human p300 cdna fragments  (Addgene inc)

Journal: iScience

Article Title: Mitochondrial hyper-acetylation induced by an engineered acetyltransferase promotes cellular senescence

doi: 10.1016/j.isci.2025.113233

Figure Lengend Snippet: Design and characterization of engineered mitochondrial acetyltransferase; eMAT (A) Schematic structure of eMAT. Mitochondria targeting sequence (MTS) of PDHA1, COX4, NDUFS1, SIRT4, and CAMP were fused with p300 HAT domain (1282–1667 a.a.) or Core domain (965–1810 a.a.). (B) Subcellular localization of the fusion constructs (anti-FLAG, colored in green). PDHA1-HAT and COX4-HAT merged with a mitochondria marker protein ETFβ (colored in red). The blue signal indicates DAPI (nuclear marker). Box with magenta; constructs that showed mitochondrial localization. Scale bar: 10 μm. (C) Immunoblot images of total cell lysates from HEK293T transfected with indicated constructs. Top; anti-AcK antibody, Middle; anti-FLAG antibody, Bottom; anti-α-tubulin antibody as a loading control. (D) Immunoblot analysis of protein acetylation in mitochondria. Mitochondria were isolated from HEK293T cells transfected with indicated constructs, and immunoblot analysis was performed with anti-AcK (top), anti-FLAG (middle), and anti-ETFβ as mitochondria loading control (bottom). (E) Structure of eMAT. Top; schematic amino acid sequence of eMAT. Bottom; Structure of eMAT protein predicted with AlphaFold2 based ColabFold program (v1.5.3).

Article Snippet: Plasmids for eMAT were constructed from human p300 cDNA fragments amplified from pCMVβ-p300-myc (addgene#30489), or pCMVβ-p300.DY-myc (addgene#30490) for the catalytic mutant.

Techniques: Sequencing, Construct, Marker, Western Blot, Transfection, Control, Isolation

Journal: Cancer Research

Article Title: Tobacco Smoking Rewires Cell Metabolism by Inducing GAPDH Succinylation to Promote Lung Cancer Progression

doi: 10.1158/0008-5472.CAN-24-3525

Figure Lengend Snippet: Acyltransferase p300 catalyzes GAPDH K251-Su. A, Expression of GAPDH K251-Su was detected after siRNA on the succinyltransferases GCN5, CPT1A, DLST, and p300/CBP. Designed three siRNAs for each gene were siRNA 1#, 2#, and 3#. The controls without added siRNA are labeled as “–." B, Confocal assay to detect the colocalization of GAPDH (green) and p300 (red) in A549 and NCI-H2170 cells. DAPI (blue), nucleus. Scale bar, 20 μm. C, Co-IP and immunoblotting assay to test the interaction of succinyltransferases GCN5, CPT1A, DLST, and p300/CBP with GAPDH in A549 cells. D, Co-IP and immunoblotting were used to analyze the interaction of expressed His-p300 and Flag-GAPDH. E and F, The levels of GAPDH K251-Su ( E ) and GAPDH enzyme activity ( F ) were detected after overexpression of p300 in GAPDH WT, K251R-mutant, and K251E-mutant A549 cells, respectively. The OD value indicates the amount of NADH catalyzed by GAPDH ( n = 3). G, Cells were treated with 2 μmol/L acyltransferase inhibitor A485 and then added with 0.5 and 6 mmol/L glutamine for 24 hours. The effect of A485 on the expression of GAPDH K251-Su was detected. Hypoxic nutrient deficiency: cells were cultured in 1% FBS and 0.5 g/L glucose medium and an incubator at 2% O 2 . H, Bioinformatics analysis from TCGA and cBioPortal database to compare the gene (violin diagram) and protein (box diagram) expression of p300 in the tumor tissues from smoker and nonsmoker patients with NSCLC. The comparison of p300 gene expression also includes the data of adjacent normal tissues. Data are presented as mean ± SD. *, P < 0.05; ****, P < 0.0001; ns, nonsignificant. Statistical significance was determined by an unpaired t test. See also Supplementary Fig. S8. WCL, whole-cell lysate.

