Journal: bioRxiv

Article Title: Medulloblastoma-Associated KBTBD4 Mutations Disrupt PP2A-A Orphan Quality Control

doi: 10.64898/2026.03.02.709011

Figure Lengend Snippet: A. PP2A subunits PP2A- 3×Flag Aα/ Strep Cα/B55α were expressed as individual proteins, in PP2A subcomplexes PP2A-AC/AB/CB, and as the heterotrimeric PP2A-ACB holoenzyme in insect cells. PP2A extracts were mixed as indicated with 6×His-MBP KBTBD4 extract and affinity purified to test for binding of KBTBD4 to PP2A subunits and subcomplexes. B. Purified PP2A subunits and (sub-)complexes were ubiquitylated in vitro with purified KBTBD4, CUL3 NEDD8 -RBX1, UBE1, UBE2D3 and fluorescently labeled ubiquitin. Representative result of n=2 independent experiments. C. Cellular extracts from HEK293T cells and Δ KBTBD4 cell lines #1and #2 were separated over a 5-20% sucrose gradient by ultracentrifugation. Fractions were probed for indicated proteins by western blot. Representative result of n=2 experiments.

Article Snippet: Ubiquitylation assays were carried out in 20 μL reactions with final concentrations of 1 μM PP2A (PP2A-A or in indicated complex), 0.1 μM UBE1 (R&D Systems, E-304), 0.2 μM UBE2D3 (R&D Systems, E2-627), 0.5 μM KBTBD4, 0.4 μM CUL3 NEDD8 -RBX1 (CRL), 7.5 μM ubiquitin IRdye680 (gift from Jake Aguirre, as published in ( )), 5 mM ATP, 2.5 mM MgCl 2 , in a buffer of 20 mM HEPES pH 7.4, 150 mM NaCl and 1 mM DTT.

Techniques: Affinity Purification, Binding Assay, Purification, In Vitro, Labeling, Ubiquitin Proteomics, Western Blot

Journal: bioRxiv

Article Title: Medulloblastoma-Associated KBTBD4 Mutations Disrupt PP2A-A Orphan Quality Control

doi: 10.64898/2026.03.02.709011

Figure Lengend Snippet: A. Flag purification of transiently expressed wild-type KBTBD4 3×Flag and medulloblastoma hotspot mutants to probe for binding to 2×HA PP2A-A. B. PP2A-A stability was monitored by flow cytometry through transient expression of the dual fluorescence reporter in HEK293T cells and Δ KBTBD4 #1 with co-expression of KBTBD4 WT or the MB mutant KBTBD4 PR construct. C. Quantification of the stability of PP2A-A as monitored by flow cytometry after expression of the dual fluorescence reporter and indicated KBTBD4 constructs (WT, MB mutant PR, CUL3-binding mutant C3). Data was measured at least in triplicates, and the median fluorescence signal ratio of GFP over mCherry was normalized to HEK293T control. D. Ubiquitin conjugates were purified under denaturing conditions from Δ KBTBD4 #1 cells, virally transduced to rescue with indicated KBTBD4 constructs, and transiently transfected to express His-tagged ubiquitin and myc PP2A-A. E. Purified PP2A-A was ubiquitylated in vitro with purified KBTBD4 (WT and MB cancer mutant PR), CUL3 NEDD8 -RBX1, UBE1, UBE2D3 and fluorescently labeled ubiquitin. Representative result of n=2 independent experiments. F. Flag purification of transiently expressed 3×Flag PP2A-A (wild-type and cancer mutants P179R and R183W) to probe for binding to KBTBD4 3×HA . G. Purified PP2A-A (WT, and cancer mutants P179R and R183W) was ubiquitylated in vitro with purified KBTBD4, CUL3 NEDD8 -RBX1, UBE1, UBE2D3 and fluorescently labeled ubiquitin. Representative result of n=2 independent experiments. H. Quantification of the stability of wild-type PP2A-A and indicated mutants as monitored by flow cytometry after viral transduction of a dual fluorescence reporter. Data representative of n=3 independent experiments.

Article Snippet: Ubiquitylation assays were carried out in 20 μL reactions with final concentrations of 1 μM PP2A (PP2A-A or in indicated complex), 0.1 μM UBE1 (R&D Systems, E-304), 0.2 μM UBE2D3 (R&D Systems, E2-627), 0.5 μM KBTBD4, 0.4 μM CUL3 NEDD8 -RBX1 (CRL), 7.5 μM ubiquitin IRdye680 (gift from Jake Aguirre, as published in ( )), 5 mM ATP, 2.5 mM MgCl 2 , in a buffer of 20 mM HEPES pH 7.4, 150 mM NaCl and 1 mM DTT.

Techniques: Purification, Binding Assay, Flow Cytometry, Expressing, Fluorescence, Mutagenesis, Construct, Control, Ubiquitin Proteomics, Transfection, In Vitro, Labeling, Transduction

