Journal: bioRxiv

Article Title: A functional map of phosphoprotein phosphatase regulation identifies an evolutionary conserved reductase for the catalytic metal ions

doi: 10.1101/2025.02.12.637884

Figure Lengend Snippet: A) Holoenzyme composition and regulators of PP2A-like phosphatases. B) Schematic of genome-wide CRISPR-Cas9 screen for genes whose knockout are synthetic lethal with okadaic acid (OA). NGS, next-generation sequencing. C) DrugZ analysis of CRISPR-Cas9 synthetic lethality screen performed in RPE1 p53-/-cells comparing treatment with a low dose of OA (2 nM) and untreated conditions. The top genes are indicated with established PP2A-like holoenzyme components and regulators in black and candidate regulators in grey. Significance threshold ( p -value of 0.05) is indicated with a dotted line at a DrugZ score of −3.3. PPP1CA is indicated as highest scoring non PP2A-like phosphatase. D) Human CYB5R4 and yeast Irc21 domain organization. Cytb5, cytochrome B5 domain. CS, CHORD/SGT domain. Cytb5-R, Cytochrome b5 reductase domain. E) Profiling of PPP composition by phosphatase inhibitor beads and mass spectrometry (PIB-MS). Volcano plot comparing the phosphatase components captured on phosphatase inhibitor beads from U2OS CYB5R4 knockout (KO) and parental (PAR) cells. PP2A holoenzyme components are indicated in green, PP4 in blue and PP6 in pink. ‘C’ indicates catalytic subunit. F) Peptide dephosphorylation assays measuring the activity of 3xFLAG-tagged PP2A C, PP4 C, and PP6 C, immunopurified from U2OS parental or CYB5R4 knockout stable cell lines. The data is representative of three independent experiments. G) Peptide dephosphorylation assays measuring the activity of HA-Pph21, myc-Pph3, and HA-Sit4 immunopurified from endogenously tagged wildtype ( wt ) and irc21 deletion strains. Data is shown for three independent experiments, and error bars represent standard deviations.

Article Snippet: Lysates were cleared by centrifugation for 30 minutes at 14,000 g whereafter supernatants were incubated with Fab-trap beads (Proteintech) for immunoprecipitation of 3xFLAG control and 3xFLAG-conjugated catalytic subunits or with GFP-trap beads (Proteintech) for immunoprecipitation of venus-tagged CYB5R4 wild-type and mutant.

Techniques: Genome Wide, CRISPR, Knock-Out, Next-Generation Sequencing, Mass Spectrometry, De-Phosphorylation Assay, Activity Assay, Stable Transfection

Journal: bioRxiv

Article Title: A functional map of phosphoprotein phosphatase regulation identifies an evolutionary conserved reductase for the catalytic metal ions

doi: 10.1101/2025.02.12.637884

Figure Lengend Snippet: A) Residue map of CYB5R4 showing synthetic lethality in OA. X-axis depicts the amino acid residue targeted for mutation and the y-axis the average log2 fold changes of gRNAs targeting the indicated residue in OA vs untreated conditions. The domains of CYB5R4 as well as H89R, H112R, and W114R are indicated. B) AlphaFold3 model of CYB5R4 with heme (salmon) and H89, H112, and W114 (yellow) indicated. C) Deep sequencing of the endogenous CYB5R4locus after transduction with a single gRNA targeting H112, followed by CRISPResso2 analysis shows the frequency of mutated alleles. Reference: the genomic sequence. The gRNA, PAM, and editing window are indicated as well as the amino acid translation. D) A colony formation assay was conducted in presence or absence of 2 nM OA with U2OS parental or CYB5R4 knockout cells. Cells were stably complemented with full length CYB5R4-venus either wildtype (WT) or with the specified mutations. The survival is calculated as the relative number of colonies in OA to untreated and represents three independent experiments. Error bares depict standard deviations and the shown p -values are based on one-way ANOVA analysis with Tukey’s multiple comparisons test.

Article Snippet: Lysates were cleared by centrifugation for 30 minutes at 14,000 g whereafter supernatants were incubated with Fab-trap beads (Proteintech) for immunoprecipitation of 3xFLAG control and 3xFLAG-conjugated catalytic subunits or with GFP-trap beads (Proteintech) for immunoprecipitation of venus-tagged CYB5R4 wild-type and mutant.

Techniques: Residue, Mutagenesis, Sequencing, Transduction, Colony Assay, Knock-Out, Stable Transfection

Journal: bioRxiv

Article Title: A functional map of phosphoprotein phosphatase regulation identifies an evolutionary conserved reductase for the catalytic metal ions

doi: 10.1101/2025.02.12.637884

Figure Lengend Snippet: A) Heatmap comparing the interactomes of venus-tagged CYB5R4 1-153 wildtype (WT), H89A/H112A, and W114A to venus control, which were immunoprecipitated from HeLa cells and analysed by mass spectrometry. PP4/6 holoenzyme components are shown. Colors represent log2 fold changes with red being increased and blue depleted compared to venus control. B) AlphaFold3 model of CYB5R4 1-153 and PP6 C with heme and residues H89, H112 and W114 indicated. The N-terminal tail of CYB5R4 is not shown in the close-up views for clarity. C) AlphaFold3 model of PP6 with annotation of CYB5R4 1-153 contacts (left) and a color gradient (right) that represents the average log2 fold changes of gRNAs targeting the indicated residue in OA vs untreated conditions. Blue values specify that mutation of the target residue causes depletion (synthetic lethality) in OA and red enrichment. Grey specifies residues not targeted. D) DiFMUP dephosphorylation assay measuring the activity of purified PP6 holoenzyme in presence or absence of pre-reduced purified CYB5R4 1-153 WT or H89A/H112A. Data is from three independent experiments, and error bars represent standard deviations. E) DiFMUP dephosphorylation assay measuring the activity of purified PP6 holoenzyme in presence or absence of the indicated reducing agents at 1 mM. Data is from three independent experiments, and error bars represent standard deviations. F) DiFMUP dephosphorylation assay measuring the activity of purified PP6 holoenzyme, which was first pre-incubated with or without EDTA to extract metal ions. Next, the enzyme was incubated in presence or absence of Mn 2+ and finally in the presence or absence of pre-reduced purified CYB5R4 1-153 WT. Activity after 30 minutes is shown (also see Supplementary Figure S7F). Data is from three independent experiments, and error bars represent standard deviations. G) Model of PP4/6 regulation by CYB5R4.

Article Snippet: Lysates were cleared by centrifugation for 30 minutes at 14,000 g whereafter supernatants were incubated with Fab-trap beads (Proteintech) for immunoprecipitation of 3xFLAG control and 3xFLAG-conjugated catalytic subunits or with GFP-trap beads (Proteintech) for immunoprecipitation of venus-tagged CYB5R4 wild-type and mutant.

Techniques: Control, Immunoprecipitation, Mass Spectrometry, Residue, Mutagenesis, De-Phosphorylation Assay, Activity Assay, Purification, Incubation