Journal: Cells

Article Title: DCUN1D1 Is an Essential Regulator of Prostate Cancer Proliferation and Tumour Growth That Acts through Neddylation of Cullin 1, 3, 4A and 5 and Deregulation of Wnt/Catenin Pathway.

doi: 10.3390/cells12151973

Figure Lengend Snippet: Figure 5. Blockage of DCUN1D1 decreases global neddylation, ubiquitination and expression of neddylation components and shows preferential neddylation activity of cullin proteins in PCa. Western blot analysis of DU145 and DU145 DCUN1D1-KD cells. Protein extracts were obtained from the cells and subjected to Western blot analysis using (a) anti-ubiquitin, (b) anti-Nedd8. Blockage of DCUN1D1 decreased the expression of the neddylation pathway components including (c) the E1 NAE heterodimer APPB1 (top panel) and the neddylation-conjugating enzyme, UBC12 (bottom panel) and (d) the cullin-associated proteins RBX1 (top panel) and CAND1 (bottom panel). Inhibition of DCUN1D1 showed preferential NEDD8 modification of the cullin family of proteins. Immunoblot analysis of DU145 and DU145 DCUN1D1 knockdown cell protein extracts using (e) anti-cullin 1, (f) anti-cullin 3, (g) anti-cullin 4A, (h) anti-cullin 4B and (i) anti-cullin 5. The GAPDH loading control was probed using the anti-GAPDH antibody. Experiments were independently repeated three times and a representative image of an independent experiment is represented.

Article Snippet: The following primary antibodies were used: DCUN1D1 (Sigma HPA035911), Nedd8 (Cell Signaling Technology, Danver, MA, USA CST2745S), Ubiquitin (Cell Signaling Technology CST39365), cullin 1 (Cell Signaling Technology CST4995), cullin 2 (Novus Biologicals, Centennial, CO, USA NBP1-67535), cullin 3 (Cell Signaling Technology CST2759S), cullin 4A (Cell Signaling Technology CST2699S), cullin 4B (Bio-Rad VMA00360) Cells 2023, 12, 1973 6 of 22 cullin 5 (Novus Biologicals NBP1-22970), APPBP1 (Cell Signaling Technology CST14321), UBA3 (Novus Biologicals NBP2-48628), UBC12 (Novus Biologicals NBP1-31459), CAND1 (Cell Signaling Technology CST8759S), RBX1 (Novus Biologicals NBP2-20113), β-Catenin (Cell Signaling Technology CST4394S) phosphor-β-Catenin (Cell Signaling Technology CST2009S) and Lef1 (Santa Cruz sc-374522).

Techniques: Ubiquitin Proteomics, Expressing, Activity Assay, Western Blot, Inhibition, Knockdown, Control

Journal: Nature chemical biology

Article Title: Targeting a Helix-in-Groove Interaction Between E1 and E2 Blocks Ubiquitin Transfer

doi: 10.1038/s41589-020-0625-7

Figure Lengend Snippet: ( a ) Structure of the binding interface between the UFD of S. pombe Uba1 (interacting residues colored pink) and the alpha-1 helix (aa 1–18, colored by amino acid property) of the E2 Ubc15 (cyan), as revealed by the x-ray structure of the S. pombe Uba1/Ubc15 complex (PDB: 5KNL) . ( b ) Amino acid sequences of the SAH-Ubc15 E7R staple-scanning library, which was generated by inserting all-hydrocarbon i, i+4 or i, i+7 staples sequentially along the length of the Ubc15 E7R peptide. X, S5-pentenyl alanine; 8, R5-octenyl alanine; B, norleucine (replacement for methionine, whose sulfur residue decreases the efficiency of ruthenium-catalyzed olefin metathesis). ( c ) Helical wheel depiction of SAH-Ubc15 E7R peptides and their staple positions (helical residues span from aa 5–18). ( d ) Inhibition of UBE1-mediated thioester transfer of ubiquitin to the E2 enzyme UBE2D2 by the indicated SAH-UBC15 E7R stapled peptides. Thioester transfer activity was monitored by the shift in E2 molecular weight from 17 kDa (free UBE2D2) to 25 kDa (UBE2D2~ubiquitin conjugate), as detected by gel electrophoresis and silver stain. Vehicle lane contained 1.5% DMSO and control lane lacked UBE1 protein. The bar graph represents mean inhibition relative to vehicle control ± s.e.m., as quantified and averaged across three independent biological replicates. Uncropped gel for panel d is available online. Source data for the thioester transfer assay plot is available online.

Article Snippet: For UBA3 thioester transfer assays, 15 nM NAE1/UBA3 was substituted for UBE1 (Boston Biochem E-313), 135 nM UBE2M for UBE2D2 (Boston Biochem E2–656), and 1 μM NEDD8 (Boston Biochem UL-812) for ubiquitin.

