Journal: Frontiers in Genetics

Article Title: SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin

doi: 10.3389/fgene.2018.00379

Figure Lengend Snippet: STUbL subunits Slx5 and Slx8 alleviate toxicity of poly-Q expanded Htt. (A) WT, slx5 Δ, and slx8 Δ strains expressing Htt-25Q, Htt-103Q, or empty vector (EV) were grown to mid-logarithmic phase and 5 μl of 10-fold serial dilutions of each culture were spotted on SC-URA medium. Plates were incubated at 30°C for 3 days. (B) Yeast transformants in A and the indicated controls were grown overnight in 5 ml of SC-URA medium. Ten OD 600 readings of cultures were recorded every hour until the OD 600 reached ∼2.0. The average doubling times of four independent experiments were graphed with +/− standard error. EV (empty vector) (C) A shuffle strain, slx5 Δ with SLX5/TRP plasmid (YOK 2990), was transformed with either Htt-25Q or Htt-103Q constructs. Transformants were patched in duplicate on selective medium (SC-TRP URA) and rich medium (YPD). Patches were then replica plated on SC-URA medium with 5FAA to counter-select against the TRP1 marked plasmid.

Article Snippet: Yeast plasmids expressing 25Q and 103Q Htt were purchased from Addgene.org (Addgene plasmid # 1177 (GPD-25Q-GFP Htt in p416), # 1180 (GPD-103Q-GFP Htt in p416)).

Techniques: Expressing, Plasmid Preparation, Incubation, Transformation Assay, Construct

Journal: Frontiers in Genetics

Article Title: SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin

doi: 10.3389/fgene.2018.00379

Figure Lengend Snippet: Plasmid-borne SLX5 reduces aggregates of poly-Q expanded huntingtin. (A) Representative images of Htt-103Q in WT cells with and without an SLX5/CEN plasmid. Example of aggregates (clearly defined, bright cytoplasmic structures – red arrow-heads) and speckles (multiple small, not clearly defined cytoplasmic granules – blue arrow-heads) (B) Quantitation of phenotypes observed in 2A. WT strain expressing Htt-103Q-GFP alone (YOK 2842) or Htt-103Q-GFP with SLX5 (YOK 2843) were grown to mid-logarithmic phase in selective medium. Images of 103Q diffuse staining, aggregates, and speckles in the indicated strains were recorded and then quantitated. Average counts for three independent experiments were graphed +/− standard deviation. Y-axis: percent of cells ( n = 100/experiment). Y-axis: phenotypes scored (C) Aggregates of poly-Q expanded Htt are localized in the cytoplasm. WT and slx5 Δ strains transformed with GAL-Htt-97Q-DsRed (YOK 3112 and YOK 3114) were grown overnight in SC-TRP medium with 2% raffinose. Cultures were diluted to ∼0.2 OD in a fresh medium with 2% galactose and incubated for an additional 16 h for expression of Htt-97Q-DsRed prior to imaging Htt aggregates using a fluorescence microscope. Nuclei were stained with Hoechst dye. Merged images indicate the absence of Htt-97Q aggregates in nuclei (yellow arrow-heads).

Article Snippet: Yeast plasmids expressing 25Q and 103Q Htt were purchased from Addgene.org (Addgene plasmid # 1177 (GPD-25Q-GFP Htt in p416), # 1180 (GPD-103Q-GFP Htt in p416)).

Techniques: Plasmid Preparation, Quantitation Assay, Expressing, Staining, Standard Deviation, Transformation Assay, Incubation, Imaging, Fluorescence, Microscopy

Journal: Frontiers in Genetics

Article Title: SUMO-Targeted Ubiquitin Ligases (STUbLs) Reduce the Toxicity and Abnormal Transcriptional Activity Associated With a Mutant, Aggregation-Prone Fragment of Huntingtin

