recombinant trim21  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: SPR assay orientations to characterize the interaction between TRIM21 and an antibody. Symmetrical and asymmetrical antibody Fc variants are investigated. In case of an asymmetrical Fc part, one Fc heavy chain contains a AAA mutation (schematically shown by red star), that completely abolishes TRIM21 binding. The used Fc variants and assay setups allow determining how Fc mutations influence the avidity-binding mode and dissecting avidity from affinity. (A) Antibody Fc variants are captured on the biosensor surface via an anti-Fab nanobody (vhh), Fc-only variants are coupled using standard amine coupling chemistry and cytokine Fc-Fusions are captured via anti-PGLALA F(ab’)2 fragment , while TRIM21 PRYSPRY domain is the analyte in solution (see Materials and Methods). Configuration (B) schematically shows the inverse to (A) while the PRYSPRY domain is captured via monovalent streptavidin. (C) To analyze the dimeric TRIM21 engagement of both IgG heavy chains, the antibody is captured via its Fab fragment, cytokine Fc-Fusions are captured via anti-PGLALA F(ab’)2 fragment (identical capture setup as in (A) and TRIM21-coiled-coil-PYRSPRY (TRIM21-CC-PS) is injected. Illustrations are created with BioRender.com .

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: SPR Assay, Mutagenesis, Binding Assay, Injection

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Interaction analysis of human IgG1 (mAb1) Fc variants and TRIM21 PRYSPRY domain. (A–C) showing sensorgrams (SPR data) where PRYSPRY was injected in five different concentrations as two-fold dilution series to immobilized mAb1 Fc variants (capture level approx. 60 RU). Each plot shows the measured raw data (colored gradient) and the global fit analysis as solid black lines. For immobilized mAb1 WT (A) and mAb1 WT-AAA (B) PRYSPRY was injected at 500 nM highest concentration and for mAb1 AAA (C) at 2000 nM. The sensorgrams show the affinity binding mode applying a mono-exponential fit model (Langmuir 1:1). The determined kinetic parameters are described in (D) . The k ON , k OFF and K D values are results from a global fit analysis ± fitting error. (E, F) show the complementary mass photometry (MP) data displaying a 2:1 binding stoichiometry confirming the SPR data. For the PRYSPRY - mAb1 WT complex, the data reveals a double bound state and for mAb1 WT-AAA a single bound state, while the control mAb1 AAA shows no binding at all. A Gaussian distribution model was used to analyze the MP data. For individual masses of the molecules, see SI Info Supplementary Figure S1 .

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Injection, Concentration Assay, Binding Assay, Control

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Kinetic characterization of TRIM21 PRYSPRY binding to immobilized antibody Fc variants and cytokine-Fc Fusion constructs, and Fc only variant (Raw data SI Info Supplementary Figure S2 ). Detailed SPR assay setup is described in materials and methods. (A) The Affinity Rate Scale Plot enables the kinetic comparison of several binding experiments at one glance. The association rate (k ON ) and corresponding dissociation rate (k OFF ) are juxtaposed in opposition, connected via a vertical line, representing the binding strength (affinity). The further apart both parameters (k ON and k OFF ) the stronger the interaction is. Compared to mAb1 Fc WT, the Fc variants YTE (M252Y, S254T, T256E), HH (T307H, N434H) and Y436A show decreased PRYSPRY affinity. The YTE affinity is 1.7-fold, HH 2.4-fold and Y436A 180-fold decreased. As shown in (B) the altered binding strength is mostly off rate driven, which becomes apparent in the overlay of normalized dissociation. The start of dissociation is normalized to 100%.

