Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: a Schematic of CRISPR/Cas9 genetic screen. MutuDCs were lentivirally transduced with the GeCKOv2 library A and puromycin-resistant cells were stained for surface MHC II. The 5% highest MHC II expressing cells were sorted by flow cytometry, genomic DNA extracted, and amplified. gRNAs were quantified by Illumina NextSeq sequencing and analyzed with edgeR. Volcano plots show detected gRNAs enriched in sorted cells with increased MHC II expression compared to the unsorted library. P values were assessed using a negative binomial generalized linear model with a two-sided likelihood ratio test, with adjustment for multiple testing using the Benjamini–Hochberg false discovery rate (FDR) method. Guide RNAs with an absolute log2 fold change >1 and FDR of <0.01 were considered significantly enriched (orange dots), with genes hit by at least two gRNAs highlighted (large red circles). b Surface expression of MHC II, CD86, or MHC I were analyzed by flow cytometry for MutuDCs lacking B2m (pool), Marchf1, or Ubl3 (single-cell clones), compared with control cells expressing non-targeting hBim gRNA. Top: representative histograms, with gray solid histograms: control MutuDCs expressing non-targeting hBim gRNA, black lined histograms: CRISPR/Cas9 modified MutuDC lines as indicated. Bottom: quantification of flow cytometry analysis from three experiments. Bars indicate mean+SD of geometric mean fluorescence intensity (gMFI), relative to untransfected cells. Statistical analysis was performed using a one-way ANOVA and Bonferroni’s multiple comparisons test, comparing each sample to hBim controls, p values shown above bars, ns (not significant) p > 0.05.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: CRISPR, Transduction, Staining, Expressing, Flow Cytometry, Amplification, Sequencing, Clone Assay, Control, Modification, Fluorescence

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: Enriched gRNAs in the MHC II high population.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: a Analysis of Ii degradation and MHC II peptide loading in control MutuDCs expressing non-targeting hBim gRNA or Δ Ubl3 MutuDCs. Cells were metabolically labeled for 30 min, washed, and cultured in a growth medium for the indicated time points before lysis. MHC II molecules were immunoprecipitated with JV1 rabbit antisera, or normal rabbit serum (NRS), and protein G-Sepharose. Samples were loaded before (NB) or after boiling (B) at 95 °C ( b ), subjected to denaturing SDS-PAGE, transferred onto PVDF membranes, and exposed to a storage phosphor screen. Sizes corresponding to free MHC II α and β, full-length invariant chain (Ii), the Ii spliced variant Ii p41 , the degradation intermediate Ii p10 , and SDS-stable complexes αβIi p10 , and αβ-peptide (pep), are indicated on the right. Data from one experiment. b Δ Ubl3 MutuDCs or Δ Ubl3 MutuDCs expressing Myc-UBL3 were lysed and post-nuclear supernatants (PNS) were subjected to immunoprecipiation (IP) with anti-Myc-Tag agarose (clone 9E10) or anti-MHC II antibody (M5/114) crosslinked to protein G-sepharose. Per lane, samples equivalent to 5 × 10 6 cells (Myc IP), 2.5 × 10 6 cells (MHC II IP), or 400,000 cells (PNS) were analyzed by non-denaturing SDS-PAGE and western blotting using antibodies against UBL3 (ab113820), MHC II β chain (JV2), Myc (71D10), and ubiquitin (P4D1). Shown are representative blots and quantification of relative ubiquitination, with bars displaying mean + 95% confidence intervals for four experiments.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Control, Expressing, Metabolic Labelling, Labeling, Cell Culture, Lysis, Immunoprecipitation, SDS Page, Variant Assay, Western Blot, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: a – d Clonal Δ Ubl3 MutuDCs were retrovirally transduced with Myc-tagged wild type UBL3 (“+Myc-UBL3 WT ”), or UBL3 C114S mutant (“+Myc-UBL3 C114S ”). a Post-nuclear supernatants of 2.5 × 10 5 cells were analyzed by non-denaturing SDS-PAGE and western blotting using anti-actin (20-33) anti-Myc (71D10) and anti-Ubl3 (ab113820) antibodies. Representative blot is shown for two independent experiments with similar results. b Proximity ligation assay (PLA). Δ Ubl3 MutuDC expressing Myc-UBL3 WT or Myc-UBL3 C114S were stained with plasma membrane (PM) CytoPainter. After washing, fixing, and permeabilization, cells were stained with Hoechst 33342, rabbit anti-MHC II (JV2) antiserum, and mouse anti-Myc mAb (9E10) and subjected to PLA. Scale bar = 10 µm. PLA spots indicate spatial proximity between MHC II and UBL3 and were enumerated within plasma membrane boundaries (violin plots). One experiment, P value as indicated, unpaired Welch’s t test (two-sided). c Immunofluorescence microscopy. Cells were fixed, permeabilized, and stained with biotinylated rat anti-MHC II mAb (M5/114), mouse anti-Myc mAb (9E10), streptavidin Alexa Fluor 647, donkey anti-mouse Alexa Fluor 594, and 0.5 μg/ml DAPI. Scale bar = 10 µm. Representative image shown for two independent experiments with similar results. d Flow cytometry analysis of MHC II and CD86. Left: Dot plots show MHC II ( x axis) and GFP ( y axis). Center: histograms show corresponding expression levels of MHC II or CD86, with gray solid histogram: control MutuDCs expressing non-targeting hBim gRNA; black line: Δ Ubl3 MutuDCs, red line: Δ Ubl3 MutuDCs expressing Myc-UBL3 WT , blue line: Δ Ubl3 MutuDCs expressing Myc-UBL3 C114S . Right: quantification of gMFI from three experiments, normalized to highest gMFI of each experiment, with bars mean ± SD and symbols. **** P < 0.0001, one-way ANOVA with Bonferroni’s test.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Transduction, Mutagenesis, SDS Page, Western Blot, Proximity Ligation Assay, Expressing, Staining, Clinical Proteomics, Membrane, Immunofluorescence, Microscopy, Flow Cytometry, Control

