Limits...
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

Enhanced protein quality control by the knockdown of UBE4B and LSD1 depends on p53.(A) Left: p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown. Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs. Right: quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control, double (UBE4B/LSD1), or triple (UBE4B/LSD1/p53) shRNAs (n = 2). (B) The degenerative TDP-43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 (p53 RNAi), or by the overexpression of a dominant-negative p53 mutant (p53.R155H). Expression of p53 RNAi, p53.R155H, and TDP-43M337V are driven by GMR-Gal4. (C) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1G85R-induced proteotoxicity. Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0.8 μM TEN1 or vehicle (VEH) DMSO, followed by infection of HSV-SOD1G85R or the HSV-LacZ control after 24 h. At day 5 post-infection, cells were fixed and stained with a motor neuron-specific anti-neurofilament H (NF-H) antibody. Left: representative images of motor neurons in each condition. Scale bar = 50 μm. Right: quantification of motor neuron survival (one-way ANOVA with multiple comparisons and Tukey correction). Data represent means ± SEM. (D) Schematic of the SUNS pathway. A reduction in UBE4B and LSD1 function synergistically activates the p53-mediated transcriptional program, promoting clearance of misfolded and aggregated proteins via increased proteasomal and autophagic clearance. The numerical data used to make this figure can be found in S1 Data.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4383508&req=5

pbio.1002114.g007: Enhanced protein quality control by the knockdown of UBE4B and LSD1 depends on p53.(A) Left: p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown. Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs. Right: quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control, double (UBE4B/LSD1), or triple (UBE4B/LSD1/p53) shRNAs (n = 2). (B) The degenerative TDP-43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 (p53 RNAi), or by the overexpression of a dominant-negative p53 mutant (p53.R155H). Expression of p53 RNAi, p53.R155H, and TDP-43M337V are driven by GMR-Gal4. (C) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1G85R-induced proteotoxicity. Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0.8 μM TEN1 or vehicle (VEH) DMSO, followed by infection of HSV-SOD1G85R or the HSV-LacZ control after 24 h. At day 5 post-infection, cells were fixed and stained with a motor neuron-specific anti-neurofilament H (NF-H) antibody. Left: representative images of motor neurons in each condition. Scale bar = 50 μm. Right: quantification of motor neuron survival (one-way ANOVA with multiple comparisons and Tukey correction). Data represent means ± SEM. (D) Schematic of the SUNS pathway. A reduction in UBE4B and LSD1 function synergistically activates the p53-mediated transcriptional program, promoting clearance of misfolded and aggregated proteins via increased proteasomal and autophagic clearance. The numerical data used to make this figure can be found in S1 Data.

Mentions: To determine whether p53 mediates the UBE4B- and LSD1-dependent clearance of the SOD1G85R proteins, we knocked down UBE4B and LSD1 with or without the removal of p53 and then examined the levels of SOD1G85R. We applied both transient and stable shRNA knockdown by creating an inducible, stable HEK293T cell line expressing tetracycline-regulated shRNAs against UBE4B, LSD1, and p53. Both transient and stable knockdown of p53 significantly reversed the SOD1G85R protein clearance conferred by the UBE4B and LSD1 knockdown (Fig. 7A and S7A Fig.). This result was confirmed with an independent set of shRNAs against UBE4B and LSD1 (S7B Fig.) and by knockdown of UBE4B and LSD1 in HCT116 cells with or without the p53 gene knocked out (S7C Fig.). Notably, unlike HEK293T cells, HCT116 cells do not express SV40 large T-antigen (LT-Ag), a regulator of p53 stability [44], suggesting that the action of UBE4B and LSD1 does not require LT-Ag. Taken together, these results demonstrate that p53 is required for the UBE4B- and LSD1-dependent clearance of the SOD1G85R proteins, and it acts downstream of UBE4B and LSD1 to positively regulate the clearance of misfolded proteins.