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Binding Assay, Construct, Variant Assay, SPR Assay, Comparison

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Affinity rate scale plot for captured TRIM21 PRYSPRY domain (ligand) and antibody (human IgG1) variable domain variants or antigen fusion constructs in solution (analyte). The injected constructs have different Fab regions but share the same Fc region. This allows the investigation of a potential Fab contribution to the PRYSPRY binding. All constructs were analyzed by applying a simple 1:1 Langmuir fit. The analyzed antibody variants do not show any Fab contribution. Notably, there is a faster on-rate (2x) for all constructs when compared to the reverse assay setup up (PRYSPRY as analyte). Raw data is shown in SI Info Supplementary Figure S3 .

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Construct, Injection, Binding Assay

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Characterization of the TRIM21 dimeric nature (TRIM21-CC-PS) applying different technologies. (A) SEC-MALS data reveals 94% TRIM21-CC-PS dimer (90 kDa). (B) Mass photometry technology shows 99% TRIM21-CC-PS with 82 kDa. (C) Selection of EM 2D classes confirming TRIM21-CC-PS dimers. The coiled coil domains facilitate dimerization whereas the C-terminal PRYSPRY domains are placed at the opposite end of each coiled-coil domain.

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Selection

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Characterizing the interaction of TRIM21-CC-PS with three different Antibody Fc variants. (A–D) Applying MP, dashed lines indicate the main peak of the respective species over all measurements. The applied 3-dimensional Gaussian Fit Distribution is shown in black lines. (A) MP of TRIM21-CC-PS with mAb 1 WT, (B) MP of TRIM21-CC-PS with mAb 1 WT-AAA (C) MP of TRIM21-CC-PS with mAb 1 AAA. Only mAb 1 WT shows binding to TRIM21-CC-PS at low nM concentration in accordance with its low nM binding strength. (D) The amount (%) of TRIM21-CC-PS - mAb1 WT complex increases with excess of TRIM21-CC-PS, while TRIM21-CC-PS - mAb1 WT-AAA shows single events of complexed species for the applied concentrations, that could not be fitted robustly. (E–H) selected 2D averages of EM data, resolving TRIM21-CC-PS with Fc WT (E) , TRIM21-CC-PS with Fc WT-AAA (F) , TRIM21-CC-PS with mAb1 WT (G) and mAb1 WT alone (H) .

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Binding Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Characterization of TRIM21-CC-PS with Antibody Fc variants. (A) shows the sensorgram (SPR) of mAb1 WT (ligand, approx. 8–10 RU) and TRIM21-CC-PS (analyte) where TRIM21-CC-PS was injected in seven different concentration, each for 180 sec as two-fold dilution series with 100 nM as highest concentration. Applied fit model is a simple 1:1 interaction reflecting 100% avid bound, 1:1 antibody - TRIM21-CC-PS species. (B) Variation in association time (10 -300 sec) injecting a constant concentration of 25 nM TRIM21-CC-PS to captured mAb1 WT reveals a biphasic binding kinetic, which can be described by applying a two state model providing fast and slow kinetic rates. (C) Rate-scale-plot comparing affinity and avidity measurements of Fc variants towards PRYSPRY or TRIM21-CC-PS. (D) The altered binding strength from affinity to avidity is mostly off rate driven, which becomes apparent in the overlay of normalized dissociation phases (k OFF,AVIDITY ) but can also occur as combination of both kinetic rate parameters, namely on and off rate.

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Injection, Concentration Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: TRIM21-CC-PS-Antibody-AAV2 Characterization. SPR Assay data is shown in (A, B) . (A1, A2) Schematic SPR assay configuration to analyze the affinity to avidity interplay of TRIM21-CC-PS, anti-capsid antibody variants A20 and rAAVv-2. Biotinylated TRIM21-CC-PS is captured via monovalent streptavidin achieving a captured level of 190 RU ( A1 , high density) and 35 RU ( A2 , low density). Subsequent, anti-AAV2 capsid antibody variants (bivalent A20 Fc WT, one-armed A20 Fc WT and Fc WT-AAA) are injected to saturate the TRIM21-CC-PS surface, followed by the injection of rAAV-2. (B) Overlay of the normalized dissociation phases (Start of Dissociation: 100%) after the injection of 3.32 nM rAAVv-2 over low and high TRIM21-CC-PS-A20 densities. At higher antibody densities, more avid complexation occurs and a higher degree of rAAVv-2 surface decoration is possible. This allows less complex to dissociate over time to due to simultaneous engagement of both, TRIM21-CC-PS and AAV2, mediated via the A20 antibody variants. (C) Electron microscopy images of rAAVv-2 interactions with antibodies alone (left column) and TRIM21 additionally (right column). The scale bars represent 50 nm.

