Mutations in the fused in sarcoma/translocated in liposarcoma(FUS/TLS)gene have been associated with amyotrophic lateral sclerosis(ALS).FUS-positive neuropathology is reported in a range of neurodegenerative diseases,...Mutations in the fused in sarcoma/translocated in liposarcoma(FUS/TLS)gene have been associated with amyotrophic lateral sclerosis(ALS).FUS-positive neuropathology is reported in a range of neurodegenerative diseases,including ALS and fronto-temporal lobar degeneration with ubiquitin-positive pathology(FTLDU).To examine protein aggregation and cytotoxicity,we expressed human FUS protein in yeast.Expression of either wild type or ALS-associated R524S or P525L mutant FUS in yeast cells led to formation of aggregates and cytotoxicity,with the two ALS mutants showing increased cytotoxicity.Therefore,yeast cells expressing human FUS protein recapitulate key features of FUSpositive neurodegenerative diseases.Interestingly,a significant fraction of FUS expressing yeast cells stained by propidium iodide were without detectable protein aggregates,suggesting that membrane impairment and cellular damage caused by FUS expression may occur before protein aggregates become microscopically detectable and that aggregate formation might protect cells from FUS-mediated cytotoxicity.The N-terminus of FUS,containing the QGSY and G rich regions,is sufficient for the formation of aggregates but not cytotoxicity.The C-terminal domain,which contains a cluster of mutations,did not show aggregation or cytotoxicity.Similar to TDP-43 when expressed in yeast,FUS protein has the intrinsic property of forming aggregates in the absence of other human proteins.On the other hand,the aggregates formed by FUS are thioflavin T-positive and resistant to 0.5%sarkosyl,unlike TDP-43 when expressed in yeast cells.Furthermore,TDP-43 and FUS display distinct domain requirements in aggregate formation and cytotoxicity.展开更多
Amyotrophic lateral sclerosis(ALS)is a fatal disease characterized by the premature loss of motor neurons.While the underlying cellular mechanisms of neuron degeneration are unknown,the cytoplasmic aggregation of seve...Amyotrophic lateral sclerosis(ALS)is a fatal disease characterized by the premature loss of motor neurons.While the underlying cellular mechanisms of neuron degeneration are unknown,the cytoplasmic aggregation of several proteins is associated with sporadic and familial forms of the disease.Both wild-type and mutant forms of the RNA-binding proteins FUS and TDP-43 accumulate in cytoplasmic inclusions in the neurons of ALS patients.It is not known if these so-called proteinopathies are due to a loss of function or a gain of toxicity resulting from the formation of cytoplasmic aggregates.Here we present a model of FUS toxicity using the yeast Saccharomyces cerevisiae in which toxicity is associated with greater expression and accumulation of FUS in cytoplasmic aggregates.We find that FUS and TDP-43 have a high propensity for co-aggregation,unlike the aggregation patterns of several other aggregation-prone proteins.Moreover,the biophysical properties of FUS aggregates in yeast are distinctly different from many amyloidogenic proteins,suggesting they are not composed of amyloid.展开更多
基金JYW is supported by NIH and James S.McDonnell Foundation.LL is supported by NIH(R01NS056086).
文摘Mutations in the fused in sarcoma/translocated in liposarcoma(FUS/TLS)gene have been associated with amyotrophic lateral sclerosis(ALS).FUS-positive neuropathology is reported in a range of neurodegenerative diseases,including ALS and fronto-temporal lobar degeneration with ubiquitin-positive pathology(FTLDU).To examine protein aggregation and cytotoxicity,we expressed human FUS protein in yeast.Expression of either wild type or ALS-associated R524S or P525L mutant FUS in yeast cells led to formation of aggregates and cytotoxicity,with the two ALS mutants showing increased cytotoxicity.Therefore,yeast cells expressing human FUS protein recapitulate key features of FUSpositive neurodegenerative diseases.Interestingly,a significant fraction of FUS expressing yeast cells stained by propidium iodide were without detectable protein aggregates,suggesting that membrane impairment and cellular damage caused by FUS expression may occur before protein aggregates become microscopically detectable and that aggregate formation might protect cells from FUS-mediated cytotoxicity.The N-terminus of FUS,containing the QGSY and G rich regions,is sufficient for the formation of aggregates but not cytotoxicity.The C-terminal domain,which contains a cluster of mutations,did not show aggregation or cytotoxicity.Similar to TDP-43 when expressed in yeast,FUS protein has the intrinsic property of forming aggregates in the absence of other human proteins.On the other hand,the aggregates formed by FUS are thioflavin T-positive and resistant to 0.5%sarkosyl,unlike TDP-43 when expressed in yeast cells.Furthermore,TDP-43 and FUS display distinct domain requirements in aggregate formation and cytotoxicity.
基金This research was supported by Uniformed Services University of the Health Sciences and the Intramural Research Program of the National Institutes of Health,National Institute of Diabetes and Digestive and Kidney Diseases.
文摘Amyotrophic lateral sclerosis(ALS)is a fatal disease characterized by the premature loss of motor neurons.While the underlying cellular mechanisms of neuron degeneration are unknown,the cytoplasmic aggregation of several proteins is associated with sporadic and familial forms of the disease.Both wild-type and mutant forms of the RNA-binding proteins FUS and TDP-43 accumulate in cytoplasmic inclusions in the neurons of ALS patients.It is not known if these so-called proteinopathies are due to a loss of function or a gain of toxicity resulting from the formation of cytoplasmic aggregates.Here we present a model of FUS toxicity using the yeast Saccharomyces cerevisiae in which toxicity is associated with greater expression and accumulation of FUS in cytoplasmic aggregates.We find that FUS and TDP-43 have a high propensity for co-aggregation,unlike the aggregation patterns of several other aggregation-prone proteins.Moreover,the biophysical properties of FUS aggregates in yeast are distinctly different from many amyloidogenic proteins,suggesting they are not composed of amyloid.
