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Regulation of protein quality control by UBE4B and LSD1 through p53-mediated transcription.

Periz G, Lu J, Zhang T, Kankel MW, Jablonski AM, Kalb R, McCampbell A, Wang J - PLoS Biol. (2015)

Bottom Line: Here, we identify two genes, ufd-2 and spr-5, that when inactivated, synergistically and robustly suppress neurotoxicity associated with misfolded proteins in Caenorhabditis elegans.Loss of human orthologs ubiquitination factor E4 B (UBE4B) and lysine-specific demethylase 1 (LSD1), respectively encoding a ubiquitin ligase and a lysine-specific demethylase, promotes the clearance of misfolded proteins in mammalian cells by activating both proteasomal and autophagic degradation machineries.An unbiased search in this pathway reveals a downstream effector as the transcription factor p53, a shared substrate of UBE4B and LSD1 that functions as a key regulator of protein quality control to protect against proteotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and Department of Neuroscience, Bloomberg School of Public Health and School of Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America.

ABSTRACT
Protein quality control is essential for clearing misfolded and aggregated proteins from the cell, and its failure is associated with many neurodegenerative disorders. Here, we identify two genes, ufd-2 and spr-5, that when inactivated, synergistically and robustly suppress neurotoxicity associated with misfolded proteins in Caenorhabditis elegans. Loss of human orthologs ubiquitination factor E4 B (UBE4B) and lysine-specific demethylase 1 (LSD1), respectively encoding a ubiquitin ligase and a lysine-specific demethylase, promotes the clearance of misfolded proteins in mammalian cells by activating both proteasomal and autophagic degradation machineries. An unbiased search in this pathway reveals a downstream effector as the transcription factor p53, a shared substrate of UBE4B and LSD1 that functions as a key regulator of protein quality control to protect against proteotoxicity. These studies identify a new protein quality control pathway via regulation of transcription factors and point to the augmentation of protein quality control as a wide-spectrum antiproteotoxicity strategy.

No MeSH data available.