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: SPR Assay, Injection, Electron Microscopy

Journal: Frontiers in Immunology

Article Title: TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization

doi: 10.3389/fimmu.2024.1401471

Figure Lengend Snippet: Schematic model suggesting how one TRIM21 dimer engages both sites of the Fc region in a two-step process. Upon Fc binding, the PRYSPRY detaches from the coiled-coil domain. The linker domain allows enough freedom of movement to allow engagement of the second PRYSPRY domain. Only after initial binding bivalent engagement of both Fc heavy chains is possible.

Article Snippet: Recombinant human TRIM21 proteins, including the PRYSPRY domain variant and the PRYSPRY-coiled-coil (TRIM21-CC-PS) variant, were produced and purified by Proteros Biostructures GmbH (Planegg, Germany).

Techniques: Binding Assay

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 1 Trim21 is elevated in osteoporotic patients, and its deficiency leads to high bone mass. a Quantitative RT‒PCR analysis of Trim21 mRNA expression in bone specimens from patients with different bone mineral densities (BMDs), which were defined as normal, osteopenia, and osteoporosis. b Correlation analysis between Trim21 mRNA expression and RF-BMD and LS-BMD. RF, right femur; LS, lumbar spine. c Immunoblotting analysis of Trim21 protein expression in the lumbar vertebra of 5-month-old sham-operated or ovariectomized mice. d Schematic diagram showing the analysis of skeletal parameters of mice at different ages. e Alcian blue/Alizarin Red staining of the whole skeleton of 1-week-old Trim21+/+, Trim21+/−, and Trim21−/−littermates. f X-ray images of Trim21+/+ and Trim21−/−mice at 1 month and 6 months (left panel). Quantitative analysis of the tibia length of mice at different ages (right panel). g Representative H&E and S/O staining images of tibial sections from 1-month-old Trim21+/+ and Trim21−/−mice (left panel). Quantitative analysis of the growth plate thickness of the indicated mice (right panel). h, i Representative immunofluorescence images (h) showing the expression of Sox9+ cells (i) in growth plates of tibial sections in 1-month-old Trim21+/+ and Trim21−/−mice. j Representative micro-CT images of the proximal tibia bone of 14-week-old mice. Quantitative measurements of bone volume per tissue volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular separation (Tb.Sp). All bar graphs are presented as the mean ± SD. *P < 0.05; ***P < 0.001; ****P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Expressing, Western Blot, Staining, Micro-CT

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 2 Loss of Trim21 enhances osteoblast activity and favors bone formation. a, b Representative immunoblotting analysis (a) and quantification of Runx2, Osterix, and Trim21 in MC3T3-E1 cells (b) treated with osteogenic medium for 0, 4, and 7 days. c Quantitative RT‒PCR analysis of osteogenic biomarker genes (Osterix, Runx2, and Trim21) in OBs with osteogenic induction. d, e Alizarin Red S (upper panel) and ALP (lower panel) staining of primary osteoblasts (OBs) after induction with osteogenic medium for different times (d). The percentage of Alizarin Red S- (n ≥3) and ALP- (n ≥3) stained area (e). f Quantitative RT‒PCR detection of osteogenic biomarker genes (Runx2, Osterix, OCN, OPG, and RANKL) in OBs derived from Trim21−/−and Trim21+/+ mice upon osteogenic induction for 7 days. g, h Representative immunoblotting analysis (g) and quantification of Runx2 and Osterix in OBs (h) after osteogenic induction for 0, 4, and 7 days. i Representative micro-CT images of calvarial bone defects in 2-month-old Trim21+/+ and Trim21−/−mice after surgical induction for 1 month (left panel). Quantitative measurements of bone volume per tissue volume (BV/TV) and bone defect diameter (right panel). All bar graphs are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Activity Assay, Western Blot, Biomarker Discovery, Staining, Derivative Assay, Micro-CT

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 3 Loss of Trim21 inhibits osteoclast formation and differentiation. a Representative image of histological sections of the tibia that were stained with TRAP (left panel). Bone marrow (BM) and trabecular bone (TB) are indicated in black. TRAP-stained osteoclasts (OCs) are denoted by the red arrow. OC. N/BPm (OC number per bone parameter) and OC. S/BS (OC surface per bone surface) were determined (right panel). Scale bar: 100 μm. b Quantification of F-actin ring number in BMM-derived OCs from immunofluorescence staining of Fig. S6e. c Representative immunoblot analysis and quantification of Ctsk expression in BMM-derived OCs from Trim21+/+ and Trim21−/−mice. d, e Quantitative RT‒PCR detection of OC differentiation genes (Ctsk, Nfatc1, Acp5, ATP6vod2, and Mmp9) in BMM-derived OCs from Trim21+/+

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Staining, Derivative Assay, Western Blot, Expressing

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 4 YAP1/β-catenin signaling is essential for Trim21-mediated osteogenic differentiation. a Schematic diagram showing TMT-based quantitative proteomics for the identification of differentially expressed proteins (DEPs) in bone marrow mesenchymal stem cells (BMSCs) derived from Trim21+/+ and Trim21−/−mice. b Volcano plots of DEPs in BMSCs from Trim21+/+ and Trim21−/−mice. c Heatmap analysis of DEPs in BMSCs. Three replicates of each group were included, and the top 29 DEPs are shown. d KEGG enrichment analysis of the DEPs in the BMSCs. e Representative immunoblotting analysis and quantification of BCL9, AXIN1, and YAP1 in BMSCs; proteomics sample: part of the samples subjected to proteomics analysis. f Representative immunoblotting analysis and quantification of BCL9, β-catenin, YAP1, and Runx2 protein expression in BMSCs after osteogenic induction for 7 days. g The endogenous interaction between Trim21, BCL9, β-catenin, and YAP1 was evaluated using a co-IP assay. h Protein‒protein interaction of YAP1 and Trim21 in living cells. The two BiFC plasmids encoding Myc- VN155-YAP1 and HA-VC155-Trim21 along with HA-cerulean were cotransfected into HEK293T cells for 24 h. Representative images showing transfected cells (cerulean) and the interaction between YAP1 and Trim21 (Venus). Nuclei were stained with DAPI. Scale bar: 20 μm. i Immunoblotting analysis of BCL9, β-catenin, YAP1, and HA-Trim21 protein expression in HEK293T cells treated with or without MG132. j Representative images showing the expression of YAP1+ OBs derived from Trim21+/+ and Trim21−/−mice. Scale bar: 20 μm. All bar graphs are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Quantitative Proteomics, Derivative Assay, Western Blot, Expressing, Co-Immunoprecipitation Assay, Transfection, Staining

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 5 Loss of Trim21 protects mice from lipopolysaccharide (LPS)-induced bone loss. a Quantitative RT‒PCR determination of IL-6, Osterix, and Runx2 mRNA expression in OBs with or without lipopolysaccharide (LPS) treatment during osteogenic induction. b Schematic diagram showing the H&E staining and micro-CT analysis of Trim21+/+ and Trim21−/−mice induced by PBS or LPS. c Representative images of H&E staining of tibia sections of 13-week-old Trim21+/+ and Trim21−/−mice induced by PBS or LPS. Bone marrow (BM) and trabecular bone (TB) are labeled with red arrows. d, e Representative micro-CT images (d) and BV/TV (e) of proximal tibia trabecular bone of 13-week-old Trim21+/+ and Trim21−/−mice induced by PBS or LPS. f The bone loss ratio after LPS treatment in global knockout mice (left panel) and conditional knockout mice (right panel). g Representative micro-CT images and BV/TV of proximal tibia trabecular bone of 13-week-old Trim21f/f and Ctsk-cre; Trim21f/f mice induced by PBS or LPS. h Representative micro-CT images of cortical bones and quantification of BV/TV (left panel) and thickness (right panel) of Trim21f/f and Ctsk-cre; Trim21f/f mice induced by either PBS or LPS. All bar graphs are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant by Student’s t test

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Expressing, Staining, Micro-CT, Labeling, Knock-Out

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 6 Trim21 orchestrates ovariectomy (OVX)-induced bone metabolism by targeting YAP1 signaling. a Determination of fat cell density in the proximal tibia of 20-week-old Trim21+/+ and Trim21−/−mice induced by sham operation or OVX. b Representative micro-CT images and quantitative data (BV/TV) of proximal tibial bone of 20-week-old Trim21+/+ and Trim21−/−mice induced by sham operation or OVX. c, d Calcein double labeling of mineral layers of tibial trabecular bone of 5-month-old mice. e Representative images of von Kossa staining of the undecalcified proximal tibia of 5-month-old mice. Scale bar: 50 μm. f IHC staining images of the proximal tibia of 5-month-old mice using an antibody against YAP1. The YAP1-stained positive cells are denoted by the red arrow. Scale bar: 50 μm. g, h Representative images of histological sections of the tibia that were stained with TRAP in Trim21+/+ and Trim21−/−mice induced by sham operation or OVX. TRAP-stained osteoclasts (OCs) are denoted by the red arrow. OC. N/BPm (OC number per bone parameter) and OC. S/BS (OC surface per bone surface) was determined. Scale bar: 100 μm. All bar graphs are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Micro-CT, Labeling, Staining, Immunohistochemistry

Journal: Bone research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling.

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Fig. 7 A schematic of Trim21 in the regulation of bone remodeling via YAP1/β-catenin signaling. Normal bone remodeling is maintained by the balance of MSC/osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Trim21, by interacting with the protein complex formed by YAP1/β-catenin/BCL9, dictates the degradation of this protein complex, which in turn inactivates YAP1 and β-catenin signaling, which is essential for osteoblast differentiation. However, Trim21 is critical for maintaining the basic expression of osteoclast biomarkers, including Nfatc1 and Ctsk. Therefore, the coupling of osteoblasts with osteoclasts is attributed to dynamic changes in bone metabolism (left panel). In contrast, the loss of Trim21 causes disassociation with the YAP1/β-catenin/BCL9 complex, which then enters the nucleus for subsequent activation of osteogenic genes, including Runx2 and Osterix. In addition, the loss of Trim21 suppresses the maturation of osteoclasts. Together, these results indicate that Trim21 deficiency alleviates pathological bone loss by activating YAP1/β-catenin signaling

Article Snippet: Establishment of global and conditional Trim21 knockout mice Heterozygous recombinant Trim21 mice (Trim21−/+) with a C57BL/ 6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Expressing, Activation Assay

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Trim21 is elevated in osteoporotic patients, and its deficiency leads to high bone mass. a Quantitative RT‒PCR analysis of Trim21 mRNA expression in bone specimens from patients with different bone mineral densities (BMDs), which were defined as normal, osteopenia, and osteoporosis. b Correlation analysis between Trim21 mRNA expression and RF-BMD and LS-BMD. RF, right femur; LS, lumbar spine. c Immunoblotting analysis of Trim21 protein expression in the lumbar vertebra of 5-month-old sham-operated or ovariectomized mice. d Schematic diagram showing the analysis of skeletal parameters of mice at different ages. e Alcian blue/Alizarin Red staining of the whole skeleton of 1-week-old Trim21 +/+ , Trim21 +/− , and Trim21 −/− littermates. f X-ray images of Trim21 +/+ and Trim21 −/− mice at 1 month and 6 months (left panel). Quantitative analysis of the tibia length of mice at different ages (right panel). g Representative H&E and S/O staining images of tibial sections from 1-month-old Trim21 +/+ and Trim21 −/− mice (left panel). Quantitative analysis of the growth plate thickness of the indicated mice (right panel). h , i Representative immunofluorescence images ( h ) showing the expression of Sox9 + cells ( i ) in growth plates of tibial sections in 1-month-old Trim21 +/+ and Trim21 −/− mice. j Representative micro-CT images of the proximal tibia bone of 14-week-old mice. Quantitative measurements of bone volume per tissue volume (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular separation (Tb.Sp). All bar graphs are presented as the mean ± SD. * P < 0.05; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Heterozygous recombinant Trim21 mice ( Trim21 −/+ ) with a C57BL/6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Expressing, Western Blot, Staining, Immunofluorescence, Micro-CT

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Loss of Trim21 enhances osteoblast activity and favors bone formation. a , b Representative immunoblotting analysis ( a ) and quantification of Runx2, Osterix, and Trim21 in MC3T3-E1 cells ( b ) treated with osteogenic medium for 0, 4, and 7 days. c Quantitative RT‒PCR analysis of osteogenic biomarker genes ( Osterix, Runx2, and Trim21 ) in OBs with osteogenic induction. d , e Alizarin Red S (upper panel) and ALP (lower panel) staining of primary osteoblasts (OBs) after induction with osteogenic medium for different times ( d ). The percentage of Alizarin Red S- ( n ≥ 3) and ALP- ( n ≥ 3) stained area ( e ). f Quantitative RT‒PCR detection of osteogenic biomarker genes ( Runx2, Osterix, OCN, OPG, and RANKL ) in OBs derived from Trim21 −/ − and Trim21 +/+ mice upon osteogenic induction for 7 days. g , h Representative immunoblotting analysis ( g ) and quantification of Runx2 and Osterix in OBs ( h ) after osteogenic induction for 0, 4, and 7 days. i Representative micro-CT images of calvarial bone defects in 2-month-old Trim21 +/+ and Trim21 −/ − mice after surgical induction for 1 month (left panel). Quantitative measurements of bone volume per tissue volume (BV/TV) and bone defect diameter (right panel). All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Heterozygous recombinant Trim21 mice ( Trim21 −/+ ) with a C57BL/6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Activity Assay, Western Blot, Biomarker Discovery, Staining, Derivative Assay, Micro-CT

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Loss of Trim21 inhibits osteoclast formation and differentiation. a Representative image of histological sections of the tibia that were stained with TRAP (left panel). Bone marrow (BM) and trabecular bone (TB) are indicated in black. TRAP-stained osteoclasts (OCs) are denoted by the red arrow. OC. N/BPm (OC number per bone parameter) and OC. S/BS (OC surface per bone surface) were determined (right panel). Scale bar: 100 μm. b Quantification of F-actin ring number in BMM-derived OCs from immunofluorescence staining of Fig. . c Representative immunoblot analysis and quantification of Ctsk expression in BMM-derived OCs from Trim21 +/+ and Trim21 −/− mice. d , e Quantitative RT‒PCR detection of OC differentiation genes ( Ctsk, Nfatc1, Acp5, ATP6vod2, and Mmp9 ) in BMM-derived OCs from Trim21 +/+ and Trim21 − / − mice. PBS indicates PBS containing 30 ng·mL −1 M-CSF, while RANKL indicates induction with 30 ng·mL −1 M-CSF and 100 ng·mL −1 RANKL f , g Quantitative RT‒PCR detection ( f ) of OC differentiation genes ( Ctsk and Nfatc1 ) and TRAP staining ( g ) in BMM-derived OCs from Trim21 f/f mice treated with 30 ng·mL −1 M-CSF plus 100 ng·mL −1 RANKL or PBS containing 30 ng·mL −1 M-CSF for 5 days and infected with lentivirus expressing EGFP or Cre recombinase (defined as LV-Con or LV-Cre, respectively). Quantification of TRAP-positive OCs and the number of nuclei per TRAP + cell (right panel) ( g ). Scale bar: 50 μm. h BMMs derived from 4-week-old Trim21 f/f and Ctsk-cre; Trim21 f/f mice were induced for OC differentiation with either 30 ng·mL −1 M-CSF plus 100 ng·mL −1 RANKL or PBS containing 30 ng·mL −1 M-CSF for 5 days (left panel). Quantification of TRAP-positive OCs and the number of nuclei per TRAP + cell (right panel). Scale bar: 50 μm. i , j Schematic diagram illustrating the coculture model of OCs with BMSCs from Trim21 +/+ and Trim21 − / − mice ( i ). Representative images (left panel) and quantification data of TRAP-positive OCs and nucleus number per TRAP + cell (right panel) ( j ). Scale bar: 50 μm. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Heterozygous recombinant Trim21 mice ( Trim21 −/+ ) with a C57BL/6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Staining, Derivative Assay, Immunofluorescence, Western Blot, Expressing, Infection

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: YAP1/β-catenin signaling is essential for Trim21-mediated osteogenic differentiation. a Schematic diagram showing TMT-based quantitative proteomics for the identification of differentially expressed proteins (DEPs) in bone marrow mesenchymal stem cells (BMSCs) derived from Trim21 +/+ and Trim21 −/− mice. b Volcano plots of DEPs in BMSCs from Trim21 +/+ and Trim21 −/− mice. c Heatmap analysis of DEPs in BMSCs. Three replicates of each group were included, and the top 29 DEPs are shown. d KEGG enrichment analysis of the DEPs in the BMSCs. e Representative immunoblotting analysis and quantification of BCL9, AXIN1, and YAP1 in BMSCs; proteomics sample: part of the samples subjected to proteomics analysis. f Representative immunoblotting analysis and quantification of BCL9, β-catenin, YAP1, and Runx2 protein expression in BMSCs after osteogenic induction for 7 days. g The endogenous interaction between Trim21, BCL9, β-catenin, and YAP1 was evaluated using a co-IP assay. h Protein‒protein interaction of YAP1 and Trim21 in living cells. The two BiFC plasmids encoding Myc-VN155-YAP1 and HA-VC155-Trim21 along with HA-cerulean were cotransfected into HEK293T cells for 24 h. Representative images showing transfected cells (cerulean) and the interaction between YAP1 and Trim21 (Venus). Nuclei were stained with DAPI. Scale bar: 20 μm. i Immunoblotting analysis of BCL9, β-catenin, YAP1, and HA-Trim21 protein expression in HEK293T cells treated with or without MG132. j Representative images showing the expression of YAP1 + OBs derived from Trim21 +/+ and Trim21 −/ − mice. Scale bar: 20 μm. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Heterozygous recombinant Trim21 mice ( Trim21 −/+ ) with a C57BL/6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Quantitative Proteomics, Derivative Assay, Western Blot, Expressing, Co-Immunoprecipitation Assay, Transfection, Staining

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Loss of Trim21 protects mice from lipopolysaccharide (LPS)-induced bone loss. a Quantitative RT‒PCR determination of IL-6 , Osterix , and Runx2 mRNA expression in OBs with or without lipopolysaccharide (LPS) treatment during osteogenic induction. b Schematic diagram showing the H&E staining and micro-CT analysis of Trim21 +/+ and Trim21 − /− mice induced by PBS or LPS. c Representative images of H&E staining of tibia sections of 13-week-old Trim21 +/+ and Trim21 −/− mice induced by PBS or LPS. Bone marrow (BM) and trabecular bone (TB) are labeled with red arrows. d , e Representative micro-CT images ( d ) and BV/TV ( e ) of proximal tibia trabecular bone of 13-week-old Trim21 +/+ and Trim21 −/− mice induced by PBS or LPS. f The bone loss ratio after LPS treatment in global knockout mice (left panel) and conditional knockout mice (right panel) . g Representative micro-CT images and BV/TV of proximal tibia trabecular bone of 13-week-old Trim21 f/f and Ctsk-cre; Trim21 f/f mice induced by PBS or LPS. h Representative micro-CT images of cortical bones and quantification of BV/TV (left panel) and thickness (right panel) of Trim21 f/f and Ctsk-cre; Trim21 f/f mice induced by either PBS or LPS. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s. not significant by Student’s t test

Article Snippet: Heterozygous recombinant Trim21 mice ( Trim21 −/+ ) with a C57BL/6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Expressing, Staining, Micro-CT, Labeling, Knock-Out

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: Trim21 orchestrates ovariectomy (OVX)-induced bone metabolism by targeting YAP1 signaling. a Determination of fat cell density in the proximal tibia of 20-week-old Trim21 +/+ and Trim21 −/− mice induced by sham operation or OVX. b Representative micro-CT images and quantitative data (BV/TV) of proximal tibial bone of 20-week-old Trim21 +/+ and Trim21 −/− mice induced by sham operation or OVX. c , d Calcein double labeling of mineral layers of tibial trabecular bone of 5-month-old mice. e Representative images of von Kossa staining of the undecalcified proximal tibia of 5-month-old mice. Scale bar: 50 μm. f IHC staining images of the proximal tibia of 5-month-old mice using an antibody against YAP1. The YAP1-stained positive cells are denoted by the red arrow. Scale bar: 50 μm. g , h Representative images of histological sections of the tibia that were stained with TRAP in Trim21 +/+ and Trim21 −/− mice induced by sham operation or OVX. TRAP-stained osteoclasts (OCs) are denoted by the red arrow. OC. N/BPm (OC number per bone parameter) and OC. S/BS (OC surface per bone surface) was determined. Scale bar: 100 μm. All bar graphs are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1; n.s. not significant by Student’s t test

Article Snippet: Heterozygous recombinant Trim21 mice ( Trim21 −/+ ) with a C57BL/6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Micro-CT, Labeling, Staining, Immunohistochemistry

Journal: Bone Research

Article Title: Trim21 depletion alleviates bone loss in osteoporosis via activation of YAP1/β-catenin signaling

doi: 10.1038/s41413-023-00296-3

Figure Lengend Snippet: A schematic of Trim21 in the regulation of bone remodeling via YAP1/β-catenin signaling. Normal bone remodeling is maintained by the balance of MSC/osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Trim21, by interacting with the protein complex formed by YAP1/β-catenin/BCL9, dictates the degradation of this protein complex, which in turn inactivates YAP1 and β-catenin signaling, which is essential for osteoblast differentiation. However, Trim21 is critical for maintaining the basic expression of osteoclast biomarkers, including Nfatc1 and Ctsk . Therefore, the coupling of osteoblasts with osteoclasts is attributed to dynamic changes in bone metabolism (left panel). In contrast, the loss of Trim21 causes disassociation with the YAP1/β-catenin/BCL9 complex, which then enters the nucleus for subsequent activation of osteogenic genes, including Runx2 and Osterix . In addition, the loss of Trim21 suppresses the maturation of osteoclasts. Together, these results indicate that Trim21 deficiency alleviates pathological bone loss by activating YAP1/β-catenin signaling

Article Snippet: Heterozygous recombinant Trim21 mice ( Trim21 −/+ ) with a C57BL/6 genetic background were purchased from Cyagen Biosciences (Guangzhou, China).

Techniques: Expressing, Activation Assay