Techniques: Binding Assay, Generated, Residue, Inhibition, Ubiquitin Proteomics, Activity Assay, Molecular Weight, Nucleic Acid Electrophoresis, Silver Staining, Control

Journal: Nature chemical biology

Article Title: Targeting a Helix-in-Groove Interaction Between E1 and E2 Blocks Ubiquitin Transfer

doi: 10.1038/s41589-020-0625-7

Figure Lengend Snippet: ( a ) Amino acid sequences of stapled peptides corresponding to the N terminus and alpha-1 helix of UBE2D2 (aa 1–16), UBE2G2 (aa 1–14), and UBE2A (aa 1–17), which incorporate the optimal staple position identified for SAH-Ubc15–11. X, S5-pentenyl alanine; 8, R5-octenyl alanine; B, norleucine. ( b ) Comparative dose-responsive inhibition of UBE1 by the indicated SAH-E2 h1 peptides, as monitored by thioester transfer assay, gel electrophoresis, and silver stain. ( c ) Quantitation of the data in b by ImageJ analysis. Data are mean percent inhibition ± s.e.m. for three independent biological replicates. ( d ) Circular dichroism spectra of UBE2A (BSTPARRRLBRDFKRLQ, where B is norleucine) and SAH-UBE2A, demonstrating induction of α-helicity by insertion of the i, i+7 staple. ( e ) Inhibition of UBE1-mediated thioester transfer of ubiquitin to the E2 enzyme UBE2D2 by SAH-UBE2A but not the corresponding unmodified template peptide, UBE2A. Data are mean inhibition ± s.e.m. for three independent biological replicates. ( f ) Comparative protease resistance of SAH-UBE2A and UBE2A upon treatment with proteinase K, as measured by HPLC analysis. SAH-UBE2A displays a 23-fold longer half-life than UBE2A. Data are mean ± s.d. for experiments performed in triplicate. ( g ) SAH-UBE2A but not UBE2A was significantly protected from deuterium exchange upon incubation with UBE1. The relative difference in deuterium uptake after 10 sec of labeling is shown. The difference in uptake was calculated from the mean deuterium level for UBE2A or SAH-UBE2A in the presence of UBE1 minus that of the peptide alone. Mean deuterium levels were obtained from at least two independent biological replicates of each condition. ( h ) C-terminally biotinylated SAH-UBE2A directly bound to recombinant human UBE1 (MW 118 kDa), as demonstrated by streptavidin capture, gel electrophoresis, and silver stain. ( i ) C-terminally FITCylated SAH-UBE2A bound to UBE1 with a K d of 384 ± 43 nM, as assessed by fluorescence polarization assay. Error bars are s.d. for assays conducted in technical triplicate and performed three times with independent preparations of peptide and protein (all 9 points for each dosing level are plotted). Uncropped gels for panels b , e , and h are available online. Source data for thioester transfer assays, CD spectra, protease resistance testing, HXMS analyses, and FP plot are available online.

Article Snippet: For UBA3 thioester transfer assays, 15 nM NAE1/UBA3 was substituted for UBE1 (Boston Biochem E-313), 135 nM UBE2M for UBE2D2 (Boston Biochem E2–656), and 1 μM NEDD8 (Boston Biochem UL-812) for ubiquitin.

Techniques: Inhibition, Nucleic Acid Electrophoresis, Silver Staining, Quantitation Assay, Circular Dichroism, Ubiquitin Proteomics, Incubation, Labeling, Recombinant, Fluorescence

Journal: Nature chemical biology

Article Title: Targeting a Helix-in-Groove Interaction Between E1 and E2 Blocks Ubiquitin Transfer