Article Snippet: The eukaryotic expression plasmid for human p300 gene (pEnCMV-6His-EP300-SV40-Neo, RRID: Addgene_228220) was obtained from MiaoLing Biology, and the plasmids and lentivirus for human GAPDH gene interference (PMT517-shGAPDH, RRID: Addgene_228221) and human WT GAPDH gene (PSE4462-GAPDH-WT-3Flag, RRID: Addgene_228221) or mutant GAPDH gene (PSE4462-GAPDH-K251R-3Flag, RRID: Addgene_228223; PSE4462-GAPDH-K251E-3Flag, RRID: Addgene_228224; and PSE4462-GAPDH-K251R/K254R-3Flag, RRID: Addgene_228225) eukaryotic expression were obtained from Sangon Biotech.

Techniques: Expressing, Labeling, Confocal Assay, Co-Immunoprecipitation Assay, Western Blot, Activity Assay, Over Expression, Mutagenesis, Cell Culture, Comparison, Gene Expression

Journal: Proteins

Article Title: Probing Enzymatic Acetylation Events in Real Time With NMR Spectroscopy: Insights Into Acyl-Cofactor Dependent p300 Modification of Histone H4.

doi: 10.1002/prot.26848

Figure Lengend Snippet: FIGURE 1 | Generation of a p300 construct with improved yield. (A) Representative expression and purification gel of p300 KAT from pETdu- et+p300 KAT suggests a total yield of ~1 mg/L of media. Lanes show (1) Whole cell lysate (2) Insoluble pellet (3) Soluble supernatant (4) NiNTA flowthrough (5) Low salt (150 mM) wash (6) High salt (1 M) wash (7) NiNTA elution alongside the protein standard ladder lane (L) (ThermoFischer 26 634). p300 KAT is the faint band ~50 kDa in lane 7. (B) Schematic representation of the histone H4 (1-25)W construct used in subsequent experi- ments. This construct can be uniformly acetylated by p300 KAT on each of its five available lysine sites. (C) Demonstration of variable activity be- tween p300 KAT preparations shown by MALDI-MS spectra of identical acetylation reactions using presumably equivalent enzyme preparations. (D) Representative expression and purification gel for p300 KAT from pET His6 MBP TEV p300(1284–1669) LIC (Addgene #233587). Total yield was typ- ically > 10 mg/L of media. Lanes show (1) Whole cell lysate (2) Insoluble pellet (3) Soluble supernatant (4) NiNTA flowthrough 1 (5) Low salt (150 mM) wash (6) High salt (1 M) wash (7) NiNTA elution 1 (8) TEV protease dialysate (9) NiNTA flowthrough 2 (10) NiNTA wash 2 (11) NiNTA elution 2 (12) Protein ladder (NEB P7719S). Cleaved p300 KAT is the band ~43 kDa in lanes 9 and 10 (expected molecular weight once cleaved is 44 684 Da). Note this is very close to the expected molecular weight of 41 584 Da for the cleaved MBP solubility tag.

Article Snippet: p300 KAT was originally obtained from pETduet+p300 KAT, a gift from Michael Rosen (Addgene #157793), which coexpresses human p300 (1284–1664) with yeast Sirt2 [22].

Techniques: Construct, Expressing, Purification, Activity Assay, Molecular Weight, Solubility

Journal: Proteins

Article Title: Probing Enzymatic Acetylation Events in Real Time With NMR Spectroscopy: Insights Into Acyl-Cofactor Dependent p300 Modification of Histone H4.

doi: 10.1002/prot.26848

Figure Lengend Snippet: FIGURE 2 | Pre-acetylation of p300 does not meaningfully alter acetyltransferase activity. (A) Overlaid 13C, 1H-HSQC NMR spectra showing re- action products after 1 h treatment of histone H4 (1-25)W with native p300 (teal) or p300 prescribed to a 2-h incubation with acetyl-CoA (purple). (B) overlaid 1D projections of the 13C, 1H-HSQC experiments demonstrate nearly equivalent peak heights for the acetyl-CoA (2.25 ppm) and acetyllysine (1.87 ppm 1H) products. (C) MALDI-MS spectra showing the distributions of reaction products.

Article Snippet: p300 KAT was originally obtained from pETduet+p300 KAT, a gift from Michael Rosen (Addgene #157793), which coexpresses human p300 (1284–1664) with yeast Sirt2 [22].