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: a Ubl3 −/− mice. b Spleen cDC surface MHC II and CD86 by flow cytometry. Representative histograms and graphs showing gMFI relative to highest signal, bars mean ± SD, symbols individual mice ( n = 9, three independent experiments). One-way ANOVA with Bonferroni’s test. c Quantitative real-time PCR of H2-Ab1 and Marchf1 mRNA in spleen cDCs. Bars mean ± SD relative to WT, n = 2 samples, two experiments. d Spleen cDCs labeled with FIP-conjugated mAb for MHC II, CD86, or MHC I were incubated for 30 mins at 37°C. Fluorescence was quenched (+Q) and internalization was calculated as described in Methods. CD86 internalization not shown for cDC2 (low expression). Data representative of two independent experiments with similar results (MHC II) or pooled from two (CD86) or three (MHC I) experiments performed in triplicate, mean ± SD, one-way ANOVA with Bonferroni’s test. e MHC II was immunoprecipitated (IP) from spleen cDCs using anti-MHC II antibody, analyzed by non-denaturing SDS-PAGE, and immunoblotted (IB) for ubiquitin and MHC II β-chain. Left: representative blot. Right: MHC II ubiquitination relative to WT, bars mean + 95% confidence interval for three biological replicates. f – i Flow cytometry of surface MHC II and CD86 of f professional antigen-presenting cells (thymus: n = 14, four experiments (MHC II), n = 5, one experiment (CD86); blood: n = 7 for WT, 10 for Ubl3 −/− , 6 for Marchf1 −/− , two experiments; peritoneal cavity: n = 5 for WT, 4 for Ubl3 −/− , one experiment), g spleen myeloid cells ( n = 5 for WT, 4 for Ubl3 −/− , one experiment), h TEC ( n = 11 for WT, 12 for Ubl3 −/− , 12 for Marchf8 −/− , four experiments), and i lung epithelial cells ( n = 5, one experiment). Graphs show gMFI relative to highest gMFI, bars mean ± SD, symbols represent individual mice. One-way ANOVA with Bonferroni’s test, unpaired t test (two-sided) with Holm–Sidak correction. SPM small peritoneal macrophages, LPM large peritoneal macrophages, Mφ macrophages, mo monocytes, AECII type II lung alveolar epithelial cells, BEC bronchial epithelial cells, EC lung endothelial cells. p values above bars, with **** p < 0.0001, ns (not significant) p > 0.05.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Flow Cytometry, Real-time Polymerase Chain Reaction, Labeling, Incubation, Fluorescence, Expressing, Immunoprecipitation, SDS Page, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: a Left: spleen cDC1 expression of CLEC9A from wild type (WT) or Ubl3 −/− mice by flow cytometry, n = 11 mice examined over three experiments, bars mean ± SD, symbols represent individual mice, ns (not significant) p > 0.05, unpaired t test (two-sided). Right: Spleen cDCs from WT or Ubl3 −/− mice were labeled with FIP-conjugated anti-CLEC9A mAb. After 30 mins at 37°C, quencher (Q) was added and percentage internalization was calculated as described in Methods. Histograms show representative CLEC9A-FIP signal. Right: quantification of internalization from one experiment performed in triplicate, with bars mean ± SD, ns (not significant) p > 0.05, unpaired t test (two-sided). b In vivo antigen presentation assay. Purified CellTrace Violet (CTV)-labeled OT-I and OT-II cells were adoptively transferred into WT and Ubl3 −/− mice. 24 hours later, mice were injected with 0.2 µg anti-CLEC9A mAb-targeted OVA and spleens harvested after 64 hours. Antigen presentation capacity was assessed by flow cytometric analysis of CTV-dilution by OT-I and OT-II proliferation. Data from n = 9 (OT-I) and n = 8 for WT, 7 for Ubl3 −/− (OT-II) mice, two experiments, bars mean ± SD, symbols represent individual mice, ns (not significant) p > 0.05, unpaired t test (two-sided). c Quantification of spleen cDC1 and cDC2 from WT or Ubl3 −/− mice. Data from n = 9 mice, three independent experiments, bars mean ± SD, symbols representing individual mice, ns (not significant) p > 0.05, unpaired t test (two-sided). d Left: C3 expression on spleen cDCs from WT, Ubl3 −/− or Marchf1 −/− mice by flow cytometry. Data from n = 5 from one experiment, bars mean ± SD, symbols represent individual mice. Two-way ANOVA with Bonferroni’s test. Right: Quantification of trogocytic B cells gated on B220 + CD19 + cells in Nycodenz-enriched splenocytes from WT, Ubl3 −/− or Marchf1 −/− mice. Representative dot plots and data from one experiment with n = 5, bars mean ± SD, and symbols representing individual mice. Significant p values shown above bars, with **** p < 0.0001, ns (not significant) p > 0.05, one-way ANOVA with Bonferroni’s test.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Expressing, Flow Cytometry, Labeling, In Vivo, Immunopeptidomics, Purification, Injection