doi: 10.1038/s41589-020-0625-7

Figure Lengend Snippet: (a) Sequence compositions of the unmodified UBE2A template peptide and stapled constructs SAH-UBE2A, SAH-UBE2A R7E , and SAH-UBE2A R7A/L9A/B10A . (b) Comparative effects of UBE2A, SAH-UBE2A, SAH-UBE2A R7E , and SAH-UBE2A R7A/L9A/B10A on UBE1-mediated thioester transfer of ubiquitin to the E2 enzyme UBE2D2. Whereas SAH-UBE2A (10 μM) completely inhibits UBE1 activity, triple mutagenesis abrogates the inhibitory effect and single R7E mutagenesis has an intermediate effect. (c) Progressive loss of UBE1 protection from deuterium exchange into SAH-UBE2A upon single R7E and triple R7A/L9A/B10A mutagenesis. The relative difference in deuterium uptake after 10 sec of labeling is shown. The difference in uptake was calculated from the mean deuterium level for each SAH-UBE2A peptide in the presence of UBE1 minus that of the peptide alone. Mean deuterium levels were obtained from at least two independent biological replicates of each condition. (d) Difference in deuterium uptake plots demonstrate the relative deuterium incorporation of SAH-UBE2A/UBE1, SAH-UBE2A R7E /UBE1, or SAH-UBE2A R7A/L9A/B10A /UBE1 minus the relative deuterium incorporation of UBE1 at 10 min of deuterium labeling. The shaded gray region indicates differences in deuteration that are below the meaningful differences threshold. Consistent with results of both the thioester transfer assay (b) and peptide deuterium exchange (c), and the predicted SAH-UBE2A/UBE1 binding mode, triple mutagenesis essentially abrogates the effect of SAH-UBE2A on UBE1 conformation, with single R7E mutagenesis having an intermediate influence. Data are representative of three independent replicates for SAH-UBE2A, and two independent replicates for SAH-UBE2A R7E and SAH-UBE2A R7A/L9A/B10A . Each peptide, from N- to C-terminus, was given a peptide number to simplify the presentation. The peptide list and residue identity of each peptide can be found in . Uncropped gel for panel b is available online. Source data for the HXMS analyses are available online.

Article Snippet: For UBA3 thioester transfer assays, 15 nM NAE1/UBA3 was substituted for UBE1 (Boston Biochem E-313), 135 nM UBE2M for UBE2D2 (Boston Biochem E2–656), and 1 μM NEDD8 (Boston Biochem UL-812) for ubiquitin.

Techniques: Sequencing, Construct, Ubiquitin Proteomics, Activity Assay, Mutagenesis, Labeling, Binding Assay, Residue

Journal: PLoS ONE

Article Title: A Metal-Based Inhibitor of NEDD8-Activating Enzyme

doi: 10.1371/journal.pone.0049574

Figure Lengend Snippet: Western blots show dose-dependent inhibition of a) Ubc-12-NEDD8 formation in a cell-free system and b) cellular Ubc12-NEDD8 levels by 1 . MLN4924 was included for comparison.

Article Snippet: NEDD8 Conjugation Initiation Kit and anti-Ubc12 rabbit polyclonal antibody was obtained from Boston BioChem (Cambridge, MA, USA).

Techniques: Western Blot, Inhibition, Comparison

Journal: Biochemical pharmacology

Article Title: The blockade of neddylation alleviates ventilator-induced lung injury by reducing stretch-induced damage to pulmonary epithelial cells.

doi: 10.1016/j.bcp.2024.116533

Figure Lengend Snippet: Fig. 1. HTV ventilation upregulates neddylation pathway in mice. The mice were subjected to HTV ventilation. (A) At various time intervals, lung tissues were collected for analysis. Western blot for NEDD8-conjugated protein, UBA3, APPBP1, and UBC12. Values represent the mean ± SD (n = 5). ***p < 0.001 compared with the initial time (0-h) of HTV ventilation. (B) Another group of mice received HTV ventilation or spontaneous breathing. DMSO or neddylation inhibitor MLN4924 was intraperitoneally administered 20 min prior to HTV ventilation. After 3 h of HTV ventilation, lung tissues were collected for analysis. Western blot for NEDD8- conjugated protein, UBA3, APPBP1, UBC12, cullin-1 neddylation, and IκB. Values represent the mean ± SD (n = 5). ***p < 0.001 versus the DMSO group, ###p < 0.001 versus the DMSO+HTV group.

Article Snippet: Antibodies against UBA3 (E-5), APPBP1 (H-187), UBC12 (D-4), cullin-1 (D-5), and β-actin (C-4) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot

Journal: Biochemical pharmacology

Article Title: The blockade of neddylation alleviates ventilator-induced lung injury by reducing stretch-induced damage to pulmonary epithelial cells.

doi: 10.1016/j.bcp.2024.116533

Figure Lengend Snippet: Fig. 2. MLN4924 impedes HTV ventilation-induced neddylation in mouse lung sections. Mice were subjected to either HTV ventilation or spontaneous breathing. DMSO or MLN4924 was intraperitoneally administered 20 min prior to HTV ventilation. After 3 h of HTV ventilation, lung tissues were collected for analysis. Immunohistochemistry analysis for NEDD8, UBA3, APPBP1, and UBC12. Scale bar, 30 μm. Values represent the mean ± SD (n = 5). ***p < 0.001 versus the DMSO group, ###p < 0.001, ##p < 0.01 versus the DMSO+HTV group.

