Regulation of Protein Quality Control by UBE4B and LSD1 through p53-Mediated Transcription

<div><p>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, <i>ufd-2</i> and <i>spr-5</i>, that when inactivated, synergistically and robustly suppress neurotoxicity associated with misfolded proteins in <i>Caenorhabditis elegans</i>. 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.</p></div

Enhanced protein quality control by the knockdown of UBE4B and LSD1 depends on p53.

<p>(<b>A</b>) Left: p53 knockdown reverses the enhanced clearance of SOD1^G85R 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 SOD1^G85R in pellet fractions from HEK293T cells transfected with control, double (UBE4B/LSD1), or triple (UBE4B/LSD1/p53) shRNAs (<i>n</i> = 2). (<b>B</b>) The degenerative TDP-43^M337V eye phenotype is exacerbated by the knockdown of the <i>Drosophila</i> 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-43^M337V are driven by GMR-Gal4. (<b>C</b>) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1^G85R-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-SOD1^G85R 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. (<b>D</b>) 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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s001" target="_blank">S1 Data</a>.</p

Suppression of neurodegeneration associated with diverse misfolded proteins in invertebrate models by <i>ufd-2</i> and <i>spr-5</i> loss-of-function mutations.

<p>(<b>A</b>) Top: schematic drawing depicts pan-neuronal expression of YFP in head and ventral neurons in the context of the <i>C</i>. <i>elegans</i> body plan. Left: micrographs show the SOD1^G85R-YFP proteins expressed in WT or the <i>spr-5(by134);ufd-2(tm1380)</i> mutant background. The double-mutant worms show a marked decrease in protein aggregation in neurons. Enlarged sections of head neurons (red framed) and ventral cord neurons (white framed) are shown. Middle: quantification of locomotion in the <i>spr-5(by134);ufd-2(tm1380)</i> and the WT <i>C</i>. <i>elegans</i> with neuronal expression of SOD1^G85R-YFP (<i>n</i> = 30). Right: a decrease in the protein levels of SOD1^G85R-YFP in the presence of <i>spr-5(by134);ufd-2(tm1380)</i> is shown by western blots of the supernatant (S) and the pellet (P) fractions. (<b>B and C</b>) Analyses of the <i>spr-5(by134);ufd-2(tm1380)</i> and the WT <i>C</i>. <i>elegans</i> with neuronal expression of TDP-43-c25-YFP (<i>n</i> = 30) or PolyQ-YFP (<i>n</i> = 12) as in (A). (<b>D</b>) Neurodegenerative rough-eye phenotype in adults is alleviated by the knockdown of the <i>Drosophila</i> homologs of <i>ufd-2</i>/UBE4B and <i>spr-5</i>/LSD1—CG9934 and Su(Var)3-3, respectively, compared to the control (CTRL). Eye-specific expression of TDP-43^M337V, FUS^R521C, and RNA interference (RNAi) was driven by GMR-Gal4. (<b>E</b>) <i>Drosophila</i> eye pigment quantitation. Data represent means ± SEM. The numerical data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s001" target="_blank">S1 Data</a>.</p

Identification and characterization of a robust suppressor that ameliorates the locomotion defects in the <i>C</i>. <i>elegans</i> model of SOD1-associated ALS.