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: a Ex vivo antigen presentation assay. WT and Ubl3 −/− mice were injected with 1 µg of anti-CLEC9A-OVA mAb. Indicated numbers of spleen cDC1 and cDC2 were purified and co-cultured with OT-I or OT-II cells, and divided OT-I or OT-II cells enumerated by flow cytometry. Data with n = 3, representative of two experiments, mean ± SD, **** p < 0.0001, two-way ANOVA with Bonferroni’s test. b Flow cytometry analysis of relative cell surface marker expression of spleen cDC1 or cDC2 isolated from WT and Ubl3 −/− mice, with n = 8 mice, two independent experiments, symbols represent individual mice, bars mean ± SD, **** p < 0.0001, unpaired t test (two-sided) with Holm–Sidak adjustment. c OVA-Cy5 uptake assay. Purified spleen cDC1 or cDC2 from WT or Ubl3 −/− mice were incubated with 50 µg/ml OVA-Cy5 for the indicated times, and washed before flow cytometry analysis. Graphs show mean ± SD, with data from one experiment performed in triplicate, two-way ANOVA with Bonferroni’s test. d Proteolysis assay. Purified spleen cDC1 or cDC2 from WT or Ubl3 −/− mice were pulsed with DQ-OVA for 15 min, washed twice and DQ-OVA signal was measured by flow cytometry at different chase time points. Graphs show mean ± SD, with data pooled from two independent experiments performed in triplicate, two-way ANOVA with Bonferroni’s test. e Quantification of cathepsin (cat) activity in spleen cDCs. Top: representative gel indicating active cathepsin X/B/S/L, and actin immunoblot. Bottom: relative protease activity normalized to actin, with bars showing mean ± SEM of one experiment with three biological replicates. Four spleens were pooled for each biological replicate, ns not significant, unpaired t test (two-sided). f Cytokine expression of purified Ubl3 −/− and wild-type spleen cDC1 and cDC2 stimulated with CpG, IFN-γ, and GM-CSF. Bars display mean ± SD of cells stimulated in duplicate, using purified cDCs pooled from eight mice, representative of two independent experiments.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Ex Vivo, Immunopeptidomics, Injection, Purification, Cell Culture, Flow Cytometry, Marker, Expressing, Isolation, Incubation, Proteolysis Assay, Activity Assay, Western Blot