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)

Enhanced protein quality control by the knockdown of UBE4B and LSD1 depends on p53.(A) Left: p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown. Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs. Right: quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control, double (UBE4B/LSD1), or triple (UBE4B/LSD1/p53) shRNAs (n = 2). (B) The degenerative TDP-43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 (p53 RNAi), or by the overexpression of a dominant-negative p53 mutant (p53.R155H). Expression of p53 RNAi, p53.R155H, and TDP-43M337V are driven by GMR-Gal4. (C) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1G85R-induced proteotoxicity. Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0.8 μM TEN1 or vehicle (VEH) DMSO, followed by infection of HSV-SOD1G85R or the HSV-LacZ control after 24 h. At day 5 post-infection, cells were fixed and stained with a motor neuron-specific anti-neurofilament H (NF-H) antibody. Left: representative images of motor neurons in each condition. Scale bar = 50 μm. Right: quantification of motor neuron survival (one-way ANOVA with multiple comparisons and Tukey correction). Data represent means ± SEM. (D) Schematic of the SUNS pathway. A reduction in UBE4B and LSD1 function synergistically activates the p53-mediated transcriptional program, promoting clearance of misfolded and aggregated proteins via increased proteasomal and autophagic clearance. The numerical data used to make this figure can be found in S1 Data.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383508&req=5

pbio.1002114.g007: Enhanced protein quality control by the knockdown of UBE4B and LSD1 depends on p53.(A) Left: p53 knockdown reverses the enhanced clearance of SOD1G85R proteins conferred by the UBE4B and LSD1 double knockdown. Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs. Right: quantification of insoluble aggregated SOD1G85R in pellet fractions from HEK293T cells transfected with control, double (UBE4B/LSD1), or triple (UBE4B/LSD1/p53) shRNAs (n = 2). (B) The degenerative TDP-43M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 (p53 RNAi), or by the overexpression of a dominant-negative p53 mutant (p53.R155H). Expression of p53 RNAi, p53.R155H, and TDP-43M337V are driven by GMR-Gal4. (C) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1G85R-induced proteotoxicity. Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0.8 μM TEN1 or vehicle (VEH) DMSO, followed by infection of HSV-SOD1G85R or the HSV-LacZ control after 24 h. At day 5 post-infection, cells were fixed and stained with a motor neuron-specific anti-neurofilament H (NF-H) antibody. Left: representative images of motor neurons in each condition. Scale bar = 50 μm. Right: quantification of motor neuron survival (one-way ANOVA with multiple comparisons and Tukey correction). Data represent means ± SEM. (D) Schematic of the SUNS pathway. A reduction in UBE4B and LSD1 function synergistically activates the p53-mediated transcriptional program, promoting clearance of misfolded and aggregated proteins via increased proteasomal and autophagic clearance. The numerical data used to make this figure can be found in S1 Data.
Mentions: To determine whether p53 mediates the UBE4B- and LSD1-dependent clearance of the SOD1G85R proteins, we knocked down UBE4B and LSD1 with or without the removal of p53 and then examined the levels of SOD1G85R. We applied both transient and stable shRNA knockdown by creating an inducible, stable HEK293T cell line expressing tetracycline-regulated shRNAs against UBE4B, LSD1, and p53. Both transient and stable knockdown of p53 significantly reversed the SOD1G85R protein clearance conferred by the UBE4B and LSD1 knockdown (Fig. 7A and S7A Fig.). This result was confirmed with an independent set of shRNAs against UBE4B and LSD1 (S7B Fig.) and by knockdown of UBE4B and LSD1 in HCT116 cells with or without the p53 gene knocked out (S7C Fig.). Notably, unlike HEK293T cells, HCT116 cells do not express SV40 large T-antigen (LT-Ag), a regulator of p53 stability [44], suggesting that the action of UBE4B and LSD1 does not require LT-Ag. Taken together, these results demonstrate that p53 is required for the UBE4B- and LSD1-dependent clearance of the SOD1G85R proteins, and it acts downstream of UBE4B and LSD1 to positively regulate the clearance of misfolded proteins.

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