Article Snippet: Antibodies against UBA3 (E-5), APPBP1 (H-187), UBC12 (D-4), cullin-1 (D-5), and β-actin (C-4) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunohistochemistry

Journal: Biochemical pharmacology

Article Title: The blockade of neddylation alleviates ventilator-induced lung injury by reducing stretch-induced damage to pulmonary epithelial cells.

doi: 10.1016/j.bcp.2024.116533

Figure Lengend Snippet: Fig. 6. CS upregulates neddylation pathway in human alveolar epithelial cells. (A) The cells were subjected to CS of 5, 10, and 20 % elongation for various durations (0, 5, 15, 60, and 90 min) at a frequency of 30 cycles/min. At different time intervals, the cells were collected to analyze. Western blot for NEDD8-conjugated protein. Values represent the mean ± SD (n = 5). ***p < 0.001 compared with the initial time (0-h) of CS. (B) Another group of cells were incubated with DMSO or MLN4924 for 1 h prior to stimulation with 10 % CS for 5 min. Thereafter, human alveolar epithelial cells were collected to analyze. Western blot for NEDD8-conjugated protein, UBA3, APPBP1, UBC12, cullin-1 neddylation, and IκB. Values represent the mean ± SD (n = 5). ***p < 0.001 versus the DMSO group, ###p < 0.001 versus the DMSO+CS group.

Article Snippet: Antibodies against UBA3 (E-5), APPBP1 (H-187), UBC12 (D-4), cullin-1 (D-5), and β-actin (C-4) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Incubation

Journal: Cell Death & Disease

Article Title: PRMT1-mediated methylation of UBE2m promoting calcium oxalate crystal-induced kidney injury by inhibiting fatty acid metabolism

doi: 10.1038/s41419-025-07888-3

Figure Lengend Snippet: A Silver-stained SDS-PAGE gel of the immunoprecipitation product. B MS/MS spectra of the peptide “FSPSGIFGAFQR-COOH.” Peaks in color are the detected b (green) and y (red) ions. C The volcano plot indicating potential target proteins of PRMT1. D Molecular docking pattern diagram of PRMT1 and UBE2m. E Representative images of immunofluorescence staining of UBE2m (green) and PRMT1 (red) in HK-2 cells treated with COM. Scale bars = 50 μm. F Reciprocal co-IP analysis of PRMT1 and UBE2m in HK-2 cells with COM treatment. IgG was used as a negative control. G Recombinant GST-PRMT1 was incubated with recombinant His-UBE2m, followed by GST-pulldown and immunoblotting analysis with GST and His antibodies. H Endogenous UBE2m was immunoprecipitated in cells from the indicated groups. The mono-methylation (me1) level of UBE2m was determined using immunoblotting. I PLA detection of PRMT1 and UBE2m interaction in HK-2 cells from the indicated groups. Scale bars = 50 μm. J The me1 level of UBE2m was determined using immunoblotting after UBE2m was immunoprecipitated in HK-2 cells with PRMT1 knockdown or overexpression.

Article Snippet: Antibodies against UBE2m (14520-1-AP, 1:1000 for WB, 1:100 for IF or IHC, 1:50 for IP), CPT1a (15184-1-AP, 1:8000 for WB), ACOX1 (10957-1-AP, 1:2000 for WB), FlAG (20543-1-AP, 1:30,000 for WB, 1 μg/mg for IP), His (66005-1-Ig, 1:10,000 for WB, 1 μg/mg for IP), GST (66001-2-Ig, 1:8000 for WB, 1 μg/mg for IP), HA (51064-2-AP, 1:8000 for WB, 1 μg/mg for IP), and Myc (60003-2-Ig, 1:4000 for WB, 1 μg/mg for IP) were purchased from Proteintech.

Techniques: Staining, SDS Page, Immunoprecipitation, Tandem Mass Spectroscopy, Immunofluorescence, Co-Immunoprecipitation Assay, Negative Control, Recombinant, Incubation, Western Blot, Methylation, Knockdown, Over Expression

Journal: Cell Death & Disease

Article Title: PRMT1-mediated methylation of UBE2m promoting calcium oxalate crystal-induced kidney injury by inhibiting fatty acid metabolism