doi: 10.3389/fgene.2018.00379

Figure Lengend Snippet: Slx5 modulates the transcriptional activity due to expression of Htt-103Q-NLS. Depiction of 398 SLX5 modulated genes identified by RNA sequencing. (A) RNA-seq analysis shows that Htt103Q-NLS leads to a global effect on the transcriptome as it affects the expression of 3438 genes (25Q [V] vs. 103Q [V]). Plasmid-borne SLX5 affects the expression of 398 genes in Htt103Q-NLS cells (103Q [V] vs. 103Q [ SLX5 ]) as shown in Supplementary Table . The overlap between Htt25Q-NLS and Htt103Q-NLS (25Q [V] vs. 103Q [V]) is 363 genes. The expanded view of A shows that expression of most of the 363 genes (99.4%) inversely correlates between 25Q [V] vs. 103Q [V] and 103Q [V] vs. 103Q [ SLX5 ]. Expression of 247 of the 363 genes (68.0%) is down-regulated by Htt103Q, and this effect is reversed by plasmid-borne SLX5 (Down – Up). Expression of 114 of the 363 genes (31.4%) is up-regulated by the Htt103Q, and this effect is reversed by plasmid-borne SLX5 (Up – Down). (B) Subcellular localization of differentially expressed genes. The localization of proteins encoded by the 398 genes indicated in Supplementary Table was analyzed using cellular components assignment from the PANTHER Classification System and the Saccharomyces Genome Database. Pie chart shows a ratio of the genes placed into cellular component categories. Individual genes are listed in Supplementary Table . (C) Schematic of gene expression in Htt25Q-NLS and Htt103Q-NLS cells and the effect of plasmid-borne SLX5 on the transcriptome of Htt103Q-NLS cells. Expression of genes A and B are downregulated and upregulated in Htt103Q-NLS cells relative to Htt25Q-NLS cells, respectively. Slx5 reduces the association of Htt with chromatin and this contributes to the reversal in gene expression such that gene A is upregulated and gene B is downregulated. (D) RT-PCR validation of gene expression analysis. Total RNAs were purified from strains expressing either Htt25Q-NLS or Htt103Q-NLS from a GAL promoter for 4 h with or without plasmid-borne SLX5 . RT-PCR analyses was performed using the same samples used for the RNA-seq. Relative intensities are reported as the mean ± SD of three biological repeats. Reactions for YDL223C/HBT1 and YPL186C/UIP4 were performed in duplicate. N = 3 for YOR339C/UBC11 and YFL039C/ACT1 , N = 6 for YDL223C/HBT1 and YPL186C/UIP4 .

Article Snippet: Yeast plasmids expressing 25Q and 103Q Htt were purchased from Addgene.org (Addgene plasmid # 1177 (GPD-25Q-GFP Htt in p416), # 1180 (GPD-103Q-GFP Htt in p416)).

Techniques: Activity Assay, Expressing, RNA Sequencing, Plasmid Preparation, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Purification

Journal: Nature structural & molecular biology

Article Title: Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors

doi: 10.1038/nsmb.3443

Figure Lengend Snippet: (a) Immunoblots showing P-eIF2α and eIF2α following a dephosphorylation reaction of 1 μM P-eIF2α by recombinant PP1 (1 μM) in the presence or absence of 1 μM of recombinant R15A, R15B or R3A. (b,c) Immunoblots of P-eIF2α and eIF2α following a dephosphorylation reaction of 1 μM P-eIF2α by PP1 (1 μM) in the presence or absence of (b) R15A and its inhibitors Guanabenz or Sephin1 and (c) calyculin A. (d) A titration curve of P-eIF2α (1 μM) dephosphorylation by increasing PP1 concentrations. A representative immunoblot corresponding to this titration is shown in Supplementary Figure 2b. Data are means ± s.e.m. (n = 3 independent experiments). (e–g) Immunoblots of P-eIF2α and eIF2α following a dephosphorylation reaction of 1 μM P-eIF2α by PP1 (10 nM) in the presence or absence of 1 μM (e) R15A, (f) R15B, or (g) R3A. All dephosphorylation reactions were carried out at 30 °C for 16 h. For all experiments, data shown is representative of one of three independent experiments. Uncropped images for blots shown are available in Supplementary Data Set 1.