<p>(<b>A</b>) Workflow of the suppressor screen identifying mutant <i>C</i>. <i>elegans</i> (red) with saliently improved movement. (<b>B</b>) Locomotor behavior, measured by thrashing rates in liquid medium, in the <i>C</i>. <i>elegans</i> strains with neuronal expression of human WT SOD1 or ALS-linked mutant SOD1^G85R, in the presence (M1/M1) or absence (+/+) of the suppressor mutation (<i>n</i> = 16). (<b>C</b>) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1^G85R strains, with (M1/M1) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1^G85R mRNA and total protein are not changed by the suppressor mutation (M1/M1). The western blot lanes are from the same gel and exposure. (<b>D</b>) Sequence analysis of the M1 strain, revealing that independent mutations in two genes, a lysine-specific demethylase, <i>spr-5 (R646Q)</i>, and a ubiquitin ligase, <i>ufd-2 (W824X)</i>, are required for the full suppressor phenotype. (<b>E</b>) <i>C</i>. <i>elegans</i> 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 <i>C</i>. <i>elegans</i> are indicated. (<b>F</b>) Locomotor behavior, measured by thrashing rates, of the <i>C</i>. <i>elegans</i> carrying the SOD1^G85R transgene on the normal background (WT), with the null mutation of either <i>ufd-2(tm1380)</i> or <i>spr-5(by134)</i>, the double mutation <i>ufd-2(tm1380);spr-5(by134)</i>, or the M1 suppressor <i>ufd-2(W824X);spr-5(R646Q</i>) (<i>n</i> = 16). (<b>G</b>) Western blotting of the supernatant (S) and pellet (P) protein fractions from the <i>C</i>. <i>elegans</i> carrying the SOD1^G85R transgene with the double mutation <i>ufd-2(tm1380);spr-5(by134)</i> compared with controls. While SOD1^G85R 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 SOD1^G85R 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. (<b>H</b>) The overexpression (OE) of WT UFD-2 or SPR-5 in the <i>C</i>. <i>elegans</i> nervous system blocks the protection conferred by the double mutation <i>ufd-2(tm1380);spr-5(by134)</i> (<i>n</i> = 16). Data represent means ± SEM. The numerical data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s001" target="_blank">S1 Data</a>.</p

UBE4B and LSD1 double-knockdown accelerates SOD1^G85R protein degradation.

<p>(<b>A</b>) Western blots of cell lysates derived from mock (CTRL), single UBE4B or LSD1, or double UBE4B and LSD1 knockdowns. Supernatant (S) and pellet (P) fractions were probed with indicated antibodies. While the LSD1 or UBE4B single-knockdown reduces the SOD1^G85R aggregates in both supernatant and pellet fractions, the combined knockdown produces the strongest reduction in the aggregates. (<b>B</b>) Quantification of SOD1^G85R protein levels by western blotting (A). <i>n</i> = 3 (supernatant); <i>n</i> = 8 (pellet). (<b>C</b>) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half-lives in the double UBE4B and LSD1 knockdown cells versus controls. (<b>D</b>) Quantification of SOD1^G85R clearance, as analyzed by western blotting in (C). The graph indicates the relative band intensity of SOD1^G85R at each chase time point. <i>n</i> = 5; Overall <i>p</i> = 0.02 (paired <i>t</i> test, CTRL versus UBE4B and LSD1 double knockdown). Individual <i>p</i> = 0.03 (3 h), <i>p</i> = 0.003 (6 h), <i>p</i> = 0.06 (9 h), and <i>p</i> = 0.004 (12–21 h). (<b>E</b>) The half-life of SOD1^G85R is reduced from 8.5 h to 5 h upon knockdown of UBE4B and LSD1. Data represent means ± SEM. The numerical data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s001" target="_blank">S1 Data</a>.</p

p53 promotes the clearance of misfolded SOD1 mutant proteins.

<p>(<b>A</b>) p53 small molecule activators Tenovin-1 and CP-31398 reduce the levels of misfolded SOD1 proteins, as determined by the SOD1^G85R solubility assay in HEK293 cells. Increasing concentrations of the p53 activators significantly decrease the levels of SOD1^G85R but not the endogenous WT SOD1 proteins in western blots of both supernatant and pellet fractions. (<b>B</b>) A decrease in p53 as the result of shRNA knockdown increases the levels of SOD1^G85R but not WT SOD1 proteins in the SOD1^G85R aggregation assay, as shown by western blots of both supernatant (S) (<i>n</i> = 2) and pellet (P) (<i>n</i> = 3) fractions. (<b>C</b>) A complete absence of p53 increases the accumulation of SOD1^G85R mutant proteins in p53–/– HCT116 cells when compared with controls. Representative western blots (left panels) and quantification of SOD1^G85R levels in the supernatant lysates are shown. The middle graph indicates the ratio of G85R to WT SOD1 proteins in the presence or absence of p53 with varying amounts of transfected mutant SOD1. The right graph panel shows the same data as shown in the middle panel, but normalized to the average SOD1^G85R level for each amount of the transfected plasmid. Data represent means ± SEM. The numerical data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s001" target="_blank">S1 Data</a>.</p

UBE4B and LSD1 double knockdown activates both proteasomes and autophagy.