Journal: Nature Communications

Article Title: Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86

doi: 10.1038/s41467-022-29524-w

Figure Lengend Snippet: Human CD14 + monocytes were infected with non-targeting control shRNA or shRNA against UBL3 (sh1 UBL3 or sh2 UBL3 ) and analyzed after five days in culture. Top left: Cell lysates were probed with antibodies against UBL3 (LS-C661402) and actin, and quantification of gene silencing was determined by UBL3 signal intensity. Immunoblot depicts a scenario of approximately 95% UBL3 knockdown. Data from n = 6 donor samples examined in a single experiment, with symbols connected with a line representing individual donors. Top right: Surface MHC II, CD86, and MHC I on monocyte-derived DCs (CD16 − CD1a + ) and monocyte-derived macrophages (CD16 + CD1a − ) were analyzed by flow cytometry. Histograms are representative of analysis from one donor. Bottom: Graphs showing data from n = 6 (DCs) or n = 4 (macrophages) donor samples examined in a single experiment, with symbols connected with a line representing individual donors, p values shown above points, ns not significant, repeated measures one-way ANOVA.

Article Snippet: To generate cell lines expressing wild type or mutant UBL3 constructs, Myc-tagged wild type or C114S mutant Ubl3 cDNA was purchased (Biomatik) and cloned into p-MSCV-IRES-eGFP II (Addgene plasmid #52107), which was a gift from Dario Vignali.

Techniques: Infection, Control, shRNA, Western Blot, Knockdown, Derivative Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a ) Schematic representation of the domain arrangement and boundaries of the full-length TGFβIp and 4 th _FAS1 domains of the wild-type, non-amyloidogenic (R555W) and amyloidogenic (H572R) mutants used in the study. ( b ) SDS-PAGE gel showing the purified fractions of the 4 th _FAS1 domains of the WT, R555W and H572R mutants. ( c–e ) Far UV CD spectra of the 4 th _FAS1 domains of WT ( d ), R555W ( e ) and H572R ( f ) incubated for 16 hours at acidic pH conditions (pH 3, 4.5, 5.5 and 7). The R555W mutant displayed clear changes in the CD spectra at 222 nm and 207 nm with decrease in pH ( f ). The CD intensity at 222 nm decreased with decrease in pH confirming the unfolding of the secondary structures. The CD spectra for the WT ( c ) and H572R ( e ) mutant remained almost unchanged. Plotting the intensities at 222 nm at varying pH ( g ) showed that the non-amyloidogenic phenotype, R555W, was more sensitive to pH and the amyloidogenic phenotype, H572R, remained more stable to pH changes at room temperature.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: SDS Page, Purification, Circular Dichroism, Incubation, Mutagenesis

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: Effects of mutation on the charge and hydrophobicity.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: Mutagenesis

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a , b ) Far UV CD spectra of the 4 th _FAS1 domain of WT at pH 7.0 and pH 8.0 before heating (black), after heating to 70 °C (red) and cooling back to 20 °C (blue). ( c,d ) Far UV CD spectra of the 4 th _FAS1 domains of R555W at pH 7 ( c ) and pH 8 ( d ) before heating (black) and after heating to 70 °C (red) and cooling back to 20 °C (blue). ( e , f ) Far UV CD spectra of the 4 th _FAS1 domains of H572R a pH 7.0 ( e ) and pH 8.0 ( f ) before heating (black) and after heating to 70 °C (red) and cooling back to 20 °C (blue). The WT and the mutants did not display any significant changes in structure at pH 7.0 and pH 8.0.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: Circular Dichroism

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a – c ) Far UV CD spectra of the 4 th _FAS1 domain of WT at pH 3.0 ( a ), pH 4.5 ( b ) and pH 5.5 ( c ) before heating (black) and after heating to 70 °C (red) and cooling back to 20 °C (blue). ( d – f ) Far UV CD spectra of the 4 th _FAS1 domain of R555W at pH 3 ( d ), pH 4.5 ( e ) and pH 5.5 ( f ) before heating (black) and after heating to 70 °C (red) and cooling back to 20 °C (blue). ( g – i ) Far UV CD spectra of the 4 th _FAS1 domain of H572R at pH 3.0 ( g ), pH 4.5 ( h ) and pH 5.5 ( i ) before heating (black) and after heating to 70 °C (red) and cooling back to 20 °C (blue). While the WT did not show any changes in structure, both the mutants displayed a very clear transition to β-sheet under acidic conditions.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: Circular Dichroism

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a – e ) Variable temperature CD curves at 222 nm of WT (black), R555W (red) and H572R (blue) proteins heated from 20 °C to 70 °C at various pH (3.0 [ a ], 4.5 [ b ], 5.5 [ c ], 7.0 [ d ] and 8.0 [ e ]) and the CD intensities at 222 nm were plotted as a function of temperature. The baseline subtracted curves of the WT (black), R555W (red) and H572R (blue) proteins show that while there was no transition observed in the WT in all the conditions as observed from the unchanged straight line in black, little or no changes were seen in pH 7 and pH 8 for the mutants. However, clear transitions to β-sheet were observed at acidic pH (pH 3, pH 4.5 and pH 5.5) for both the mutants. In all cases, we observe transition (Tt) is higher for R555W compared to H572R. A clear shift in their thermal denaturation curves between the mutants at acidic pH (pH 3.0, 4.5 and 5.5) is observed. A difference in T t of 5–12 °C is observed at various pH conditions.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques:

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a – b ) Transmission Electron Microscopy. TEM images of the β-oligomers of the 4 th _FAS1 domains of R555W ( a ) and H572R ( b ) mutants were acquired with a JEOL JEM-1010 transmission electron microscope using Digital Micrograph™ 1.81.78 for GMS 1.8.0. The β-oligomers of the amyloidogenic phenotype were larger measuring between 10–40 nm, mean size ~19.1 nm ± 4.9 nm ( a ) compared to the non-amyloidogenic β-oligomers that measured 4–8 nm, with a mean size ~5.1 nm ± 1.79 nm ( b ). Inset figures – particle size distribution of the β-oligomers. While the amyloidogenic β-oligomers were larger and displayed rugged edges and varying diameters, the non-amyloidogenic β-oligomers were smaller in size with smoother edges and were more homogenous. ( c ) ThT assay. Emission fluorescence intensities at 485 nm recorded after incubating the WT TGFβIp and the β-oligomers of R555W and H572R with ThT dye. An amyloid forming peptide (611-633aa - pN622K) of TGFβIp was used as the positive control. The β-oligomers of the amyloidogenic mutant H572R showed significant fluorescence intensity (**P < 0.01) almost 3 times more fluorescence compared to the non-amyloidogenic R555W. ( d ) DLS. %intensity plots plotted against the apparent hydrodynamic radii (R H ). The R H values calculated from the distribution curves for R555W (~39.58 nm| pH 3.0 , ~51.9 nm| pH 4.5 and ~68.2 nm| pH 5.5 ) and H572R (~89.95 nm| pH 3.0 , ~69 nm| pH 4.5 and ~155 nm| pH 5.5 ) show that H572R b-oligomers are larger in size and slightly more heterogeneous. ( e , f ) Mass Spectrometric analyses - Identification of the aggregation hotspots in the mutants and β-oligomers. ( d ) Peptide map generated following the insilico trypsin digestion of TGFβIp displaying a series of peptides. ( e ) The β-oligomers formed from the mutant 4 th _FAS1 domains were digested with trypsin and the resulting fragments were analyzed by LC–MS/MS. The regions inaccessible for trypsin digestion have been underlined in red. It is clearly seen that the non-amyloidogenic R555W shows more regions inaccessible for trypsin digestion.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: Transmission Assay, Electron Microscopy, Microscopy, ThT Assay, Fluorescence, Positive Control, Mutagenesis, Generated, Liquid Chromatography with Mass Spectroscopy

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a – d ) Thermal stability. Variable temperature CD values at 222 nm of the 4 th _FAS1 domains of R555W ( a ) and H572R ( c ) in buffer at pH 5.5 when heated from 20 °C to 70 °C. Far UV CD spectra of the 4 th _FAS1 domains of R555W ( b ) and H572R ( d ) after heating (red) to 70 °C and cooling (blue) back to 20 °C. ( e , f ) Stability at physiological pH. Far UV CD spectra of the 4 th _FAS1 domains (black) of R555W ( e ) and H572R ( f ) at pH 5.5 under native conditions (black), after heating to 70 °C (red) and after reconstituting in buffer at pH 7.0 (blue) after incubation at room temperature for 4 weeks.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: Circular Dichroism, Incubation

Journal: Scientific Reports

Article Title: pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

doi: 10.1038/srep23836

Figure Lengend Snippet: ( a ) The growth and proliferation of the pHCSF cells following the treatment with the β-oligomers and the mutant 4 th _FAS1 domains monitored for 48 hours in an xCELLigence system. ( b ) The cell survival after treatment for 24 hours plotted as area under the curve (AUC). While the WT and R555W show low cytotoxicity, almost similar to the media control, the amyloidogenic H572R mutant was cytotoxic compared to the control (**P < 0.01). Interestingly, the β-oligomers of both the amyloidogenic and non-amyloidogenic mutants showed high cytotoxicity (**P < 0.01) with the approximately 7 times more for the non-amyloidogenic mutant and 12 times more for the amyloidogenic mutant. To obtain detailed information on the cytotoxicity of the soluble and insoluble fractions, β-oligomers of the two mutants were prepared at various acidic pH conditions (pH 3.0, pH 4.5, pH 5.5) and their cytotoxicity were examined on pHCSFs from 3 different donors (n = 3) using xCELLigence ( c ) and MTT assays ( d ). ( c ) xCELLigence assay. The WT shows low cytotoxicity, almost similar to the control. The soluble β-oligomers of both the mutants showed high cytotoxicity (**P < 0.01) compared to the controls. The insoluble β-oligomers were relatively less cytotoxic compared to the soluble β-oligomers. ( d ) MTT assay. The cytotoxicity of the β-oligomers was also tested using an MTT assay. Similar to xCELLigence, we could see that soluble β-oligomers derived from both the mutants displayed potent cytotoxic effect (**P < 0.01) compared to the controls and insoluble aggregates. Though the insoluble oligomers were relatively less cytotoxic than the soluble oligomers, they displayed cytotoxicity compared to the controls.

Article Snippet: The cDNA constructs of the 4 th _FAS1 domains of the WT TGFβIp, the mutants R555W and H572R were bought from Genscript (Piscataway, NJ) in pUC57 vectors.

Techniques: Mutagenesis, Control, MTT Assay, Derivative Assay