doi: 10.1038/s41419-025-07888-3

Figure Lengend Snippet: A The scores of potential methylation sites of UBE2m predicted using the GPS-MSP tool. B MS identification of R169 mono-methylation of HA-UBE2m immunopurified by HA beads. C Sequence alignment of UBE2m protein from indicated species. D HA-UBE2m WT or different point mutants were co-transfected with FLAG-PRMT in HEK293T cells. The me1 level of UBE2m was measured by immunoblotting following immunopurification. E The me1 of immunopurified HA-UBE2m was measured using a site-specific antibody against R169 mono-methylation (meUBE2m (R169me1)). Antibody efficacy and specificity were examined by pre-incubating with the R169me1 peptide or the unmodified peptide prior to application. F Dot blot analysis of different amounts of R169me1 peptide or unmodified peptide by a site-specific antibody against meUBE2m (R169me1). G Endogenous UBE2m was immunoprecipitated in cells from the indicated groups. The R169me1 level of UBE2m was determined using immunoblotting. H The R169me1 level of UBE2m was determined using immunoblotting after UBE2m was immunoprecipitated in HK-2 cells with PRMT1 knockdown or overexpression. I Recombinant GST-PRMT1 and His-UBE2m proteins were incubated with or without SAM to detect the methylation of UBE2m mediated by PRMT1 in vitro. The reaction mixture was then subjected to CBB staining and immunoblotting with meUBE2m (R169me1) antibody. J Mitochondrial oxidative capacity was measured in real time after reintroduced UBE2m WT or RK treatment in UBE2m KO HK-2 cells; basal respiration, ATP production-coupled respiration, and maximal respiration were quantified. Significance was assessed using two-way ANOVA tests. Data are shown as mean ± SD.

Article Snippet: Antibodies against UBE2m (14520-1-AP, 1:1000 for WB, 1:100 for IF or IHC, 1:50 for IP), CPT1a (15184-1-AP, 1:8000 for WB), ACOX1 (10957-1-AP, 1:2000 for WB), FlAG (20543-1-AP, 1:30,000 for WB, 1 μg/mg for IP), His (66005-1-Ig, 1:10,000 for WB, 1 μg/mg for IP), GST (66001-2-Ig, 1:8000 for WB, 1 μg/mg for IP), HA (51064-2-AP, 1:8000 for WB, 1 μg/mg for IP), and Myc (60003-2-Ig, 1:4000 for WB, 1 μg/mg for IP) were purchased from Proteintech.

Techniques: Methylation, Sequencing, Transfection, Western Blot, Immu-Puri, Dot Blot, Immunoprecipitation, Knockdown, Over Expression, Recombinant, Incubation, In Vitro, Staining

Journal: Cell Death & Disease

Article Title: PRMT1-mediated methylation of UBE2m promoting calcium oxalate crystal-induced kidney injury by inhibiting fatty acid metabolism

doi: 10.1038/s41419-025-07888-3

Figure Lengend Snippet: A Representative western blot banding and quantitative analysis of the expression levels of NEDD4 from the indicated groups in HK-2 cells. B Detection of endogenous neddylation levels of NEDD4 from the indicated groups in mice or in HK-2 cells ( C ). D , E Detection of exogenous neddylation levels of NEDD4 in HEK193T cells with UBE2m knockdown or overexpression. F Detection of exogenous neddylation levels of NEDD4 in HEK193T cells transfecting UBE2m WT or RK. G Detection of endogenous neddylation levels of NEDD4 in UBE2mKO HK-2 cells re-introducing UBE2m WT or RK. H PLA detection of NEDD4 and NEDD8 interaction in HK-2 cells from the indicated groups. Scale bars = 50 μm. I HK-2 cells were treated with MLN4924. Representative western blot banding and quantitative analysis of the expression levels of UBE2m, NEDD4, and PPARγ from the indicated groups in HK-2 cells; and the expression levels of NEDD4, PPARγ, CD36, FATP2, CPT1α, and ACOX1 from the indicated groups in HK-2 cells ( J ). K Mitochondrial oxidative capacity was measured in real time after reintroduced UBE2m WT or RK treatment in UBE2m KO HK-2 cells, with or without MLN. L FAO blue staining demonstrates the degree of FAO in HK-2 cells from the indicated groups. Significance was assessed using two-way ANOVA tests. Data are shown as mean ± SD.

Article Snippet: Antibodies against UBE2m (14520-1-AP, 1:1000 for WB, 1:100 for IF or IHC, 1:50 for IP), CPT1a (15184-1-AP, 1:8000 for WB), ACOX1 (10957-1-AP, 1:2000 for WB), FlAG (20543-1-AP, 1:30,000 for WB, 1 μg/mg for IP), His (66005-1-Ig, 1:10,000 for WB, 1 μg/mg for IP), GST (66001-2-Ig, 1:8000 for WB, 1 μg/mg for IP), HA (51064-2-AP, 1:8000 for WB, 1 μg/mg for IP), and Myc (60003-2-Ig, 1:4000 for WB, 1 μg/mg for IP) were purchased from Proteintech.

