Journal: Nature medicine

Article Title: Asfotase-α improves bone growth, mineralization and strength in mouse models of neurofibromatosis type-1

doi: 10.1038/nm.3583

Figure Lengend Snippet: ( a ) BMSC differentiation analyzed by Alizarin red–S (differentiation/mineralization, CFU–Ob), crystal violet staining (cell number, CFU–F, left panel), soluble Alizarin redS/crystal violet optical density ratio (middle panel) and ALP activity/crystal violet ratio (right panel) ( n = 6). ( b ) Runx2, Alpl and Ocn mRNA expression in BMSCs differentiated for 7, 14 and 21 days ( n = 4). ( c ) Runx2 and Alpl mRNA expression in serum–starved BMSCs treated with vehicle (DMSO) or U0126 for 24 h ( n = 6). ( d ) Extracellular PPi concentration/protein concentration in BMSCs differentiated for 7, 14 and 21 days ( n = 4). ( e ) Normalized Ank, Enpp1 and Opn mRNA expression in BMSCs differentiated for 7, 14 and 21 days ( n = 4). Blue bars: WT mice, grey bars: Col2-Nf1 KO mice, *: p < 0.05.

Article Snippet: The probe and primer sets for mouse Runx2 (Mm00501578_m1); Alpl (Mm00475834_m1); Ank (Mm00445047_m1); Enpp1 (Mm00501097_m1); Opn (Mm00436767_m1), Igf1 (Mm01228180_m1), human ANKH (Hs00219798_m1) and human ENPP1 (Hs01054040_m1) and the normalizers Hprt (Mm00446968_m1); human GAPDH (Hs99999905_m1) were obtained from Applied Biosystems (Foster City, CA, USA).

Techniques: Staining, Activity Assay, Expressing, Concentration Assay, Protein Concentration

Journal: Nature medicine

Article Title: Asfotase-α improves bone growth, mineralization and strength in mouse models of neurofibromatosis type-1

doi: 10.1038/nm.3583

Figure Lengend Snippet: ( a ) BMSC differentiation analyzed by Alizarin red–S (differentiation/mineralization, CFU–Ob) and crystal violet (cell number, CFU–F) staining ( n = 3) and ALP activity ( n = 3), following vehicle or BMP2 treatment. ( b ) Phospho–Smad1/5 induction in serum–starved BMSCs following BMP2 treatment for 1 h. Smad1/5 and β–actin served as loading control. ( c and d ) Alpl, Runx2, Col1a1, Ank, Enpp1 and Opn mRNA expression following BMP2 treatment for 2 weeks ( n = 3). ( e ) Extracellular PPi relative concentration (normalized to protein concentration) in the conditioned medium of BMSCs treated with BMP2 for 24 h ( n = 3). ( f and g ) BMSC differentiation analyzed by Alizarin red–S (differentiation/mineralization, CFU–Ob) and crystal violet (cell number, CFU–F) staining ( f , n = 3) and ALP activity ( g , n = 3) following treatment with vehicle or BMP2 or U0126 or both for 2 weeks. Blue bars: WT mice; grey bars: Col2-Nf1 KO mice. *: p < 0.05 versus WT in the same treatment group; #: p < 0.05 versus vehicle in the same genotype group.

Article Snippet: The probe and primer sets for mouse Runx2 (Mm00501578_m1); Alpl (Mm00475834_m1); Ank (Mm00445047_m1); Enpp1 (Mm00501097_m1); Opn (Mm00436767_m1), Igf1 (Mm01228180_m1), human ANKH (Hs00219798_m1) and human ENPP1 (Hs01054040_m1) and the normalizers Hprt (Mm00446968_m1); human GAPDH (Hs99999905_m1) were obtained from Applied Biosystems (Foster City, CA, USA).

Techniques: Staining, Activity Assay, Control, Expressing, Concentration Assay, Protein Concentration