Techniques: Activity Assay, Incubation

Journal: Proteins

Article Title: Probing Enzymatic Acetylation Events in Real Time With NMR Spectroscopy: Insights Into Acyl-Cofactor Dependent p300 Modification of Histone H4.

doi: 10.1002/prot.26848

Figure Lengend Snippet: FIGURE 3 | Comparison of p300 and p300Δ acetyltransferase activity towards the histone H4 tail. (A) Circular dichroism spectra for p300 (teal) and p300Δ (pink) used to estimate secondary structure content with BestSel. (B) MALDI-MS time courses comparing relative acetyltransferase ef- ficiency of p300 (teal) and p300Δ (pink) using the histone H4 tail as a substrate. (C) Mono-exponential fit of acetyltransferase reaction curves from a 1H-13C, HSQC based NMR time course for p300 (teal) and p300Δ (pink) acetyltransferase reactions with the histone H4 tail. Progress curves show the increase in intensity for the acetyllysine resonance centered at 1.86 ppm 1H, 22.1 ppm 13C over time, each data point represents the maximum acetyllysine peak intensity from a 3 min and 36 s experiment. Progress curves were fit with a mono-exponential kinetic model to estimate relative turnover rates (k) and maximum intensity (Imax). (D) Progress curves for p300 (teal) and p300Δ (pink) acetyltransferase reactions with the histone H4 tail fit with a bi-exponential kinetic model to estimate relative turnover rates (k1 and k2) and relative contributions to the maximum intensity (A1 and A2). R2 values are included in C and D to indicate the goodness of fit, and fit residuals for each data point are displayed at scale relative to 20% of the maximum data intensity.

Article Snippet: p300 KAT was originally obtained from pETduet+p300 KAT, a gift from Michael Rosen (Addgene #157793), which coexpresses human p300 (1284–1664) with yeast Sirt2 [22].

Techniques: Comparison, Activity Assay, Circular Dichroism

Journal: Proteins

Article Title: Probing Enzymatic Acetylation Events in Real Time With NMR Spectroscopy: Insights Into Acyl-Cofactor Dependent p300 Modification of Histone H4.

doi: 10.1002/prot.26848

Figure Lengend Snippet: FIGURE 4 | Validation of 12C propionyl-CoA synthesis and con- trol reactions to enable 13C propionylation resonance assignments. (A) Proton 1D NMR spectra showing the reaction product of the propionyl- CoA (green) synthesis reaction from propionic anhydride precursor (gray). The peaks at 1.06 and 2.56 ppm, respectively, were assigned to the methyl and methylene protons of the CoA conjugated propionyl moiety based on comparison to reference proton 1D spectra provided by CoALA Biosciences (SKU PC01). (B) Overlaid 13C, 1H-HSQC NMR spectra of the propionyltransferase reaction mixture (without enzyme) before adding 13C propionyl-CoA (gray) and after adding 13C propionyl- CoA (green). (C) Overlaid 13C, 1H-HSQC NMR spectra for p300 cata- lyzed propionylation of the histone H4 tail (green) overlaid with the ap- propriate no enzyme control (gray).

Article Snippet: p300 KAT was originally obtained from pETduet+p300 KAT, a gift from Michael Rosen (Addgene #157793), which coexpresses human p300 (1284–1664) with yeast Sirt2 [22].

Techniques: Biomarker Discovery, Comparison, Control

Journal: Proteins

Article Title: Probing Enzymatic Acetylation Events in Real Time With NMR Spectroscopy: Insights Into Acyl-Cofactor Dependent p300 Modification of Histone H4.

doi: 10.1002/prot.26848

Figure Lengend Snippet: FIGURE 5 | Comparison of p300 and p300Δ propionyltransferase activity towards the histone H4 tail. (A) MALDI-MS time course com- paring relative propionyltransferase efficiency of p300 (teal) and p300Δ (pink) using the histone H4 tail as a substrate. (B) Progress curves for p300 (teal) and p300Δ (pink) propionyltransferase reactions with the histone H4 tail fit with a mono-exponential kinetic model to estimate relative turnover rates (k) and maximum intensity (Imax). (C) Progress curves for p300 (teal) and p300Δ (pink) propionyltransferase reactions with the histone H4 tail fit with a lagged mono-exponential kinetic model to estimate relative turnover rate (k1), amplitude (A), lag time (t0), and slope around t0 (α). R2 values are included in B and C to indicate the goodness of fit, and fit residuals for each data point are displayed at scale relative to 20% of the maximum data intensity.