Journal: The Journal of Biological Chemistry

Article Title: Regulation of the MDM2-p53 nexus by a nuclear phosphoinositide and small heat shock protein complex

doi: 10.1016/j.jbc.2025.110527

Figure Lengend Snippet: PIP 2 and sHSPs regulate the ubiquitination function of MDM2. A , for MDM2 autoubiquitination, 100 nM of E1 enzyme, 1 μM of E2 enzyme, 1 μM of MDM2, E3 ligase reaction buffer, 10 mM of MgATP solution and 100 μM of ubiquitin were incubated with different concentrations of αBC, HSP27 (0.9, 1.8, or 2.7 μM), or PIP 2 (10, 20, or 30 μM) for 1 h. Ubiquitin, MDM2, αBC and HSP27 were analyzed by IB, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. B , for in vitro ubiquitination, 100 nM of E1 enzyme, 1 μM of E2 enzyme, 1 μM of His- and FLAG-tagged MDM2, E3 ligase reaction buffer, 10 mM of MgATP solution and 100 μM of ubiquitin were incubated with 1 μM of His- and FLAG-tagged p53 for 1 h. For IP, samples were incubated with anti-p53-conjugated agarose overnight. Ubiquitin, MDM2, and p53 were analyzed by IB, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. C , MDA-MB-231 cells were transfected with siRNAs for PIPKIα and treated with vehicle or 30 μM cisplatin for 24 h. Empty vector (Mock) was used as a negative control. After cells were treated with 10 μM of MG132 for 4 h, cells were harvested for IP of MDM2 and p53. Expression of the indicated proteins was analyzed by IB, and ubiquitin IBs were quantified. The graph is shown as mean ± s.d. of n = 3 independent experiments. KD, knockdown; UT, untreated; Cis, cisplatin treated; Cis/MG132, Cisplatin/MG132 treated. D , MDA-MB-468 cells were transfected with siRNAs for αBC and HSP27 for 48 h in absence or presence of MG132. Empty vector (Mock) was used as a negative control. Cells were processed for IP of MDM2. IB was used to analyze indicated proteins, and ubiquitin IBs were quantified. The graphs are shown as mean ± s.d. of n = 3 independent experiments. KD, knockdown. E , a model of PIP 2 regulation of the interaction between MDM2 and sHSPs, which differentially control the stability of MDM2 and function of of MDM2. In the presence of PIPKIα, PIP 2 is linked to MDM2, which recruits αBC to MDM2 to stabilize it and enhances p53 binding. Conversely, in the absence of PIPKIα and PIP 2 , HSP27 is recruited to MDM2, which increases the ubiquitin E3 ligase activity of MDM2.

Article Snippet: For the in vitro ubiquitination assay targeting MDM2 autoubiquitination, 100 nM of E1 enzyme (#E304, R&D Systems), 1 μM of E2 enzyme (#E2627, R&D Systems), 1 μM of MDM2, E3 ligase reaction buffer (#B71, R&D Systems), 10 mM of MgATP solution (#B20, R&D Systems) and 100 μM of ubiquitin (#U100H, R&D Systems) were incubated with different concentrations of αBC, HSP27 (0.9, 1.8, or 2.7 μM), or PIP 2 (10, 20, or 30 μM).

Techniques: Ubiquitin Proteomics, Incubation, In Vitro, Transfection, Plasmid Preparation, Negative Control, Expressing, Knockdown, Control, Binding Assay, Activity Assay

Journal: bioRxiv

Article Title: Catalytic pocket of Clr4 (Suv39h) methyltransferase serves as a substrate receptor for Cullin 4-dependent histone H3 ubiquitination

doi: 10.1101/2025.08.28.672867

Figure Lengend Snippet: (A) Map of Recombinant Bacmid Vector Encoding CLRC -Clr4 . Diagram of the recombinant bacmid vector used for insect cell expression, encoding all CLRC components except Clr4. Key vector features and regulatory elements are indicated. (B) SEC chromatogram of CLRC complexes using Superose 6 Increase 3.2/300 column (Cytiva). Absorbance at 280 nm (mAU) is shown. The grey line indicated the elution profile of CLRC complex lacking Clr4, with the peak fractions highlighted. The blue line indicated the elution profile of the full CLRC complex with peak fractions highlighted in blue and fractions containing excess unbound Clr4 highlighted in pink. (C) SDS-PAGE analysis of SEC fractions from panel B. Coomassie-stained SDS–PAGE gel showing input (CLRC -Clr4 and Clr4) and eluted fractions from size-exclusion chromatography. Fractions corresponding to the blue peak (1.45–1.55 mL) contain all 5 CLRC complex subunits, while later fractions highlighted in pink (1.65–1.85 mL) contain excess unbound Clr4. *, MBP-Raf1 degradation products. (D) Mass spectrometry analysis of the reconstituted CLRC complex. Quantitative LC-MS/MS analysis of the reconstituted CLRC complex showing normalized spectral counts relative to MBP–Raf1 (set to 1). The calculated molar ratios of 0.66 for Clr4, 0.33 for Rik1, 0.31 for Cul4, and 0.22 for Raf2 are consistent with SDS–PAGE band intensities. (E) Schematic representation of Clr4 truncation constructs used in this study. Domain architecture of Clr4 and its truncation variants, with the chromodomain shown in yellow, the hinge region in green, and the SET domain in blue. (F) SDS-PAGE analysis of in vitro H3 ubiquitination by reconstituted CLRC complex. Ubiquitination reactions were performed using recombinant human E1, E2, HA–ubiquitin (Ub), and the N-terminal peptide of histone H3 (residues 1– 20), in the presence of reconstituted CLRC complexes with or without Clr4. Both CLRC and CLRC -Clr4 efficiently ubiquitinated the H3 peptide, whereas E1 and E2 alone showed no activity. *, MBP-Raf1 degradation products; HO, histone octamer.

Article Snippet: A 5× ubiquitination mix was prepared in reaction buffer [50 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 , 0.5 mM DTT] and contained 500 nM recombinant human E1 (UBE1), 1 μM recombinant E2 (UbcH5c/UBE2D3), 20 mM ATP, and 50 μM recombinant human HA-Ubiquitin (all from R&D Systems, Bio-Techne) together with 100 μM S-adenosylhomocysteine (SAH).

Techniques: Recombinant, Plasmid Preparation, Expressing, SDS Page, Staining, Size-exclusion Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Construct, In Vitro, Ubiquitin Proteomics, Activity Assay