Journal: bioRxiv

Article Title: PDZ-directed substrate recruitment is the primary determinant of specific 4E-BP1 dephosphorylation by PP1-Neurabin

doi: 10.1101/2024.09.23.614477

Figure Lengend Snippet: A. Structures of fusion proteins. N-terminally Flag-tagged PP1α(7-304) is linked to sequences from each of the four families of RVxF-ΦΦ-R-W PIPs, shown as an open box. Each fusion contains sequences immediately C-terminal to the PP1 interaction motif (coloured lines) including known protein interaction domains previously implicated in potential substrate interactions (coloured blocks). For PIP sequences in each fusion see and Methods. Middle, sequences of the RVxF-ΦΦ-R-W string in each PIP, with motifs coloured. Each fusion contains the sequences C-terminal to the dashed line, represents the position of PP1-SGSGS linker insertion. Bottom, structures of PP1/PIP complexes. Crystal structures of different PIP/PP1 complexes superimposed, aligned on PP1. Grey: PP1 (PDB: 4MOV), with PIP sequences as follows: green, Phactr1 (PDB: 6ZEE); magenta, Neurabin (PDB: 3HVQ); orange, R15A (PDB: 7NZM); blue, PNUTS (PDB: 4MOY). Dashed line, GSGSG linker. B. Activity of PP1-Phactr1 expressed in Flp-In T-Rex 293 cells. PP1-Phactr1 expression was induced by tetracycline as indicated. Phosphorylation of Phactr1/PP1 substrates IRSp53 S455 and Afadin S1275 is shown below. C. Analysis of Phactr1/PP1 substrate Afadin pS1275 phosphorylation in Flp-In TRex 293 cells expressing PP1 and PP1-fusion proteins.

Article Snippet: His-tagged Neurabin and Spinophilin PDZ domains were produced in BL21 (DE3) E. coli cells (Invitrogen).

Techniques: Activity Assay, Expressing

Journal: bioRxiv

Article Title: PDZ-directed substrate recruitment is the primary determinant of specific 4E-BP1 dephosphorylation by PP1-Neurabin

doi: 10.1101/2024.09.23.614477

Figure Lengend Snippet: A-C . Identification of PP1-R15A ( A ), PP1-R15B ( B ) and PP1-PNUTS ( C ) substrates by depletion of phosphorylation sites in the corresponding samples as opposed to average abundance in the dataset. Dashed line, 5% false-discovery threshold; red, significantly depleted phosphorylation sites; blue, PNUTS phosphorylation sites arising from overexpression of PP1-PNUTS. D. Sequences of significantly depleted phosphorylation sites from PP1-PNUTS samples. E. Comparison of total protein levels in cells PP1-Neurabin cells with or without induction. Neurabin and 4E-BPs are highlighted in red. Dashed line, 5% false-discovery threshold. F. Specificity analysis of the commercial anti-phospho-S65 antibody. G. mTORC1 pathway schematic (see ( ; )).

Article Snippet: His-tagged Neurabin and Spinophilin PDZ domains were produced in BL21 (DE3) E. coli cells (Invitrogen).

Techniques: Over Expression, Comparison

Journal: bioRxiv

Article Title: PDZ-directed substrate recruitment is the primary determinant of specific 4E-BP1 dephosphorylation by PP1-Neurabin

doi: 10.1101/2024.09.23.614477

Figure Lengend Snippet: A-B . Identification of PP1-Neurabin ( A ) and PP1-Spinophilin ( B ) substrates by depletion of phosphorylation sites in the corresponding samples compared to average abundance in the dataset (excluding PP1-Neurabin and PP1-Spinophilin). Dashed line, 5% false-discovery threshold; significantly depleted sites shown in red. C . Sequences of significantly depleted phosphorylation sites identified in A and B . D . Immunoblot analysis of 4E-BP1 phosphorylation sites in 293 Flp-In T-Rex cells upon expression of PP1-Neurabin or empty vector. E . Protein synthesis quantification assay. 293 Flp-In T-Rex cells expressing vector alone, PP1-Neurabin, or PP1, were induced with tetracycline (50 nM) and/or treated with rapamycin (50 nM) for 16h as indicated before treatment with O -propargyl puromycin to label nascent polypeptides, which were conjugated to AlexaFluor-488 azide and quantified by flow cytometry. Fluorescence intensities were normalized to untreated cells.

Article Snippet: His-tagged Neurabin and Spinophilin PDZ domains were produced in BL21 (DE3) E. coli cells (Invitrogen).

Techniques: Western Blot, Expressing, Plasmid Preparation, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: PDZ-directed substrate recruitment is the primary determinant of specific 4E-BP1 dephosphorylation by PP1-Neurabin

doi: 10.1101/2024.09.23.614477

Figure Lengend Snippet: A . mCherry-tagged wild-type 4E-BP1 or 4E-BP1(118+A) were expressed and purified from 293 cells, incubated with increasing amounts of recombinant PP1-Neurabin. Phosphorylation of the indicated sites was analysed by immunoblotting. B . Quantification of A . C . Left, sequence alignment of potential Neurabin/Spinophilin PDZ domain ligands. Grey shading, hydrophobic residues; pink, acidic residues; cyan, basic residues; orange, hydrophilic residues. Underlining shows sequences N-terminally linked to 6-carboxyfluorescein (FAM) for use in fluorescence polarisation (FP) assay. Right, binding affinities for the Neurabin and Spinophilin PDZ domains as determined in the FP assay. D . FP assay. FAM-labelled peptides (see C) were titrated with increasing concentrations of recombinant Neurabin PDZ domain and affinity estimated from change in fluorescence anisotropy. For Spinophilin data see .

Article Snippet: His-tagged Neurabin and Spinophilin PDZ domains were produced in BL21 (DE3) E. coli cells (Invitrogen).

Techniques: Purification, Incubation, Recombinant, Western Blot, Sequencing, Fluorescence, FP Assay, Binding Assay

Journal: bioRxiv

Article Title: PDZ-directed substrate recruitment is the primary determinant of specific 4E-BP1 dephosphorylation by PP1-Neurabin

doi: 10.1101/2024.09.23.614477

Figure Lengend Snippet: A. Immunoblotting analysis of wildtype mCherry-4E-BP1 or mutants either lacking the 6 C-terminal residues (ΔCter), or containing an additional C-terminal alanine (118+A) upon expression in 293 cells with or without PP1-Neurabin expression as indicated. B. Left, sequence alignment of potential Neurabin/Spinophilin PDZ domain ligands. Grey shading, hydrophobic residues; pink, acidic residues; cyan, basic residues; orange, hydrophilic residues. Underlining shows sequences N-terminally linked to 6-carboxyfluorescein (FAM) for use in fluorescence polarisation (FP) assay. FAM-labelled peptides were titrated with increasing concentrations of recombinant Spinophilin PDZ domain and affinity estimated from change in fluorescence anisotropy (for summary see ). C. Immunoblotting analysis of S6K phosphorylation 293 Flp-In T-Rex cells upon expression of PP1-Neurabin or empty vector.

Article Snippet: His-tagged Neurabin and Spinophilin PDZ domains were produced in BL21 (DE3) E. coli cells (Invitrogen).

Techniques: Western Blot, Expressing, Sequencing, Fluorescence, FP Assay, Recombinant, Plasmid Preparation

Journal: bioRxiv

Article Title: PDZ-directed substrate recruitment is the primary determinant of specific 4E-BP1 dephosphorylation by PP1-Neurabin

doi: 10.1101/2024.09.23.614477

Figure Lengend Snippet: A. Top, synthetic substrate peptides contain either the 4E-BP1 T70 or IRSp53 S455 phosphorylation sites, joined by a GSG linker to the Neurabin PDZ-binding C-terminal sequences. PBM, PDZ-binding motif. Below, sequences of the different peptides analysed; highlights indicate the dephosphorylation site (yellow), the +4/+6 region (orange), and the PDZ-binding sequence (cyan), with alanine and other substitutions indicated in red. Right, K M and catalytic efficiencies; for catalytic efficiency quantification see . B-E . Peptides were treated with recombinant PP1-Neurabin, PP1-Phactr1 or PP1 in the presence of the phosphate sensor, and K M and catalytic efficiencies determined. Panels show relative catalytic efficiencies as determined from data displayed in . B. Comparison of Neurabin-PP1 and Phactr1-PP1 substrates 4E-BP1 and IRSp53. C. Role of the +4/+6 region in 4E-BP1 substrate recognition. D. Role of the +5 residue in IRSp53 substrate recognition. E. Role of 4E-BP1 +1/+2 residues.

Article Snippet: His-tagged Neurabin and Spinophilin PDZ domains were produced in BL21 (DE3) E. coli cells (Invitrogen).

Techniques: Binding Assay, De-Phosphorylation Assay, Sequencing, Recombinant, Comparison, Residue

Journal: bioRxiv

Article Title: PDZ-directed substrate recruitment is the primary determinant of specific 4E-BP1 dephosphorylation by PP1-Neurabin

doi: 10.1101/2024.09.23.614477

Figure Lengend Snippet: A. Catalytic efficiencies for the various peptide dephosphorylation reactions by PP1-Neurabin, PP1-Phactr1 and PP1 are shown. B-E . Dephosphorylation reaction rates plotted against substrate concentration for different sets of phosphopeptides with PP1-Neurabin, PP1-Phactr1 or PP1.

Article Snippet: His-tagged Neurabin and Spinophilin PDZ domains were produced in BL21 (DE3) E. coli cells (Invitrogen).

Techniques: De-Phosphorylation Assay, Concentration Assay

Journal: bioRxiv

Article Title: PDZ-directed substrate recruitment is the primary determinant of specific 4E-BP1 dephosphorylation by PP1-Neurabin

doi: 10.1101/2024.09.23.614477

Figure Lengend Snippet: A. Schematic of the PP1-4E-BP1 chimera and of Neurabin PP1-interacting and PDZ domain sequences. B. Crystal structure of the PP1-4E-BP1/Neurabin complex. PP1 in white surface representation, Neurabin in lilac surface representation, 4E-BP1 in blue stick representation, with unresolved sequences indicated by dashed line. PP1 active site presumptive Mn 2+ ions in purple. C. Comparison of PP1-4E-BP1/Neurabin complex structure with previous Neurabin/PP1 holophosphatase structure . PP1 in white surface representation, Neurabin in ribbon representation (lilac, PP1-4E-BP1/Neurabin; red, Neurabin/PP1). 4E-BP1 in blue stick representation, unresolved sequences not shown. Structures are superimposed on PP1 residues 7-298 (rmsd=0.21 Å, 277 alpha carbons). D. Close-up view of interactions between 4E-BP1 C-terminal sequences (blue sticks) with the Neurabin PDZ domain (lilac cartoons). E. AlphaFold3 model of the phosphorylated PP1-4E-BP1 chimera / Neurabin(423-593) interaction. A close-up view of predicted interaction of pT70 with the PP1 catalytic site is shown. For PAE and pLDDT plots, see . PP1 and Neurabin are shown respectively in white and lilac surface representation, with PP1 active site Mn 2+ ions in purple. 4E-BP1 sequences are in stick representation, colour-coded according to the AlphaFold3 pLDDT score (inset). See also . F. AlphaFold3 modelling of the Neurabin(423-593)/PP1 - 5x phospho-4E-BP1 interaction. PP1 and Neurabin are shown respectively in white and lilac surface representation, with PP1 active site Mn 2+ ions in purple. 4E-BP1 sequences are in ribbon and stick representation, colour-coded according to the AlphaFold3 pLDDT score (inset), with the 4E-BP1 phosphorylations at T37, T46, S65, T70 and S101 shown in spheres. For PAE and pLDDT plots, see .

Article Snippet: His-tagged Neurabin and Spinophilin PDZ domains were produced in BL21 (DE3) E. coli cells (Invitrogen).

Techniques: Comparison

Journal: bioRxiv

Article Title: PDZ-directed substrate recruitment is the primary determinant of specific 4E-BP1 dephosphorylation by PP1-Neurabin

doi: 10.1101/2024.09.23.614477

Figure Lengend Snippet: A, B. AlphaFold3 models of the phosphorylated (A) and unphosphorylated (B) PP1-4E-BP1 chimera / Neurabin(423-593) interaction. Left, PAE plots; right, pLDDT plots, with confidence boundaries indicated by dashed lines (>90%, very high (side-chains); 70-90%, high (main-chain); 50-70%, low). C, D. AlphaFold3 models of the phosphorylated (C) and unphosphorylated (D) PP1-4E-BP1 chimera / Neurabin(423-593) interaction. PP1 and Neurabin are shown respectively in white and lilac surface representation with PP1 active site Mn 2+ ions in purple. 4E-BP1 sequences are in stick representation, colour coded according to the AlphaFold3 pLDDT score (inset), with pT70 and T70 in space-fill; linker residues are in black. Below are shown close-up views of predicted interactions with the PP1 catalytic site. For PAE and pLDDT plots, see A, B . E. Comparison of crystal structure and AlphaFold3 model of 4E-BP1/PDZ interactions in phosphorylated and unphosphorylated PP1-4E-BP1 chimera / Neurabin(423-593) interaction. Predicted structures are oriented by superposition of the PDZ domain, shown in lilac ribbon representation. 4E-BP1 sequences are in stick representation, colour coded according to the AlphaFold3 pLDDT score (inset) F. AlphaFold3 modelling of the Neurabin(423-593)/PP1 - 5x phospho-4E-BP1 interaction. Left, PAE plots; right, pLDDT plots, with confidence boundaries indicated by dashed lines (>90%, very high (side-chains); 70-90%, high (main-chain); 50-70%, low).

Article Snippet: His-tagged Neurabin and Spinophilin PDZ domains were produced in BL21 (DE3) E. coli cells (Invitrogen).

Techniques: Comparison