Related in: MedlinePlus

Identification and characterization of a robust suppressor that ameliorates the locomotion defects in the C. elegans model of SOD1-associated ALS.(A) Workflow of the suppressor screen identifying mutant C. elegans (red) with saliently improved movement. (B) Locomotor behavior, measured by thrashing rates in liquid medium, in the C. elegans strains with neuronal expression of human WT SOD1 or ALS-linked mutant SOD1G85R, in the presence (M1/M1) or absence (+/+) of the suppressor mutation (n = 16). (C) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1G85R strains, with (M1/M1) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1G85R mRNA and total protein are not changed by the suppressor mutation (M1/M1). The western blot lanes are from the same gel and exposure. (D) Sequence analysis of the M1 strain, revealing that independent mutations in two genes, a lysine-specific demethylase, spr-5 (R646Q), and a ubiquitin ligase, ufd-2 (W824X), are required for the full suppressor phenotype. (E) C. elegans SPR-5 and UFD-2 and their mammalian homologs lysine-specific demethylase 1 (LSD1) and ubiquitination factor E4 B (UBE4B) share all major protein domains. These include the Swi3-Rsc8-Moira (SWIRM), amine oxidase-like (AOL), and TOWER domains in LSD1/SPR-5 and the Ufd2 Core and U-box domains in UBE4B/UFD-2. Positions of the missense, nonsense, and deletion mutations in mutant C. elegans are indicated. (F) Locomotor behavior, measured by thrashing rates, of the C. elegans carrying the SOD1G85R transgene on the normal background (WT), with the  mutation of either ufd-2(tm1380) or spr-5(by134), the double mutation ufd-2(tm1380);spr-5(by134), or the M1 suppressor ufd-2(W824X);spr-5(R646Q) (n = 16). (G) Western blotting of the supernatant (S) and pellet (P) protein fractions from the C. elegans carrying the SOD1G85R transgene with the double mutation ufd-2(tm1380);spr-5(by134) compared with controls. While SOD1G85R protein levels are unchanged in the S fraction, those in the P fraction are decreased in the double suppressor mutant. The P fraction represents only about 1.7% of total SOD1G85R in the WT sample; therefore, a higher ratio of the P fraction relative to the S fraction (approximately 2:1) is used for the western analysis. (H) The overexpression (OE) of WT UFD-2 or SPR-5 in the C. elegans nervous system blocks the protection conferred by the double mutation ufd-2(tm1380);spr-5(by134) (n = 16). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
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pbio.1002114.g001: Identification and characterization of a robust suppressor that ameliorates the locomotion defects in the C. elegans model of SOD1-associated ALS.(A) Workflow of the suppressor screen identifying mutant C. elegans (red) with saliently improved movement. (B) Locomotor behavior, measured by thrashing rates in liquid medium, in the C. elegans strains with neuronal expression of human WT SOD1 or ALS-linked mutant SOD1G85R, in the presence (M1/M1) or absence (+/+) of the suppressor mutation (n = 16). (C) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1G85R strains, with (M1/M1) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1G85R mRNA and total protein are not changed by the suppressor mutation (M1/M1). The western blot lanes are from the same gel and exposure. (D) Sequence analysis of the M1 strain, revealing that independent mutations in two genes, a lysine-specific demethylase, spr-5 (R646Q), and a ubiquitin ligase, ufd-2 (W824X), are required for the full suppressor phenotype. (E) C. elegans SPR-5 and UFD-2 and their mammalian homologs lysine-specific demethylase 1 (LSD1) and ubiquitination factor E4 B (UBE4B) share all major protein domains. These include the Swi3-Rsc8-Moira (SWIRM), amine oxidase-like (AOL), and TOWER domains in LSD1/SPR-5 and the Ufd2 Core and U-box domains in UBE4B/UFD-2. Positions of the missense, nonsense, and deletion mutations in mutant C. elegans are indicated. (F) Locomotor behavior, measured by thrashing rates, of the C. elegans carrying the SOD1G85R transgene on the normal background (WT), with the mutation of either ufd-2(tm1380) or spr-5(by134), the double mutation ufd-2(tm1380);spr-5(by134), or the M1 suppressor ufd-2(W824X);spr-5(R646Q) (n = 16). (G) Western blotting of the supernatant (S) and pellet (P) protein fractions from the C. elegans carrying the SOD1G85R transgene with the double mutation ufd-2(tm1380);spr-5(by134) compared with controls. While SOD1G85R protein levels are unchanged in the S fraction, those in the P fraction are decreased in the double suppressor mutant. The P fraction represents only about 1.7% of total SOD1G85R in the WT sample; therefore, a higher ratio of the P fraction relative to the S fraction (approximately 2:1) is used for the western analysis. (H) The overexpression (OE) of WT UFD-2 or SPR-5 in the C. elegans nervous system blocks the protection conferred by the double mutation ufd-2(tm1380);spr-5(by134) (n = 16). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.

Mentions: To better understand the regulatory mechanisms that mitigate an increased load of misfolded proteins, we conducted a forward genetic screen for cellular factors that alleviate such stress and relieve cells from proteotoxic insults. This screen took advantage of a C. elegans model of ALS, in which the neuron-directed expression of the ALS-linked, G85R mutant human SOD1 (SOD1G85R) protein leads to its aggregation into misfolded soluble oligomers and larger insoluble aggregates [14,15]. Misfolded SOD1G85R protein is highly toxic, leading to age-dependent synaptic dysfunction, neurodegeneration, and severely impaired movement in the worms [14]. This severe locomotor defect allowed us to perform a large-scale screen for genes that suppress neurodegeneration and improve worm locomotion. In these experiments, we treated homozygous transgenic SOD1G85RC. elegans with ethyl methanesulfonate (EMS) to induce genomic mutations, and the mutagenized P0 hermaphrodites were allowed to self-reproduce for two generations (Fig. 1A). Next, in the F2 offspring, which contain both heterozygous and homozygous suppressor mutations, we selected individual C. elegans based on a salient improvement in the locomotion on a background of poorly moving populations. The potential suppressor clones were bred through until 100% of progeny showed phenotypic improvements and were then subjected to further analysis (Fig. 1A).