<p>(<b>A</b>) Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T cells. (<b>B</b>) The UBE4B and LSD1 single or double knockdowns increase the proteasomal degradation of a reporter substrate, Suc-LLVY-Luciferin, whose degradation is measured in a luciferase release assay. The double knockdown shows synergistic proteasomal activation when compared with the individual knockdowns (<i>n</i> = 6). (<b>C</b>) The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double knockdown. The in vivo activity of the ATG4B protease, which cleaves LC3 precursors, was quantified via a <i>Gaussia</i> luciferase release assay (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s006" target="_blank">S5D Fig., S5E Fig</a>., and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#sec012" target="_blank">Materials and Methods</a> for details). The HCT116 cells were analyzed 48 h after the initiation of the knockdown. (<b>D</b>) Quantification of LC3-II levels in HEK293T cells with the UBE4B and LSD1 double knockdown or the mock control. The cells were treated with or without 10 mM 3-methyladenine (3-MA) for 48h before lysis. Western blots indicate an increase in LC3 levels (top panels) in the double UBE4B and LSD1 knockdown cells (middle panels), while the actin control is unchanged (bottom panels). The graph shows the quantification of LC3-II levels as normalized to actin levels (<i>n</i> = 3). A one-way ANOVA test with matched multiple comparisons and Tukey correction was used for statistics. Data represent means ± SEM. The numerical data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s001" target="_blank">S1 Data</a>.</p

UBE4B and LSD1 knockdown activates transcription mediated by p53.

<p>(<b>A</b>) Venn diagram of upstream activators (z-score ≥ 2) that are differentially activated in single—UBE4B or LSD1—knockdowns and double UBE4B and LSD1 knockdowns, compared with the control. Activation state of an upstream regulator is predicted from differential mRNA levels of its downstream target genes. (<b>B</b>) The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSD1 double knockdown versus control shRNA. Gray spots represent 22,148 annotated transcripts. Red spots are predicted p53-activated targets, and blue spots are predicted p53-inhibited targets. The enrichment of red spots in the up-regulated genes (right upper quadrant, >1.2-fold change, <i>p</i> ≤ 0.04) and blue spots in the down-regulated genes (left upper quadrant) indicates that the p53-mediated transcription is activated by the UBE4B and LSD1 double knockdown. (<b>C</b>) RT-qPCR validation of the expression levels of representative p53 target genes in the UBE4B and LSD1 double-knockdown samples. <i>n</i> = (2 to 6), <i>p</i> ≤ 0.04. (<b>D</b>) The p53 protein level is significantly increased by UBE4B and LSD1 double knockdown. Left: representative western blots showing p53 levels, the knockdown of UBE4B and LSD1, and the actin control in HEK293T cells. Right: quantification of the p53 protein levels normalized against actin (<i>n</i> = 3). (<b>E</b>) The p53-mediated transcriptional activity is measured by a luciferase reporter under the control of a p53-response element promoter, which was transfected into HEK293T cells 72–96 h after the initiation of the UBE4B and LSD1 single or double knockdowns (<i>n</i> = 6). (<b>F</b>) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B and LSD1 knockdown. Left: representative western blots of 53BP1 co-immunoprecipitation. Right: quantification of the 53BP1 co-immunoprecipitation (<i>n</i> = 2). Data represent means ± SEM. (<b>G</b>) RT-qPCR validation of the expression levels of FOXO3a, FOXO4, and PSMD11 (<i>n</i> = 6). (<b>H</b>) The FOXO3a-mediated transcriptional activity was measured with a luciferase reporter under the control of a FOXO-response element promoter. HEK293T cells were transfected with shRNAs for control, LSD1, UBE4B, or both LSD1 and UBE4B, followed by transfection of the luciferase reporter together with a constitutively active form of FOXO3a (TM) 72–96 h later (<i>n</i> = 6). Data represent means ± SEM. The numerical data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002114#pbio.1002114.s001" target="_blank">S1 Data</a>.</p