and to be heavily mutated in ∼5% of familial amyotrophic lateral sclerosis (ALS) (3-5), a progressive and fatal neurodegenerative disorder. ALS mutations in the PY-NLS disrupted nuclear import of FUS, causing its mislocalization and aggregation in the cytoplasm, as evidenced by cytoplasmic FUS inclusions in motor neurons of ALS patients (2, 6-9). The PY-NLS of FUS is also mutated in another neurodegenerative disease, frontotemporal lobar dementia (FTLD), which also is characterized by cytoplasmic mislocalization and aggregation of FUS (10, 11). The pathogenic role of FUS PY-NLS mutants was further confirmed by observations that expression of such proteins in Drosophila, Caenorhabditis elegans, zebrafish, and rats caused neurodegeneration (12-15). Proper FUS localization is important in maintaining neuronal homeostasis, and it is thus important to understand the factors that govern localization of both wild-type and mutant proteins.PY-NLSs are recognized by the import-karyopherin Kapβ2 (also known as Transportin) for transport through the nuclear pore complex into the nucleus (1, 16-18). These 15-to 100-residue-long sequences are large and diverse and cannot be sufficiently described by a traditional consensus sequence but are instead described by a collection of physical rules that include requirements for intrinsic structural disorder, overall basic character, and a set of sequence motifs. PY-NLS motifs consist of an N-terminal hydrophobic or basic motif and a C-terminal RX 2-5 PY motif (Fig. ...

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“…The cells were lysed, and the cytosolic and nuclear fractions of cells were prepared as described previously [28] .…”

Section: Sod Assaymentioning

confidence: 99%

Characterization of a novel curcumin analog P1 as potent inhibitor of the NF-κB signaling pathway with distinct mechanisms

et al. 2013

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Aim: Curcumin has shown promising anticancer activity, which relies on its inhibition on NF-κB pathway. In this study, we characterized the pharmacological profile of a novel curcumin analog P1 and elucidate the related mechanisms. Methods: HEK293/NF-κB cells, stably transfected with an NF-κB-responsive luciferase reporter plasmid, were generated for highthroughput screen (HTS). Eight cancer cell lines, including PC3, COLO 205, HeLa cells etc. were tested. Cell viability was assessed using the sulforhodamine B (SRB) assays. Cell apoptosis was evaluated using FACS, immunocytochemistry, and Western blotting. H 2 -DCFDA and MitoSOX Red were used to detect cellular and mitochondrial reactive oxygen species (ROS). The mitochondrial function was evaluated using mitochondrial oxygen consumption assay. Results: P1, a tropinone curcumin, was found in HTS targeting the NF-κB pathway. Its IC 50 value in inhibition of TNF-α-induced NF-κB activation was 0.8 μmol/L, whereas its IC 50 values in inhibiting the growth of A549 and HeLa cells were 1.24 and 0.69 μmol/L, respectively, which was 20-to 30-fold more potent than curcumin. The inhibition of P1 on the NF-κB pathway was further addressed in HeLa cells. The compound up to 10 µmol/L did not affect the binding of NF-κB to DNA, but markedly inhibited NF-κB nuclear translocation, IκB degradation and IκB kinase phosphorylation. The compound (1 and 3 µmol/L) concentration-dependently induced ROS generation, whereas curcumin up to 20 µmol/L had no effect. P1-induced ROS generation was mainly localized in mitochondria, and reversed by NAC. Moreover, the compound significantly enhanced TNF-α-induced apoptosis. Conclusion: P1 is a novel curcumin analog with potent anticancer activities, which exerts a distinct inhibition on the NF-κB pathway.

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Nuclear transport impairment of amyotrophic lateral sclerosis‐linked mutations in FUS/TLS

Cited by 154 publications

References 43 publications

Roles of Ataxin-2 in Pathological Cascades Mediated by TAR DNA-binding Protein 43 (TDP-43) and Fused in Sarcoma (FUS)

Roles of Ataxin-2 in Pathological Cascades Mediated by TAR DNA-binding Protein 43 (TDP-43) and Fused in Sarcoma (FUS)

Structural and energetic basis of ALS-causing mutations in the atypical proline–tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS)

Characterization of a novel curcumin analog P1 as potent inhibitor of the NF-κB signaling pathway with distinct mechanisms

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