Article Snippet: p300 KAT was originally obtained from pETduet+p300 KAT, a gift from Michael Rosen (Addgene #157793), which coexpresses human p300 (1284–1664) with yeast Sirt2 [22].

Techniques: Comparison, Activity Assay

Journal: Nature Communications

Article Title: Zfp260 choreographs the early stage osteo-lineage commitment of skeletal stem cells

doi: 10.1038/s41467-024-54640-0

Figure Lengend Snippet: a Workflow of in vivo labeling strategy using TAM and the experimental design. b PCA indicating the variations of transcriptomes among Lin - ZsGreen + cells isolated from BF-Ctrl, BF-cKO, MSFL-Ctrl and MSFL-cKO groups. c GO and KEGG enrichment analysis of FACS-RNA-seq data. d Heatmap of replicate data for H3K4me1 and H3K27ac enrichment as detected by CUT&Tag. n = 3 from 3 biological replicates. e GO-biological process enrichment analysis of differentially enriched super-enhancers. f Heatmap of replicate data for Zfp260-V5 enrichment detected by ChIP-seq. n = 2 from 2 biological replicates. g Top enriched de novo motifs of Zfp260-V5 enriched genes. h Distribution of peaks in the genome. i GO and KEGG enrichment analysis of Zfp260-V5 enriched genes. j Screening strategy for the potential master downstream regulator. k Transcripts Per Kilobase (TPM) of Runx2 expression level from fracture and MSFL derived Lin - ZsGreen + cells. n = 3 from 3 biological replicates of RNA-seq data. l Genome browser view of peaks enriched for H3K4me1, Brd4, H3K27ac, and Zfp260-V5 over the Runx2 gene locus on chromosome 17 (left) with the magnified super-enhancer region displayed on the right. Primers 1 and 2 indicated the primer sets for the subsequent ChIP-qPCR detection. m Co-IP was performed to examine the condensates for the super-enhancer via immortalized PSCs. n = 3 from 3 biological replicates. n , o mIHC co-staining for Zfp260 (purple) with Brd4 (gold), Med1 (cyan), and P300 (gray) in the homeostatic and osteogenic states of PSCs. The yellow dotted line indicated the route for the subsequent fluorescence intensity measurements. n = 3 from 3 biological replicates. p Fluorescence intensity measurements along the route, with black triangles indicating the merged signals of the four channels. q , r ChIP-qPCR assays for H3K27ac and Brd4 binding via immortalized PSCs. n = 6 from 2 biological replicates. Two-way ANOVA. Scalebars: 5 μm. All data in this figure are represented as mean ± SD. Source data and exact p values are provided in the Source Data file.

Article Snippet: The primary antibodies used in mIHC (dilution 1:400 for all antibodies) included goat anti-mouse/human/rat Itgav (AF1219, Novus Biologicals), mouse anti-mouse/rat CD90 (NB100-65543, Novus Biologicals), mouse anti-mouse/human CD105 (NBP2-22122, Novus Biologicals), rabbit anti-human/mouse/rat CD200 (AF2724, Novus Biologicals), rabbit anti-mouse/human/rat Runx2 (ab236639, Abcam), rabbit anti-mouse/human/rat Sox9 (ab185966, Abcam), rabbit anti-mouse/human Alpl (MA5-24845, Invitrogen), rabbit anti-mouse/human/rat Zfp260 (ABE295, Merck), mouse anti-human/mouse/rat p300 (NB100-616, Novus Biologicals), rabbit anti-human/mouse MED1 (NB100-2574, Novus Biologicals), rabbit anti-human/mouse BRD4 (NBP2-76393, Novus Biologicals), mouse anti-human/mouse/rat Prkca (NB600-201, Novus Biologicals), rabbit anti-V5 tag (13202, CST), mouse anti-Collagen type I (67288-1-Ig, proteintech), rabbit anti-Collagen type II (28459-1-AP, proteintech).

Techniques: In Vivo, Labeling, Isolation, RNA Sequencing, ChIP-sequencing, Expressing, Derivative Assay, ChIP-qPCR, Co-Immunoprecipitation Assay, Staining, Fluorescence, Binding Assay