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Staining

Journal: Cell Death & Disease

Article Title: PRMT1-mediated methylation of UBE2m promoting calcium oxalate crystal-induced kidney injury by inhibiting fatty acid metabolism

doi: 10.1038/s41419-025-07888-3

Figure Lengend Snippet: A Representative immunohistochemical staining images (400X) and quantitative analysis of the expression of M-UBE2m in the nonfunctioning kidney of patients with stones or the control. Scale bars = 50 μm. B Pearson correlation analysis of the expression of PRMT1 and M-UBE2m. C Representative immunohistochemical staining images (400X) and quantitative analysis of the expression of KIM-1 in the nonfunctioning kidney of patients with stones or the control. Scale bars = 50 μm. D – G Pearson correlation analysis of the expression of PRMT1 and eGFR ( D ), PRMT1 and KIM-1 ( E ), M-UBE2m and eGFR ( F ), M-UBE2m and KIM-1 ( G ). Significance was assessed using t-tests. Data are presented as mean ± SD.

Article Snippet: Antibodies against UBE2m (14520-1-AP, 1:1000 for WB, 1:100 for IF or IHC, 1:50 for IP), CPT1a (15184-1-AP, 1:8000 for WB), ACOX1 (10957-1-AP, 1:2000 for WB), FlAG (20543-1-AP, 1:30,000 for WB, 1 μg/mg for IP), His (66005-1-Ig, 1:10,000 for WB, 1 μg/mg for IP), GST (66001-2-Ig, 1:8000 for WB, 1 μg/mg for IP), HA (51064-2-AP, 1:8000 for WB, 1 μg/mg for IP), and Myc (60003-2-Ig, 1:4000 for WB, 1 μg/mg for IP) were purchased from Proteintech.

Techniques: Immunohistochemical staining, Staining, Expressing, Control

Journal: The Journal of Neuroscience

Article Title: Superior Colliculus Controls the Activity of the Rostromedial Tegmental Nuclei in an Asymmetrical Manner

doi: 10.1523/JNEUROSCI.1556-20.2021

Figure Lengend Snippet: RMTg neurons monosynaptically innervated by the contralateral SC project to the midbrain dopaminergic system. A , Exemplary brain image showing the site of unilateral injection of anterograde transsynaptic AAV (AAV1-hSyn-Cre-hGH) containing the gene for Cre recombinase into the SC. Immunohistochemical staining was performed to visualize Cre-recombinase (Alexa Fluor 647; yellow color). B , Transsynaptically labeled Cre-positive neurons within RMTg. The orange dashed line indicates the RMTg boundaries based on the anti-FoxP1 immunostaining. C , Representative image of RMTg after the follow-up injection of AAV (AAV2-EF1α-DIO-mCherry) carrying Cre-dependent gene for fluorescent protein (mCherry; red color). The orange dashed line indicates the RMTg boundaries based on the anti-FoxP1 immunostaining. D , Top, Representative image of VTA and SNc after the injections of anterograde transsynaptic AAV (containing Cre recombinase gene) into the SC and AAV with Cre-dependent gene for mCherry into the contralateral (right in this case) RMTg. Immunohistochemical staining was performed to visualize TH-positive neurons (Alexa Fluor 488; green color). Bottom panels, Magnified regions of VTA and SNc with mCherry-expressing axons originating in the RMTg neurons innervated by the contralateral SC. E , Top, Exemplary image of binarized mCherry-positive axons within the VTA and SNc used for area fraction calculation. Bottom, Mediolateral distribution of normalized area fraction of mCherry-expressing axons originating from the RMTg neurons innervated by the contralateral SC. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: In the transsynaptic tract-tracing experiments, 80–200 nl of AAV1-hSyn-Cre-hGH (AAV; viral titer: 1.8 × 10 13 vg/ml; Addgene) was injected into the SC unilaterally (−6.4 to −6.6 mm caudally, ±1.8 to ±2.0 mm laterally, and −4.4 to −4.6 mm ventrally from the bregma point), and, in some of these rats, 300 nl of AAV2-EF1α-DIO-mCherry (viral titer: 5.3 × 10 12 vg/ml; UNC Vector Core) was injected into the contralateral RMTg (10° lateral angle, −6.8 to −7.0 mm caudally, +1.7 mm laterally, and −7.4 mm ventrally from the bregma point).

Techniques: Injection, Immunohistochemical staining, Staining, Labeling, Immunostaining, Expressing

Journal: The Journal of Neuroscience

Article Title: Superior Colliculus Controls the Activity of the Rostromedial Tegmental Nuclei in an Asymmetrical Manner