Article Snippet: GST-tagged (amino-terminal) murine PERK kinase domain (amino acids 537–1114) (Addgene #21817, https://www.addgene.org/21817/ ) and His 6 -tagged (carboxy-terminal) human eIF2α (amino acids 1–185) solubility-enhanced mutant 42 were expressed in BL21 pLysS cells in LB at 37 °C for 6 h. GST-PERK was purified on GST Sepharose beads (GE Healthcare) followed by size exclusion chromatography on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) using kinase buffer (50 mM Tris pH 7.4, 100 mM NaCl, 10 mM MgCl 2 , 5 mM DTT). eIF2α was purified by affinity chromatography on a HisTrap excel column (GE Healthcare), followed by size exclusion chromatography on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) using buffer A. eIF2α phosphorylation eIF2α was phosphorylated on residue Ser51 using purified PERK kinase, as described above.

Techniques: Western Blot, De-Phosphorylation Assay, Recombinant, Titration

Journal: Nature structural & molecular biology

Article Title: Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors

doi: 10.1038/nsmb.3443

Figure Lengend Snippet: (a,b) Schematics of the proteins studied here: (a) R15A and (b) R15B. Amino acid residues delimiting the amino-terminal and carboxy-terminal regions and the presence of an amino-terminal MBP-tag and carboxy-terminal His6-tag are shown. The location of the PP1-binding region is indicated. (c,d) Thermophoresis binding curves of labeled PP1 binding to titrations of unlabeled (c) R15A (amino acids 325–636), R15AN (amino acids 325–512), R15AC (amino acids 513–636) or (d) R15B (amino acids 340–698), R15BN (amino acids 340–635), R15BC (amino acids 636–698). Dissociation constants (KD) are means ± s.e.m. (n = 3 independent experiments). Thermophoresis raw data are available in Supplementary Table 2. (e,f) Immunoblots of P-eIF2α and eIF2α following a dephosphorylation reaction by PP1 (10 nM) in the presence or absence of (e) R15A, R15AN, R15AC or (f) R15B, R15BN, R15BC. Dephosphorylation reactions were carried out at 30 °C for 16 h. Data shown are representative immunoblots of three independent experiments; uncropped images for blots shown are in Supplementary Data Set 1.

Article Snippet: GST-tagged (amino-terminal) murine PERK kinase domain (amino acids 537–1114) (Addgene #21817, https://www.addgene.org/21817/ ) and His 6 -tagged (carboxy-terminal) human eIF2α (amino acids 1–185) solubility-enhanced mutant 42 were expressed in BL21 pLysS cells in LB at 37 °C for 6 h. GST-PERK was purified on GST Sepharose beads (GE Healthcare) followed by size exclusion chromatography on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) using kinase buffer (50 mM Tris pH 7.4, 100 mM NaCl, 10 mM MgCl 2 , 5 mM DTT). eIF2α was purified by affinity chromatography on a HisTrap excel column (GE Healthcare), followed by size exclusion chromatography on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) using buffer A. eIF2α phosphorylation eIF2α was phosphorylated on residue Ser51 using purified PERK kinase, as described above.

Techniques: Binding Assay, Labeling, Western Blot, De-Phosphorylation Assay

Journal: Nature structural & molecular biology

Article Title: Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors

doi: 10.1038/nsmb.3443

Figure Lengend Snippet: (a) Thermophoresis binding curve of labeled P-eIF2α binding to titrations of unlabeled PP1D95A. KD is the mean ± s.e.m. (n = 3; biological replicates). (b–d) Thermophoresis-binding curves of labeled P-eIF2α binding to titrations of unlabeled (b) R15A, R15AN, R15AC (c) R15B, R15BN, R15BC, or (d) R3A. KD are means ± s.e.m. (n = 3 independent experiments). Thermophoresis raw data are available in Supplementary Table 2. (e) KD of labeled P-eIF2α to titrations of unlabeled PP1D95A, in the presence of saturating, and unlabeled, functional R15 (R15A or R15B), their nonfunctional carboxy-terminal fragments (R15AC or R15BC), their amino-terminal fragments (R15AN or R15BN), or R3A. The value of P-eIF2α binding to PP1D95A corresponds to that shown in a. KD values are means ± s.e.m. (n = 3 independent experiments). Details in Supplementary Table 1. Statistical significances, relative to PP1D95A binding to P-eIF2α, are shown. **P ≤ 0.01, ***P ≤ 0.001, n.s., not significant (one-way ANOVA).

Article Snippet: GST-tagged (amino-terminal) murine PERK kinase domain (amino acids 537–1114) (Addgene #21817, https://www.addgene.org/21817/ ) and His 6 -tagged (carboxy-terminal) human eIF2α (amino acids 1–185) solubility-enhanced mutant 42 were expressed in BL21 pLysS cells in LB at 37 °C for 6 h. GST-PERK was purified on GST Sepharose beads (GE Healthcare) followed by size exclusion chromatography on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) using kinase buffer (50 mM Tris pH 7.4, 100 mM NaCl, 10 mM MgCl 2 , 5 mM DTT). eIF2α was purified by affinity chromatography on a HisTrap excel column (GE Healthcare), followed by size exclusion chromatography on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) using buffer A. eIF2α phosphorylation eIF2α was phosphorylated on residue Ser51 using purified PERK kinase, as described above.

Techniques: Binding Assay, Labeling, Functional Assay

Journal: Nature structural & molecular biology

Article Title: Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors

doi: 10.1038/nsmb.3443

Figure Lengend Snippet: (a) Binding of R15A to biotinylated Guanabenz and biotinylated Sephin1 immobilized on streptavidin beads. Immunoblots of input and bound samples, probed with α-MBP (to reveal R15s) are shown. Bound samples (lane 3) were eluted with an excess of Guanabenz (top) or Sephin1 (bottom). Data shown are representative of three independent experiments. (b) The chemical structures of Guanabenz, Sephin1 and C3. (c,d) Coomassie-stained gels showing limited trypsin digestion of (c) R15A and (d) R15B in the presence or absence of Guanabenz, Sephin1 or C3. Trypsin digestions were carried out using 2.5 nM of trypsin, and reactions were allowed to proceed for 0 h, 30 min, 1 h, 2 h and 3 h (left to right lanes of each gel, respectively) at 22 °C and terminated by the addition of 4% SDS Laemmli sample buffer. Data shown are representative of three independent experiments. (e–h) Binding of P-eIF2α to MBP-tagged R15s immobilized on magnetic amylose beads in the presence or absence of (e) Guanabenz or (g) Sephin1. Immunoblots of input and bound samples, probed with α-MBP (to reveal R15s) or α-eIF2α antibodies are shown. Data shown are representative of three independent experiments. (f,h) Levels of eIF2α bound to R15s in the presence or absence of (f) Guanabenz or (h) Sephin1. eIF2α immunoblots of three independent pull down experiments, as shown in e and g were quantified and normalized independently for R15A and R15B against DMSO control samples (lanes 10 and 13, for R15A and R15B, respectively, in e and g). Means ± s.e.m. (n = 3 independent experiments) are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s., not significant (two-way ANOVA). Uncropped images of blots shown are in Supplementary Data Set 1.

Article Snippet: GST-tagged (amino-terminal) murine PERK kinase domain (amino acids 537–1114) (Addgene #21817, https://www.addgene.org/21817/ ) and His 6 -tagged (carboxy-terminal) human eIF2α (amino acids 1–185) solubility-enhanced mutant 42 were expressed in BL21 pLysS cells in LB at 37 °C for 6 h. GST-PERK was purified on GST Sepharose beads (GE Healthcare) followed by size exclusion chromatography on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) using kinase buffer (50 mM Tris pH 7.4, 100 mM NaCl, 10 mM MgCl 2 , 5 mM DTT). eIF2α was purified by affinity chromatography on a HisTrap excel column (GE Healthcare), followed by size exclusion chromatography on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) using buffer A. eIF2α phosphorylation eIF2α was phosphorylated on residue Ser51 using purified PERK kinase, as described above.

Techniques: Binding Assay, Western Blot, Staining

Journal: Nature structural & molecular biology

Article Title: Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors

doi: 10.1038/nsmb.3443

Figure Lengend Snippet: (a,b) Immunoblots of P-eIF2α and eIF2α following a dephosphorylation reaction of 1 μM P-eIF2α by R15A–PP1 and R15B–PP1 holoenzymes in the presence or absence of (a) Guanabenz or (b) Sephin1. (c) Immunoblots of P-eIF2α and eIF2α following a dephosphorylation reaction of 1 μM P-eIF2α by R15AN–R15BC–PP1 and R15BN–R15AC–PP1 chimeric holoenzymes in the presence or absence of Guanabenz or Sephin1. (d) Immunoblots of P-eIF2α and eIF2α following a dephosphorylation reaction of 1 μM P-eIF2α by R15A–PP1 and R15B–PP1 holoenzymes in the presence or absence of C3. All dephosphorylation reactions were carried out using R15 at 50 nM and PP1 at 10 nM at 30 °C for 16 h. For all experiments, data shown are representative of three independent experiments. P-eIF2α levels were quantified and normalized against levels in lane 2 (P-eIF2α alone) and expressed as percentage relative to levels in lane 2. Uncropped images of blots shown are in Supplementary Data Set 1.

Article Snippet: GST-tagged (amino-terminal) murine PERK kinase domain (amino acids 537–1114) (Addgene #21817, https://www.addgene.org/21817/ ) and His 6 -tagged (carboxy-terminal) human eIF2α (amino acids 1–185) solubility-enhanced mutant 42 were expressed in BL21 pLysS cells in LB at 37 °C for 6 h. GST-PERK was purified on GST Sepharose beads (GE Healthcare) followed by size exclusion chromatography on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) using kinase buffer (50 mM Tris pH 7.4, 100 mM NaCl, 10 mM MgCl 2 , 5 mM DTT). eIF2α was purified by affinity chromatography on a HisTrap excel column (GE Healthcare), followed by size exclusion chromatography on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare) using buffer A. eIF2α phosphorylation eIF2α was phosphorylated on residue Ser51 using purified PERK kinase, as described above.

Techniques: Western Blot, De-Phosphorylation Assay

Journal: Disease Models & Mechanisms

Article Title: Integrated multi-omics analysis of Huntington disease identifies pathways that modulate protein aggregation

doi: 10.1242/dmm.049492

Figure Lengend Snippet: Metabolomic study using an HD yeast model. (A-F) Heat maps of targeted metabolomics in Htt yeast models 46Q, 72Q and 103Q compared to that of 25Q in positive and negative modes. Altered metabolites in 46Q compared to 25Q in positive (A) and negative (B) mode. Altered metabolites in 72Q compared to 25Q in positive (C) and negative mode (D). (E) Altered metabolites in 103Q compared to 25Q in positive (E) and negative (F) mode.

Article Snippet: A total of 8×10 8 cells was taken for transformation with p426 25Q GPD, p426 46Q GPD, p426 72Q GPD and p426 103Q GPD (Addgene plasmids #1181 , #1182 , #1183 and #1184 , respectively).

Techniques:

Journal: Disease Models & Mechanisms

Article Title: Integrated multi-omics analysis of Huntington disease identifies pathways that modulate protein aggregation

doi: 10.1242/dmm.049492

Figure Lengend Snippet: Metabolite set enrichment analysis (MESA) and shared deregulated pathways. (A) Yeast metabolomics (present study) show significantly different levels of metabolites in yeasts 46Q, 72Q and 103Q compared to 25Q. (B) MSEA showing shared pathways deregulated in 25Q vs 46Q, 25Q vs 72Q and 25Q vs 103Q. (C) Metabolomics pathway overlaps determined in our study (yeasts 25Q vs 46Q, 25Q vs 72Q and 25Q vs 103Q) and literature show pathways commonly deregulated in yeast 103Q compared to yeast 25Q. (D,E) Comparative analysis of significantly enriched metabolic pathways identified in a HD mouse model (D) and in HD patients (E). (F) Metabolomics of previously published deregulated metabolic pathways in human (literature) and deregulated pathways identified in our HD patient cohort (present study), showing an overlap of three deregulated pathways. (G) Plotted is the overlap of deregulated metabolic pathways identified in yeast and mouse HD models, and HD patients. BS, brain stem; CB, cerebellum; CSF, cerebrospinal fluid; CT, cortex; FL, frontal lobe; ST, striatum.

Article Snippet: A total of 8×10 8 cells was taken for transformation with p426 25Q GPD, p426 46Q GPD, p426 72Q GPD and p426 103Q GPD (Addgene plasmids #1181 , #1182 , #1183 and #1184 , respectively).

Techniques: