protein crystal structures  (Databank Inc)

Journal: Molecules

Article Title: Recent Applications of In Silico Approaches for Studying Receptor Mutations Associated with Human Pathologies

doi: 10.3390/molecules29225349

Figure Lengend Snippet: Summary of molecular modeling techniques and software employed in reviewed studies.

Article Snippet: Molecular docking simulations of the selected Azalamellarin N derivatives were performed after retrieving the crystal structure of the EGFR protein (PDB code: 4I22) using AutoDock, after the preparation of the ligand and EGFR pdb files using the AutoDock Tools program [ ].

Techniques: Software

Journal: Nature Communications

Article Title: QSOX2 Deficiency-induced short stature, gastrointestinal dysmotility and immune dysfunction

doi: 10.1038/s41467-024-52587-w

Figure Lengend Snippet: A Inheritance of QSOX2 variants delineated across two generations for each respective kindred. B Height, weight, and BMI centile growth charts (2–9 yrs) of probands 1 and 2, generated by Growth XP (PC PAL version 2.8). GH indicates when recombinant growth hormone therapy (0.025 mg/kg/day) was commenced. Most recent measurements suggest a modest improvement in height trajectories. C Colonic marker transit studies for probands 1 and 2 were performed after bowel dis-impaction. Patients ingested 10 differently shaped markers for three consecutive days. Plain abdominal X-rays were performed on days 4 and 6 post-first marker ingestion. Colonic marker transit study of proband 1 was indicative of rectal outlet dysfunction. D Abdominal X-rays for colonic marker transit study in proband 2 indicate a mixed type of rectal outlet dysfunction and slow colonic transit (retention of innumerable markers).

Article Snippet: Protein 3D modelling of the Alpha Fold Protein Structure Database QSOX2 crystal structure Q6ZRP7 was performed using the tool PyMOL (Schrodinger, LLC.

Techniques: Generated, Recombinant, Marker

Journal: Nature Communications

Article Title: QSOX2 Deficiency-induced short stature, gastrointestinal dysmotility and immune dysfunction

doi: 10.1038/s41467-024-52587-w

Figure Lengend Snippet: The clinical and biochemical profiles of the probands harbouring bi-allelic QSOX2 variants

Article Snippet: Protein 3D modelling of the Alpha Fold Protein Structure Database QSOX2 crystal structure Q6ZRP7 was performed using the tool PyMOL (Schrodinger, LLC.

Techniques: Reflux

Journal: Nature Communications

Article Title: QSOX2 Deficiency-induced short stature, gastrointestinal dysmotility and immune dysfunction

doi: 10.1038/s41467-024-52587-w

Figure Lengend Snippet: A Schematic of QSOX2 protein with key domains, including the relative location of QSOX2 variants identified in probands P1-P4. B Immunoblotting of FLAG-tagged QSOX2 cDNA constructs showed that expression of both variants was reduced compared to wild-type (WT)-QSOX2, with expected truncated protein due to early protein termination observed for p.V325Wfs*26. C Immunofluorescent microscopy demonstrated a reduction in QSOX2 peri-nuclear expression for both variants when compared to WT-QSOX2. D Immunoblot analyses of transfected HEK 293-hGHR cell lysates demonstrated that p-STAT5 was markedly enhanced in the presence of both variants following GH stimulation. E Nuclear and cytoplasmic fractions of transfected HEK 293-hGHR cells demonstrated a reduction of p-STAT5 in nuclear fractions of both variants with concomitant cytoplasmic abundance of p-STAT5 when compared to wild type. Nuclear levels of p-STAT3 and p-STAT1 were indistinguishable between both variants and the wild type. F Immunofluorescent microscopic analysis of GH-stimulated transfected HEK 293-hGHR cells showed nuclear translocation impairment of p-STAT5 for both QSOX2 variants but not with WT-QSOX2. G Co-immunoprecipitation and immunoblot analysis of WT-QSOX2-STAT5B interactions showed a direct protein-protein interaction between unstimulated WT-QSOX2 and STAT5B. H NanoBit complementation assays showed that the robust interaction seen between QSOX2-WT and STAT5B-WT was attenuated for both p.T352M ( p < 0.0001) and p.V325Wfs*26 ( p < 0.0001). Ordinary one-way-ANOVA was used for statistical analysis with multiple testing corrections performed using Sidak’s test. I NanoBit complementation assays showed a significant reduction in interaction affinity for the pathogenic variant p.Q177P known to abrogate nuclear STAT5B import ( p = 0.0004), supporting the importance of QSOX2 for STAT5B nuclear localisation. Ordinary one-way-ANOVA was used for statistical analysis with multiple testing corrections performed using Dunnett’s test. J In vitro STAT5B transcriptional activities were evaluated by dual luciferase growth hormone response element (GHRE) reporter assay. The 4-fold increase in GH-induced luciferase activities in the presence of WT-QSOX2 (WT), was significantly blunted in the presence of QSOX2 variants (“T352M”, p = 0.0001; “V325Wfs*26”, p = 0.0001). Ordinary one-way-ANOVA was used for statistical analysis with multiple testing corrections performed using Sidak’s test. Source data are provided as a Source Data file. Data are presented as the mean ± SD of three repeated measurements (3 independent replicates).

Article Snippet: Protein 3D modelling of the Alpha Fold Protein Structure Database QSOX2 crystal structure Q6ZRP7 was performed using the tool PyMOL (Schrodinger, LLC.

Techniques: Western Blot, Construct, Expressing, Microscopy, Transfection, Translocation Assay, Immunoprecipitation, Variant Assay, In Vitro, Luciferase, Reporter Assay

Journal: Nature Communications

Article Title: QSOX2 Deficiency-induced short stature, gastrointestinal dysmotility and immune dysfunction

doi: 10.1038/s41467-024-52587-w

Figure Lengend Snippet: A Expression of p.F474del was reduced compared to wild-type (WT)-QSOX2. Molecular weights (MW), in kiloDaltons, indicated left of the immunoblot. B Immunofluorescent microscopy demonstrated a reduction in QSOX2 peri-nuclear expression when compared to WT-QSOX2. C Immunoblot analyses of transfected HEK 293-hGHR cell lysates showed that in the presence of p.F474del, STAT5 was robustly phosphorylated following GH stimulation. D Immunofluorescent microscopic analysis of GH-stimulated transfected HEK 293-hGHR cells revealed nuclear translocation impairment of p-STAT5 and perinuclear accumulation for p.F474del when compared to WT-QSOX2. E Nuclear and cytoplasmic fractionation of transfected HEK 293-hGHR cells were probed by immunoblotting for p-STAT5, nuclear marker HDAC1, and cytoplasmic GAPDH. A reduction of p-STAT5 in p.F474del nuclear fractions was noted when compared to wild type. F NanoBit complementation assays demonstrated blunted interaction between unstimulated STAT5B and QSOX2 p.F474del ( p = 0.0019). Ordinary one-way-ANOVA was used for statistical analysis with multiple testing corrections performed using Sidak’s test. Source data are provided as a Source Data file. Data are presented as the mean ± SD of three repeated measurements (3 independent replicates).

Article Snippet: Protein 3D modelling of the Alpha Fold Protein Structure Database QSOX2 crystal structure Q6ZRP7 was performed using the tool PyMOL (Schrodinger, LLC.

Techniques: Expressing, Western Blot, Microscopy, Transfection, Translocation Assay, Fractionation, Marker

Journal: Nature Communications

Article Title: QSOX2 Deficiency-induced short stature, gastrointestinal dysmotility and immune dysfunction

doi: 10.1038/s41467-024-52587-w

Figure Lengend Snippet: A Immunoblot analysis of control (C), proband 2 (P2) and parental dermal fibroblasts (M, F) revealed a global reduction in QSOX2 protein in patient-derived fibroblasts. B Robust tyrosine phosphorylation of STAT5 was elicited in patient fibroblasts when compared to control and heterozygote parents. C Immunofluorescent microscopy indicated GH-stimulated p-STAT5 translocated to the nucleus in ( C ), M and F fibroblasts but not in P2 fibroblasts. P2 fibroblasts demonstrated diffused cytoplasmic staining for p-STAT5 with nuclear sparing. D Immunoblot analysis of IGF-1 stimulated (100 ng/ml, 30 min) signalling pathways. IGF-1 activated pAKT, and pERK1/2 was comparable between P2, C and parental fibroblasts. E MitoTracker immunostaining of patient P2 fibroblasts, compared to control fibroblasts, indicate disrupted mitochondrial morphology upon GH, but not IGF-1, stimulation. Alterations in patient mitochondrial morphology seen with GH stimulation were consistent with mitochondrial fragmentation. F Immunofluorescent microscopy of P2 fibroblasts demonstrated an increase in GH-induced phospho-S616-DRP1 when compared to control (C). G Cytoplasmic accumulated p-STAT5B appeared to localise to the mitochondria in P2 fibroblasts co-immunostained for outer mitochondrial membrane marker, Tom20 and p-STAT5B. H Unstimulated and GH stimulated control (C), patient (P2), and parental (M, F) fibroblasts were immunoblot analysed for expression of mitochondrial oxidative phosphorylation complexes I–V. In P2 fibroblasts, a stark reduction in complex profiles was observed upon GH stimulation. IGF-1 stimulation, in contrast, did not alter complex profiles. I Mitochondrial membrane potential measurements of untreated and GH-treated primary fibroblasts with carbonyl cyanide p-triflouromethoxyphenylhydrazone, FCCP (20 μM) added to control fibroblasts as a depolarisation control. Reproducible reduction in mitochondrial membrane potential was detected in GH-treated patient fibroblasts compared to control fibroblasts ( p = 0.0013). FCCP depolarisation control effectively showed reduced mitochondrial membrane potential when compared to GH-treated control fibroblasts ( p = 0.0105). Ordinary one-way-ANOVA was used for statistical analysis with multiple testing corrections performed using Sidak’s test. Source data are provided as a Source Data file. Data are presented as the mean ± SD of three repeated measurements (3 independent replicates).

Article Snippet: Protein 3D modelling of the Alpha Fold Protein Structure Database QSOX2 crystal structure Q6ZRP7 was performed using the tool PyMOL (Schrodinger, LLC.

Techniques: Western Blot, Control, Derivative Assay, Microscopy, Staining, Immunostaining, Membrane, Marker, Expressing