Regulation of protein quality control by UBE4B and LSD1 through p53-mediated transcription.

Periz G, Lu J, Zhang T, Kankel MW, Jablonski AM, Kalb R, McCampbell A, Wang J - PLoS Biol. (2015)

Identification and characterization of a robust suppressor that ameliorates the locomotion defects in the C. elegans model of SOD1-associated ALS.(A) Workflow of the suppressor screen identifying mutant C. elegans (red) with saliently improved movement. (B) Locomotor behavior, measured by thrashing rates in liquid medium, in the C. elegans strains with neuronal expression of human WT SOD1 or ALS-linked mutant SOD1G85R, in the presence (M1/M1) or absence (+/+) of the suppressor mutation (n = 16). (C) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1G85R strains, with (M1/M1) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1G85R mRNA and total protein are not changed by the suppressor mutation (M1/M1). The western blot lanes are from the same gel and exposure. (D) Sequence analysis of the M1 strain, revealing that independent mutations in two genes, a lysine-specific demethylase, spr-5 (R646Q), and a ubiquitin ligase, ufd-2 (W824X), are required for the full suppressor phenotype. (E) C. elegans SPR-5 and UFD-2 and their mammalian homologs lysine-specific demethylase 1 (LSD1) and ubiquitination factor E4 B (UBE4B) share all major protein domains. These include the Swi3-Rsc8-Moira (SWIRM), amine oxidase-like (AOL), and TOWER domains in LSD1/SPR-5 and the Ufd2 Core and U-box domains in UBE4B/UFD-2. Positions of the missense, nonsense, and deletion mutations in mutant C. elegans are indicated. (F) Locomotor behavior, measured by thrashing rates, of the C. elegans carrying the SOD1G85R transgene on the normal background (WT), with the  mutation of either ufd-2(tm1380) or spr-5(by134), the double mutation ufd-2(tm1380);spr-5(by134), or the M1 suppressor ufd-2(W824X);spr-5(R646Q) (n = 16). (G) Western blotting of the supernatant (S) and pellet (P) protein fractions from the C. elegans carrying the SOD1G85R transgene with the double mutation ufd-2(tm1380);spr-5(by134) compared with controls. While SOD1G85R protein levels are unchanged in the S fraction, those in the P fraction are decreased in the double suppressor mutant. The P fraction represents only about 1.7% of total SOD1G85R in the WT sample; therefore, a higher ratio of the P fraction relative to the S fraction (approximately 2:1) is used for the western analysis. (H) The overexpression (OE) of WT UFD-2 or SPR-5 in the C. elegans nervous system blocks the protection conferred by the double mutation ufd-2(tm1380);spr-5(by134) (n = 16). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
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pbio.1002114.g001: Identification and characterization of a robust suppressor that ameliorates the locomotion defects in the C. elegans model of SOD1-associated ALS.(A) Workflow of the suppressor screen identifying mutant C. elegans (red) with saliently improved movement. (B) Locomotor behavior, measured by thrashing rates in liquid medium, in the C. elegans strains with neuronal expression of human WT SOD1 or ALS-linked mutant SOD1G85R, in the presence (M1/M1) or absence (+/+) of the suppressor mutation (n = 16). (C) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1G85R strains, with (M1/M1) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1G85R mRNA and total protein are not changed by the suppressor mutation (M1/M1). The western blot lanes are from the same gel and exposure. (D) Sequence analysis of the M1 strain, revealing that independent mutations in two genes, a lysine-specific demethylase, spr-5 (R646Q), and a ubiquitin ligase, ufd-2 (W824X), are required for the full suppressor phenotype. (E) C. elegans SPR-5 and UFD-2 and their mammalian homologs lysine-specific demethylase 1 (LSD1) and ubiquitination factor E4 B (UBE4B) share all major protein domains. These include the Swi3-Rsc8-Moira (SWIRM), amine oxidase-like (AOL), and TOWER domains in LSD1/SPR-5 and the Ufd2 Core and U-box domains in UBE4B/UFD-2. Positions of the missense, nonsense, and deletion mutations in mutant C. elegans are indicated. (F) Locomotor behavior, measured by thrashing rates, of the C. elegans carrying the SOD1G85R transgene on the normal background (WT), with the mutation of either ufd-2(tm1380) or spr-5(by134), the double mutation ufd-2(tm1380);spr-5(by134), or the M1 suppressor ufd-2(W824X);spr-5(R646Q) (n = 16). (G) Western blotting of the supernatant (S) and pellet (P) protein fractions from the C. elegans carrying the SOD1G85R transgene with the double mutation ufd-2(tm1380);spr-5(by134) compared with controls. While SOD1G85R protein levels are unchanged in the S fraction, those in the P fraction are decreased in the double suppressor mutant. The P fraction represents only about 1.7% of total SOD1G85R in the WT sample; therefore, a higher ratio of the P fraction relative to the S fraction (approximately 2:1) is used for the western analysis. (H) The overexpression (OE) of WT UFD-2 or SPR-5 in the C. elegans nervous system blocks the protection conferred by the double mutation ufd-2(tm1380);spr-5(by134) (n = 16). Data represent means ± SEM. The numerical data used to make this figure can be found in S1 Data.
Mentions: To better understand the regulatory mechanisms that mitigate an increased load of misfolded proteins, we conducted a forward genetic screen for cellular factors that alleviate such stress and relieve cells from proteotoxic insults. This screen took advantage of a C. elegans model of ALS, in which the neuron-directed expression of the ALS-linked, G85R mutant human SOD1 (SOD1G85R) protein leads to its aggregation into misfolded soluble oligomers and larger insoluble aggregates [14,15]. Misfolded SOD1G85R protein is highly toxic, leading to age-dependent synaptic dysfunction, neurodegeneration, and severely impaired movement in the worms [14]. This severe locomotor defect allowed us to perform a large-scale screen for genes that suppress neurodegeneration and improve worm locomotion. In these experiments, we treated homozygous transgenic SOD1G85RC. elegans with ethyl methanesulfonate (EMS) to induce genomic mutations, and the mutagenized P0 hermaphrodites were allowed to self-reproduce for two generations (Fig. 1A). Next, in the F2 offspring, which contain both heterozygous and homozygous suppressor mutations, we selected individual C. elegans based on a salient improvement in the locomotion on a background of poorly moving populations. The potential suppressor clones were bred through until 100% of progeny showed phenotypic improvements and were then subjected to further analysis (Fig. 1A).