doi: 10.1523/JNEUROSCI.1556-20.2021

Figure Lengend Snippet: Stimulation of RMTg neurons innervated by the contralateral SC inhibit DA-like neurons in the VTA and SNc. A , Scheme of the experiment. SC was unilaterally injected with the transsynaptic viral vector (AAV1-hSyn-Cre-hGH) carrying the gene for Cre recombinase and contralateral RMTg was injected with AAV2-EF1α-DIO-ChR2(H134R)-mCherry carrying Cre-dependent genes for ChR2 and mCherry fluorescent protein. At least 2 weeks later, extracellular recordings of the activity of midbrain DA-like neurons was conducted while RMTg neurons, monosynaptically innervated by the contralateral SC, were optogenetically stimulated. The shape of a typical action potential of a DA-like neuron is shown in the inset on the left side. B , C , Top panels, Heatmaps of peristimulus firing rates (normalized to baseline) showing the responses of all recorded DA-like neurons to RMTg stimulation with trains of laser light pulses (473 nm, <20 mW, 5 ms pulses at 40 Hz over 1 s; B ) or single laser light pulses (473 nm, 100 ms, <20 mW; C ). Responses of individual neurons are shown in rows and are sorted by the amplitude of the response during the stimulation time (marked by vertical dashed line). Bottom panels, Peristimulus mean firing rate (normalized to baseline; ±SEM marked with gray color) and peristimulus mean change in firing rate (±SEM marked with gray color). The stimulation time is marked with a blue vertical bar. D , Localization of all recorded DA-like neurons with color-coded type of response to the optogenetic stimulation of RMTg neurons monosynaptically innervated by the contralateral SC. E , G , Pie charts showing the proportions of response types (inhibition, no effect, excitation) of VTA (marked by green outer ring) and SNc (marked by purple outer ring) DA-like neurons elicited by train (5 ms at 40 Hz; E ) or single-pulse (100 ms; G ) optogenetic stimulation of RMTg neurons innervated by the contralateral SC. F , H , Mean firing rates before, during, and after the train (5 ms at 40 Hz; F ) or single-pulse (100 ms; H ) optogenetic stimulation of RMTg neurons innervated by the contralateral SC (100 ms bins). I , J , From left to right: percentage change in firing rate, duration, and latency to the minimum of the response of DA-like neurons both in VTA (green bars) and SNc (purple bars) caused by train (5 ms at 40 Hz; I ) or single-pulse (100 ms; J ) optogenetic stimulation of RMTg neurons innervated by the contralateral SC. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, Nonsignificant.

Article Snippet: In the transsynaptic tract-tracing experiments, 80–200 nl of AAV1-hSyn-Cre-hGH (AAV; viral titer: 1.8 × 10 13 vg/ml; Addgene) was injected into the SC unilaterally (−6.4 to −6.6 mm caudally, ±1.8 to ±2.0 mm laterally, and −4.4 to −4.6 mm ventrally from the bregma point), and, in some of these rats, 300 nl of AAV2-EF1α-DIO-mCherry (viral titer: 5.3 × 10 12 vg/ml; UNC Vector Core) was injected into the contralateral RMTg (10° lateral angle, −6.8 to −7.0 mm caudally, +1.7 mm laterally, and −7.4 mm ventrally from the bregma point).

Techniques: Injection, Plasmid Preparation, Activity Assay, Inhibition

Journal: bioRxiv

Article Title: N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

doi: 10.1101/2022.09.01.505523

Figure Lengend Snippet: (A) MS-spectra for the N-terminal peptide of endogenous IST1 (P53990) from HAP1 WT and NatC KO cells following trypsin digestion and strong cation exchange (SCX) enrichment. (B) Bar graph showing the degree of Nt-acetylation of IST1 in HAP1 WT and NatC KO cells as determined by proteomics. Data are shown as mean ± SD (n = 4). (C) Endogenous IST1 protein levels from indicated HAP1 cells determined by immunoblotting. (D) Immunoblot analysis of confirmed and putative NatC substrates using total cell extract from HAP1 WT and NatC KO cells. (E) N-terminal variants of UBE2M-FLAG were expressed in HAP1 NAA30 -KO cells and protein levels were determined by immunoblotting. (F) HAP1 WT and NAA30 -KO cells were transfected with the indicated UBE2M-V5-P2A-GST-GFP reporter construct, and protein levels were determined by immunoblot analysis. UBE2M-V5 levels were normalized to GST-GFP and expressed relative to WT sample. Data are shown as mean ± SD of four independent experiments. ***p < 0.0004; two-tailed unpaired t test. (G) NAA30-WT-V5 and NAA30-mut-V5 was immunoprecipitated from HeLa cell extracts and used in Nt-acetylation assays with [ 14 C]-acetyl-CoA and synthetic peptides representing the NatC substrates UBE2M (MIKL) and ARFRP1 (MYTL), and the NAA80/NatH substrate β-actin (DDDI). The experiment was performed three independent times with three technical replicates each. Data of one representative setup is shown as mean ± SD. (H) NatC regulates the protein level of UBE2M, UBE2F ARFRP1, and CAPNS1. Immunoblot analysis of HAP1 WT and NAA30 -KO cells transfected with control V5 plasmid, NAA30-V5 or the catalytically dead mutant NAA30-mut-V5. (I) HAP1 WT and NAA30 -KO cells were treated with proteasomal (MG132 and bortezomib (BMZ)) and lysosomal inhibitors (bafilomycin A (BafA), leupeptin (LP) or ammonium chloride (NH4Cl)) for 6 h followed by immunoblot analysis using the indicated antibodies. DMSO served as vehicle control.

