Intranuclear Aggregation of Mutant FUS/TLS as a Molecular Pathomechanism of Amyotrophic Lateral Sclerosis

Nomura, Takao; Watanabe, Shôji; Kaneko, Kazuyuki; Yamanaka, Koji; Nukina, Nobuyuki; Furukawa, Yoshiaki

doi:10.1074/jbc.m113.516492

Cited by 120 publications

(102 citation statements)

References 48 publications

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“…In contrast, our group has shown that a fulllength FUS protein dual-tagged with an N-terminal GST and a C-terminal 6x His remained soluble and we also found that an ALS-causing mutation, G156E, triggered the formation of insoluble aggregates with amyloid-like fibrillar morphologies (Nomura et al 2013). Furthermore, the fibrils of GST-FUS(G156E)-His reacted with Thioflavin T, suggesting distinct properties from those of the hydrogels prepared from the LC domain as mentioned above.…”

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confidence: 69%

“…In many of aggregation-prone proteins, fusion with glutathione-S-transferase (GST) can increase their solubility; indeed, soluble FUS proteins can be prepared when tagged with an N-terminal but not C-terminal GST (Sun et al 2011). As shown by our group, however, most fractions of GST-FUS expressed in E. coli were collected as insoluble inclusions, and significant amounts of the truncated GST-FUS were also contaminated even after the affinity purification using glutathione Sepharose resins (Nomura et al 2013). We thus further attached a 6x His tag at the C-terminus of GST-FUS and then successfully removed the truncated proteins by additional purification using Ni 2+ -affinity resins.…”

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confidence: 94%

“…Very interestingly, the affinity of PY-NLS with its nuclear import receptor, transportin, has been reported to significantly decrease with a series of ALS-causing mutations and appears to inversely correlate with the disease severity (Dormann et al 2012;Zhang and Chook 2012), while the cytoplasmic accumulation of FUS with mutations in the regions outside PY-NLS has not been well described. Instead, mutations in RGG1 of FUS, RGG2/3 of EWSR1, and RGG3 of TAF15 have been proposed to accelerate the protein aggregation (Couthouis et al 2011(Couthouis et al , 2012Nomura et al 2013); however, because FET proteins are quite prone to aggregation, effects of mutations on aggregation kinetics need to be investigated in more detail. FET proteins can bind single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and RNA.…”

Section: Introductionmentioning

confidence: 99%

“…We thus further attached a 6x His tag at the C-terminus of GST-FUS and then successfully removed the truncated proteins by additional purification using Ni 2+ -affinity resins. Furthermore, purified GST-FUS-His samples were found to contain E. coli endogenous nucleotides (Nomura et al 2013); therefore, dissociation of those nucleotides with a buffer containing high concentrations of salts (1 M NaCl) was required to prepare nucleotide-free GST-FUS-His samples.…”

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confidence: 99%

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Aggregation of FET Proteins as a Pathological Change in Amyotrophic Lateral Sclerosis

Furukawa

Tokuda

2016

Advances in Experimental Medicine and Biology

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Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease that is characterized by the formation of abnormal inclusions in neurons. While the pathomechanism of ALS remains obscure, a number of proteins have been identified in the inclusion bodies, and the pathological roles of RNA-binding proteins have been increasingly emphasized. Among those, the FET proteins (FUS, EWSR1, TAF15) were recently identified as RNA-binding proteins in pathological inclusions of ALS and other neurodegenerative diseases; moreover, mutations in the genes encoding the FET proteins were found to be associated with familial forms of ALS. FET proteins are normally localized in the nucleus, but the introduction of pathogenic mutations in FET proteins leads to their abnormal redistribution to the cytoplasm, where they form aggregates. While further investigation will be required to understand the intracellular factors controlling the aggregation propensities of FET proteins, they are thought to lose their physiological functions and become toxic through their misfolding/aggregation. Here, we will briefly review recent advances of our understanding of the physiological functions and aggregation behavior of FET proteins in vivo as well as in vitro.

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confidence: 69%

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confidence: 94%

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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Aggregation of FET Proteins as a Pathological Change in Amyotrophic Lateral Sclerosis

Furukawa

Tokuda

2016

Advances in Experimental Medicine and Biology

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“…The prion-like domain consists of an intrinsically disordered QGSY (glutamine-glycine-serine-tyrosine)-rich region (amino acids 1-164) and a glycine-rich region (amino acids 165-239). A missense mutation (G156E) in the QGSY-rich region has been found in patients with familial ALS and has been reported to cause intranuclear aggregation of FUS (17). However, the role of the QGSY-rich region in maintaining FUS intranuclear distribution and function under physiological conditions is unknown.…”

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confidence: 99%

Self-assembled FUS binds active chromatin and regulates gene transcription

Yang

Gál

Chen

et al. 2014

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

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Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. Fused in sarcoma (FUS) is a DNA/RNA binding protein and mutations in FUS cause a subset of familial ALS. Most ALS mutations are clustered in the C-terminal nuclear localization sequence of FUS and consequently lead to the accumulation of protein inclusions in the cytoplasm. It remains debatable whether loss of FUS normal function in the nucleus or gain of toxic function in the cytoplasm plays a more critical role in the ALS etiology. Moreover, the physiological function of FUS in the nucleus remains to be fully understood. In this study, we found that a significant portion of nuclear FUS was bound to active chromatin and that the ALS mutations dramatically decreased FUS chromatin binding ability. Functionally, the chromatin binding is required for FUS transcription activation, but not for alternative splicing regulation. The N-terminal QGSY (glutamine-glycine-serine-tyrosine)-rich region (amino acids 1-164) mediates FUS self-assembly in the nucleus of mammalian cells and the self-assembly is essential for its chromatin binding and transcription activation. In addition, RNA binding is also required for FUS self-assembly and chromatin binding. Together, our results suggest a functional assembly of FUS in the nucleus under physiological conditions, which is different from the cytoplasmic inclusions. The ALS mutations can cause loss of function in the nucleus by disrupting this assembly and chromatin binding. FUS is a multifunctional protein and has been reported to play a role in various aspects of RNA metabolism (6), including transcription regulation and alternative splicing. FUS was initially identified in liposarcomas as part of a fusion protein (7,8) in which the N-terminal domain of FUS (amino acid 1-266) is recombined to transcription factor CHOP at its N terminus. The FUS-CHOP fusion protein activates the transcription of oncogenes and promotes tumorigenesis (9, 10). In familial ALS, most mutations are clustered in the C-terminal nuclear localization sequence (NLS) of FUS and consequently cause the mislocalization of FUS protein from the nucleus to the cytoplasm and the accumulation of protein inclusions (11-13). Such observations suggest two potential disease-causing mechanisms: loss of FUS normal function in the nucleus and gain of toxic function in the cytoplasm. It remains to be determined which mechanism plays a more critical role in ALS etiology and the two mechanisms are not necessarily exclusive of each other.Cytoplasmic FUS inclusions resemble stress granules, indicated by colocalization of FUS with different stress granule components (11,12). Stress granules are temporary cellular structures containing RNAs and proteins from suspended translation apparatus (14). Stress granule formation promotes cell survival under stressed conditions by redistributing translation resources. Compromised stress granule response in the presence of FUS mutants is considered a contributing factor to motor neuron dysfunction (15).Although t...

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Valosin‐containing protein regulates the stability of fused in sarcoma granules in cells by changing ATP concentrations

et al. 2022

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Fused in sarcoma (FUS), a DNA/RNA‐binding protein, undergoes liquid‐liquid phase separation to form granules in cells. Aberrant FUS granulation is associated with neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We found that FUS granules contain a multifunctional AAA ATPase, valosin‐containing protein (VCP), which is known as a key regulator of protein degradation. FUS granule stability depends on ATP concentrations in cells. VCP ATPase changes the FUS granule stability time‐dependently by consuming ATP to reduce its concentrations in the granules: VCPs in de novo FUS granules stabilize the granules, while long‐lasting VCP colocalization destabilizes the granules. The proteolysis‐promoting function of VCP may subsequently dissolve the unstabilized granules. We propose that VCP colocalized to the FUS granules acts as a timer to limit the residence time of the granules in cells.

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Intranuclear Aggregation of Mutant FUS/TLS as a Molecular Pathomechanism of Amyotrophic Lateral Sclerosis

Cited by 120 publications

References 48 publications

Aggregation of FET Proteins as a Pathological Change in Amyotrophic Lateral Sclerosis

Aggregation of FET Proteins as a Pathological Change in Amyotrophic Lateral Sclerosis

Self-assembled FUS binds active chromatin and regulates gene transcription

Valosin‐containing protein regulates the stability of fused in sarcoma granules in cells by changing ATP concentrations

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