Bottom Line: Here, we identify two genes, ufd-2 and spr-5, that when inactivated, synergistically and robustly suppress neurotoxicity associated with misfolded proteins in Caenorhabditis elegans.Loss of human orthologs ubiquitination factor E4 B (UBE4B) and lysine-specific demethylase 1 (LSD1), respectively encoding a ubiquitin ligase and a lysine-specific demethylase, promotes the clearance of misfolded proteins in mammalian cells by activating both proteasomal and autophagic degradation machineries.An unbiased search in this pathway reveals a downstream effector as the transcription factor p53, a shared substrate of UBE4B and LSD1 that functions as a key regulator of protein quality control to protect against proteotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology and Department of Neuroscience, Bloomberg School of Public Health and School of Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America.

ABSTRACT
Protein quality control is essential for clearing misfolded and aggregated proteins from the cell, and its failure is associated with many neurodegenerative disorders. Here, we identify two genes, ufd-2 and spr-5, that when inactivated, synergistically and robustly suppress neurotoxicity associated with misfolded proteins in Caenorhabditis elegans. Loss of human orthologs ubiquitination factor E4 B (UBE4B) and lysine-specific demethylase 1 (LSD1), respectively encoding a ubiquitin ligase and a lysine-specific demethylase, promotes the clearance of misfolded proteins in mammalian cells by activating both proteasomal and autophagic degradation machineries. An unbiased search in this pathway reveals a downstream effector as the transcription factor p53, a shared substrate of UBE4B and LSD1 that functions as a key regulator of protein quality control to protect against proteotoxicity. These studies identify a new protein quality control pathway via regulation of transcription factors and point to the augmentation of protein quality control as a wide-spectrum antiproteotoxicity strategy.

No MeSH data available.


Related in: MedlinePlus