Article Snippet: The plasmid pCMV6-UBE2M-Myc-DDK (DDK is the same as FLAG®) was obtained from OriGene Technologies (Rockville, Maryland, USA, # RC208946). pcDNA3.1-NAA30-V5 was previously described and was constructed using cDNA from a human cell line ( ).

Techniques: Western Blot, Transfection, Construct, Two Tailed Test, Immunoprecipitation, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

doi: 10.1101/2022.09.01.505523

Figure Lengend Snippet: (A and B) The protein level of endogenous UBE2M in HAP1 WT and NAA30 -KO cells transfected with the indicated siRNAs for 72 h was assessed by immunoblotting. (C) Schematic representation of protein capture by peptide pulldown. A set of 11-mer peptides derived from the N-terminal sequence of UBE2M were C-terminally labelled with K-biotin ( MI KLFSLKQQK(K-biotin)) and conjugated to streptavidin magnetic beads. The first two residues were replaced to represent different N-termini (XY-UBE2M). Biotinylated UBE2M peptides were incubated with cell extracts and the pulled-down proteins were identified by immunoblot analysis. ( D) In vitro peptide pulldown assay of UBR4-V5 expressed in HeLa cells using acetylated and non-Nt-acetylated UBE2M peptide. (E) XY-UBE2M peptide pulldown assay with UBE2M peptides bearing different N-terminal amino acids and UBR4-V5 expressed in HeLa cells. (F) In vitro peptide pulldown assay of UBR4-N-FLAG expressed in HAP1 WT cells using acetylated and non-Nt-acetylated UBE2M peptide and X-nsP4 controls peptides. The UBR4-N construct contains the UBR-box (yellow), which is the substrate recognition domain of the UBR proteins. * indicates saturated UBE2A band. (G) HAP1 WT and NAA30 -KO cells were transfected with siCtrl or siUBR4 for 72 h and protein abundance was determined by TMT-based quantitative proteomics (see Table S5 ). Intensity profile plot showing protein levels of the top 100 proteins with abundance profiles most similar to UBE2M (blue trace) (FDR = 0.01, S0 = 0.1). The intensity profiles of RGS10, ARLB, CAPSN1, DIS3, and HK2 are highlighted in orange. (H) UBR4 knockdown stabilizes the protein levels of non-Nt-acetylated RGS10, HK1, DIS3, UBE2F, ARFRP1 and CAPNS1 in NAA30 -KO cells. HAP1 WT and NAA30 -KO cells were transfected with siCtrl or siUBR4 for 72 h followed by immunoblotting using the indicated antibodies.

Article Snippet: The plasmid pCMV6-UBE2M-Myc-DDK (DDK is the same as FLAG®) was obtained from OriGene Technologies (Rockville, Maryland, USA, # RC208946). pcDNA3.1-NAA30-V5 was previously described and was constructed using cDNA from a human cell line ( ).

Techniques: Transfection, Western Blot, Derivative Assay, Sequencing, Magnetic Beads, Incubation, In Vitro, Construct

Journal: bioRxiv

Article Title: N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

doi: 10.1101/2022.09.01.505523

Figure Lengend Snippet: ( Left ) The NatC complex co-translationally acetylates proteins harboring a hydrophobic residue in the second position (MΦ-). Following acetylation, the NEDD8 E2 ligases Ac-UBE2M and Ac-UBE2F promote cullin neddylation, resulting in proteasomal degradation of targeted substrates, Ac-ARFRP1 is targeted to the Golgi where it plays a role in the secretory pathway, while the hypothetical proteins Ac-X and Ac-Y are thought to affect the secretory pathway and mitochondria, respectively. ( Right ) Loss of NatC leads to proteasomal and, in some cases, lysosomal degradation of non-Nt-acetylated substrates primarily via the Arg/N-recognin UBR4-KCMF1 and to some extent via UBR1 and UBR2. Targeted degradation of unacetylated non-Nt-acetylated NatC substrates leads to decreased cullin neddylation, increased mitochondrial elongation and fragmentation, and is thought to affect intracellular trafficking.

Article Snippet: The plasmid pCMV6-UBE2M-Myc-DDK (DDK is the same as FLAG®) was obtained from OriGene Technologies (Rockville, Maryland, USA, # RC208946). pcDNA3.1-NAA30-V5 was previously described and was constructed using cDNA from a human cell line ( ).

Techniques: