Journal: Military Medical Research

Article Title: Glucocorticoids trigger muscle-liver crosstalk to attenuate acute liver injury and promote liver regeneration via the FGF6-FGFBP1 axis

doi: 10.1186/s40779-025-00618-y

Figure Lengend Snippet: FGFBP1 undergoes liquid–liquid phase separation (LLPS) and is interfered with heparin. a The domain structure and the intrinsically disordered tendency of mouse FGFBP1 (lower). IUPred ( https://iupred.elte.hu/ ) assigned scores of the disordered tendencies of the sequences between 0 and 1, with scores higher than 0.5 indicating disorder. The amino acid sequence of FGFBP1 contains two intrinsically disordered regions (IDRs). b Confocal microscopy images of the assembly status of 2 μmol/L purified rFGFBP1-EGFP with 0–5–10% PEG. Scale bar = 5 µm. c Representative images of the fusion of FGFBP1-EGFP condensates (arrowheads) over time in AML12 cells expressing FGFBP1-EGFP. Scale bar = 5 µm. d Representative micrographs of FGFBP1-EGFP condensates before and after photobleaching. Fluorescence recovery after photobleaching (FRAP) quantification of FGFBP1-EGFP condensates over a period of 3 min. Scale bar = 2.5 µm. e Schematic of the FGFBP1 domains and FGFBP1 mutants. f Fluorescence microscopy analysis of purified rFGFBP1-EGFP mutants (2 μmol/L, 10% PEG) mixed with heparin (10 μg/ml), and the size of the droplets was quantified. Scale bar = 5 µm. g HEK 293T cells were transfected with pCDH-CMV-FGFBP1 (WT, ΔHBS, ΔIDR1, or ΔIDR2)-Flag-EF1α-EGFP for 24 h and treated with or without heparin (5.5 μg/ml) for another 2 h. Western blotting analysis of flag levels in the cell culture medium and cell lysate. EGFP and α-tubulin levels in the cell lysate were used as a control. h Fluorescence microscopy analysis of purified rFGFBP1-EGFP (2 μmol/L, 10% PEG) mixed with rFGF6 (1 μmol/L) with or without heparin (10 μg/ml), and the size of the droplets was quantified. Scale bar = 5 µm. i HEK 293T cells were transfected with pCDH-CMV-FGFBP1-flag-EF1α-EGFP for 24 h and treated with rFGF6 (1 or 2 μmol/L) with or without heparin (5.5 μg/ml) for another 2 h. Western blotting analysis of flag levels in the cell culture medium and cell lysate was performed. EGFP and α-tubulin levels in the cell lysate were used as a control. j Concentration of FGFBP1 in the culture medium of differentiated primary myoblasts treated with or without heparin (5.5 μg/ml) and rFGF6 (1 or 2 μmol/L) ( n = 3). k Proposed model for the regulation of FGFBP1 release from cell surface via LLPS. Image created with BioRender.com. * P < 0.05, ** P < 0.01. FGFBP1 fibroblast growth factor binding protein 1, SP signal peptide, WT wild-type, HBS heparin-binding site, rFGF6 recombinant FGF6 protein, PEG polyethylene glycol, EGFP enhanced green fluorescent protein, pCDH-CMV plasmid cloning and delivery vector with cytomegalovirus promoter, EF1α elongation factor 1-alpha, HSPG heparan sulfate proteoglycan, HS heparan sulfate, FGF fibroblast growth factor, FGFRs fibroblast growth factor receptors

Article Snippet: For the cell proliferation assay, AML12 cells were seeded into 96-well plates and transfected with 18 FGF expression vectors (200 ng per well) using Lipofectamine 3000 (Invitrogen, CA, USA). rFGFBP1 (10 ng/ml; 1413-FB, R&D Systems, MN, USA) or FGFBP1Ab (2 μg/ml; AF1413, R&D Systems, MN, USA) was then added to cells 24 h after transfection.

Techniques: Sequencing, Confocal Microscopy, Purification, Expressing, Fluorescence, Microscopy, Transfection, Western Blot, Cell Culture, Control, Concentration Assay, Binding Assay, Recombinant, Plasmid Preparation, Cloning

Journal: Military Medical Research

Article Title: Glucocorticoids trigger muscle-liver crosstalk to attenuate acute liver injury and promote liver regeneration via the FGF6-FGFBP1 axis

doi: 10.1186/s40779-025-00618-y

Figure Lengend Snippet: FGF5 and FGF5s compete for binding to FGFBP1 via LLPS and regulate liver regeneration. a BrdU cell proliferation assay results to detect cell proliferation ability in AML12 cells overexpressing the 18 FGFs and treated with rFGFBP1 (10 ng/ml) or rFGFBP1 and FGFBP1Ab (2 μg/ml) ( n = 3). b Hepatic protein levels of FGF5 and FGF5s at the indicated times after partial (2/3) hepatectomy (PHx), with the corresponding quantification ( n = 2). c Representative liver FGFBP1 and FGF5 immunofluorescence results 24 h after acetaminophen (APAP) or saline administration. d Domain structure and the intrinsically disordered tendency of mouse FGF5 and FGF5s. IUPred assigned scores of the disordered tendencies of the sequences between 0 and 1, with scores higher than 0.5 indicating disorder. The amino acid sequence of FGF5 contains one IDR. e Confocal microscopy images of the assembly status of 5 μmol/L purified rFGF5-mCherry, rFGF5s-mCherry, or rΔIDR-mCherry with 0 or 10% PEG. Scale bar = 5 µm. f Images of purified protein colocalization assay on the cell surface via confocal microscopy and representative curves describing the distribution of the relative fluorescence intensities of FGFBP1 (green) and FGF5 or FGF5s (red). Top two panels: rFGFBP1-EGFP (5 nmol/L) with rFGF5-mCherry (20 nmol/L) or rFGF5s-mCherry (10 nmol/L). Bottom three panels: rFGFBP1-EGFP (5 nmol/L) with rFGF5-mCherry (20 nmol/L) and different amounts of rFGF5s (10, 20, or 40 nmol/L). Scale bar = 5 µm. g Representative albumin (ALB, a hepatocellular function marker) immunofluorescence of human liver organoids (HLOs) in the indicated groups. h Measurement of ALB secretion in HLOs ( n = 3). i Size calculation of HLOs in the indicated groups (CON, n = 146; rFGFBP1, n = 157; rFGFBP1 + rFGF5, n = 184). * P < 0.05, *** P < 0.001. FGF5 fibroblast growth factor 5, FGF5s short form of FGF5, FGFBP1 fibroblast growth factor binding protein 1, LLPS liquid–liquid phase separation, IDR intrinsically disordered region, PEG polyethylene glycol, EGFP enhanced green fluorescent protein, DIC differential interference contrast, DAPI 4’,6-diamidino-2-phenylindole, SP signal peptide

Article Snippet: For the cell proliferation assay, AML12 cells were seeded into 96-well plates and transfected with 18 FGF expression vectors (200 ng per well) using Lipofectamine 3000 (Invitrogen, CA, USA). rFGFBP1 (10 ng/ml; 1413-FB, R&D Systems, MN, USA) or FGFBP1Ab (2 μg/ml; AF1413, R&D Systems, MN, USA) was then added to cells 24 h after transfection.

Techniques: Binding Assay, BrdU Cell Proliferation Assay, Immunofluorescence, Saline, Sequencing, Confocal Microscopy, Purification, Fluorescence, Marker

Journal: EMBO Reports

Article Title: Selective cargo and membrane recognition by SNX17 regulates its interaction with Retriever

doi: 10.1038/s44319-024-00340-1

Figure Lengend Snippet: ( A ) Schematic representation of the domains from SNX17 and the cargos studied in this work. The recycling signaling motif NPxY is highlighted in the diagram. The intracellular domain (ICD) of the physiological cargos and the FERM binding region (FBR) of the L2 protein that were fused with GST for pull-down assays are also highlighted. The length of the proteins is scaled according to their respective number of amino acids, except for LRP1. The depicted triangle in LRP1 corresponds to the missing sequence of 3700 amino acids. CT C-terminal domain, ED extracellular domain, TM transmembrane domain, CPP cell-penetrating peptide. ( B ) Alignment of the sequences of cargo peptides used in fluorescence anisotropy assays. The conserved NPxY motif is highlighted by pink bars. ( C ) Fluorescence anisotropy assays were performed to study the interaction of SNX17 FERM-CT , with the peptides outlined in panel ( B ). Peptides were labeled with the fluorescent reagent 5-FAM at the N-terminus. The data points on the graph represent the mean ± standard deviation (SD) across three technical replicates. The line represents the fit to the data. The values for the dissociation constants are presented in the table. The Retromer-dependent cargo DMT1 was used as a negative control (Tabuchi et al, ). NB no detectable binding. ( D ) The interaction of the Retriever complex with MBP-SNX17 was evaluated in the presence and absence of the cargos LRP1 ICD , APP ICD , ITGB1 ICD , and L2 FBR , each fused with GST, in MBP pull-down assays. Non-fused MBP protein was used as a negative control. Proteins were visualized by Coomassie Blue staining. The right panel shows the quantification of the Retriever binding to SNX17. Quantification was carried out using ImageJ, measuring VPS35L as a representative band of the Retriever complex. The ratio of the VPS35L pull-down band to the MBP-SNX17 band was calculated in each lane, assuming a one-to-one binding stoichiometry. Non-specific binding of VPS35L to MBP was subtracted from the VPS35L band intensities. The results are expressed as mean ± SD ( n = 4 biological replicates). Statistical analysis was performed using an unpaired Student’s t -test, with cargo vs. without cargo. ** p = 0.004. M, protein marker. .

Article Snippet: Peptide SNX17 CT-18 (MNVEIGNPTYKMYE) , Genscript , N/A.

Techniques: Binding Assay, Sequencing, Fluorescence, Labeling, Standard Deviation, Negative Control, Staining, Marker

Journal: EMBO Reports

Article Title: Selective cargo and membrane recognition by SNX17 regulates its interaction with Retriever

doi: 10.1038/s44319-024-00340-1

Figure Lengend Snippet: ( A ) The Retriever complex was incubated with MBP-SNX17 in the presence of GST-LRP1 ICD , GST-APP ICD , GST-ITGB1 ICD , and GST-L2 FBR in GST pull-down assays. Non-fused GST protein was used as a negative control. Purified proteins and pull-down samples were separated by SDS-PAGE and visualized by Coomassie Blue staining (a representative gel shown). The right panel presents the densitometry-based quantification of the amount of SNX17 or Retriever retained in the cargo-GST pull-down assays. VPS35L was used as a representative band of the Retriever complex. The band intensities of SNX17 and VPS35L were normalized to the GST or GST-cargo band intensity. Non-specific binding to GST was subtracted. The percentage of SNX17 or VPS35L binding to GST-cargos was calculated as the ratio of the pull-down protein to the input protein (lanes 2 and 3). Values represent the mean ± SD of two independent experiments. ( B ) The effect of mutating the conserved NPxY motif to APxA in LRP1 and L2 on the cargo-dependent Retriever-SNX17 interaction. Coomassie-stained SDS-PAGE gel of pull-down assays with MBP-SNX17 and Retriever in the presence of GST-LRP1 ICD , GST-LRP1 ICD-mut (N4470A + Y4473A), GST-L2 FBR , and GST-L2 FBR-mut (N254A + Y257A). Retriever binding to MBP-SNX17 was quantified as described in Fig. . Values represent the mean ± SD of three independent experiments. ( C ) MBP pull-down assays to examine the impact of salt concentration on the SNX17-Retriever interaction in the presence or absence of cargo. The Coomassie-stained SDS-PAGE gel shown is a representative image of three independent experiments. MBP was included as a control for nonspecific binding. Retriever binding to MBP-SNX17 was quantified as described in Fig. . Error bars represent the standard deviation of three technical replicates. M protein marker, R Retriever.

Article Snippet: Peptide SNX17 CT-18 (MNVEIGNPTYKMYE) , Genscript , N/A.

Techniques: Incubation, Negative Control, Purification, SDS Page, Staining, Binding Assay, Concentration Assay, Control, Standard Deviation, Marker

Journal: EMBO Reports

Article Title: Selective cargo and membrane recognition by SNX17 regulates its interaction with Retriever

doi: 10.1038/s44319-024-00340-1

Figure Lengend Snippet: ( A ) Cartoon representation of the AF2 model of the SNX17:Retriever complex. VPS26C is colored in red, VPS35L in light pink, SNX17 in green, and the hinge region of VPS35L (residues 135–178) is colored in magenta. The confidence estimation for the AF2 model is detailed in Appendix Fig. S . ( B ) Top panel: Detailed view of the SNX17 CT-18 interaction with the VPS35L-VPS26C interface in the AF2 model of the complex SNX17 CT-18 :VPS26C:VPS35L 110-598 . Proteins are color-coded as in ( A ). Residues involved in H-bonds (yellow dashed lines), salt bridges (green dashed lines), and the SNX17 CT-18 chain are shown as sticks. The mutated residues are highlighted with a different color: SNX17 residues with light green, residues of the VPS35L C-terminal binding pocket in orange, and residues of the VPS35L hinge region in magenta. Middle panel: The sequence of SNX17 CT-18 colored according to sequence conservation calculated with the ConSurf server using a green-through-purple scale, corresponding to variable (grade 1) through conserved (grade 9) positions. Bottom panel: Zoomed-in-view of the binding surface of SNX17 CT-18 . SNX17 residues are shown as cartoon and sticks, and the hinge region of VPS35L (residues 135–178) is in magenta. Mutated residues are colored as in the top panel. ( C ) Analysis of the interaction between Retriever and SNX17 mutants in the presence and absence of the cargos LRP1 or L2. MBP pull-down assays were performed with wild-type MBP-SNX17 or indicated mutants, Retriever, GST-LRP1 ICD or GST-L2 FBR . Non-fused MBP protein was used as a negative control. Samples were loaded onto an SDS-PAGE gel and stained with Coomassie Blue. Quantification was carried out as detailed in Fig. . The graph represents the mean ± SD of technical replicates (LRP1: n = 3; L2: n = 2). ( D ) Fluorescence anisotropy assays measuring direct interaction between 5-FAM-labeled SNX17 CT-18 peptide and the indicated Retriever constructs to delimit the SNX17 binding region in Retriever. BSA protein was used as a negative control. Data points are the mean ± SD of two technical replicates. Bottom panel: K D values ± SD calculated using GraphPad Prism, unless too weak to be determined. M protein marker, R Retriever, C cargo, WT wild-type, NB no detectable binding, LB low binding (poor fit quality). .

Article Snippet: Peptide SNX17 CT-18 (MNVEIGNPTYKMYE) , Genscript , N/A.

Techniques: Binding Assay, Sequencing, Negative Control, SDS Page, Staining, Fluorescence, Labeling, Construct, Marker

Journal: EMBO Reports

Article Title: Selective cargo and membrane recognition by SNX17 regulates its interaction with Retriever

doi: 10.1038/s44319-024-00340-1

Figure Lengend Snippet: ( A ) GST pull-down assays to map the region of SNX17 that interacts with Retriever. GST-L2 FBR was incubated with the indicated combinations of SNX17 PX , SNX17 FERM-CT and MBP-SNX17 FL . Non-fused GST protein was used as a negative control. Samples were loaded onto an SDS-PAGE gel and stained with Coomassie Blue. Densitometry-based quantification was carried out with ImageJ, measuring VPS35L as a representative band of the Retriever complex. The band intensities of VPS35L were normalized to the GST or GST-cargo band intensity. Non-specific binding to GST was subtracted. The percentage of VPS35L bound in the presence of MBP-SNX17 FL and GST-L2 FBR was set to 100%, and the values for the other conditions were calculated relative to this. Values represent mean ± SD based on four technical replicates. M protein marker, R Retriever complex, FL full-length. ( B ) Effect of SNX17 mutants of the Retriever-binding region on cargo binding affinity. Fluorescence anisotropy binding curves of 5-FAM-labeled LRP1 14-mer or L2 14-mer peptide titrated with indicated SNX17 mutants. Data points are the mean ± SD of two biological replicates, with MBP-SNX17 and its mutants obtained from two independent protein purifications. The estimated K D ± SD of each mutant is listed in the right panel. MBP is used as a negative control. NB no detectable binding.

Article Snippet: Peptide SNX17 CT-18 (MNVEIGNPTYKMYE) , Genscript , N/A.

Techniques: Incubation, Negative Control, SDS Page, Staining, Binding Assay, Marker, Fluorescence, Labeling, Mutagenesis

Journal: EMBO Reports

Article Title: Selective cargo and membrane recognition by SNX17 regulates its interaction with Retriever

doi: 10.1038/s44319-024-00340-1

Figure Lengend Snippet: ( A ) SNX17 residues involved in the autoinhibition mechanism. The CT region of SNX17 (residues 389 to 470) is depicted in dark green. Key amino acids are highlighted with sticks; W321 and V390 residues belong to the cargo-binding pocket (cyan), whereas H457, N459, and F462 residues are part of the cargo-mimicking region (light green). ( B ) Alignment of the SNX17 FERM :P-selectin crystal structure (PDB ID: 4GXB) with the SNX17-L2 FBR AF2 model. SNX17 (gray) aligns with SNX17 FERM (pink), and the L2 FBR peptide (orange) occupies the same position in the cargo-binding pocket as the P-selectin peptide (blue). In the presence of L2 cargo, the CT region of SNX17 (green) is positioned distantly from the cargo-binding pocket, thus making the CT region accessible for potential interactions with other proteins. ( C ) Sequence alignment of SNX17 CT-18 with the FERM binding motifs of LRP1 and L2. Residues targeted in mutagenesis studies are marked with green squares. ( D ) Diagram illustrating the potential autoinhibition mechanism of SNX17. In the absence of cargo, SNX17 exhibits minimal affinity for Retriever, because the cargo-mimicking region (depicted as a triangle) is bound to the cargo-binding pocket rendering the Retriever-binding region poorly accessible (left scene). Cargo binding displaces the inhibitory cargo-mimicking region from the pocket, freeing the Retriever-binding region (middle scene) and facilitating its association with Retriever (right scene). The Retriever-binding region and the cargo-mimicking region partially overlap. ( E ) Fluorescence anisotropy binding curves upon titration of indicated SNX17 mutants to 5-FAM-labeled LRP1 14-mer , L2 14-mer , or SNX17 CT-18 peptide to validate the autoinhibition mechanism. Data points represent the mean ± SD from three biological replicates, with MBP-SNX17 and its mutants obtained from three independent protein purifications. MBP protein was used as a negative control. K D values ± SD were determined from individual binding curves. ( F ) The purified Retriever complex was incubated with the indicated MBP-SNX17 mutants in the presence or absence of GST-LRP1 ICD or GST-L2 FBR in MBP pull-down assays. Non-fused MBP protein was used as a negative control. Quantification of the Coomassie-stained SDS-PAGE gel was carried out in ImageJ, measuring VPS35L as a representative band of the Retriever complex. The level of Retriever binding to MBP-SNX17 was quantified as described in Fig. . Values represent mean ± SD ( n = 2 biological replicates, with MBP-SNX17 and its mutants obtained from two independent protein purifications). R retriever, C cargo, WT wild-type, NB no detectable binding, LB low binding (poor fit quality). .

Article Snippet: Peptide SNX17 CT-18 (MNVEIGNPTYKMYE) , Genscript , N/A.

Techniques: Binding Assay, Sequencing, Mutagenesis, Fluorescence, Titration, Labeling, Negative Control, Purification, Incubation, Staining, SDS Page

Journal: EMBO Reports

Article Title: Selective cargo and membrane recognition by SNX17 regulates its interaction with Retriever

doi: 10.1038/s44319-024-00340-1

Figure Lengend Snippet: ( A ) Confocal fluorescence imaging of GUVs incubated with Retriever-mKate2 (red), GFP-SNX17 (green), or both in the presence or absence of the cargo His 10 -L2 FBR . GUV membranes were stained with Marina Blue DHPE lipid dye (cyan). Scale bar, 5 μm. ( B ) Diagram of the potential mechanism of Retriever recruitment via SNX17 in membranes. In the absence of endosomal membrane and cargo, the CT region of SNX17 interacts with itself and masks the Retriever-binding region. Under this condition, SNX17 has a negligible affinity for Retriever. After membrane attachment, the CT region of SNX17 becomes exposed, allowing both Retriever recruitment and cargo binding to facilitate the assembly of the recycling machinery. ( C ) A co-sedimentation assay was performed by incubating PI3P-containing liposomes with His-Sumo3-SNX17, Retriever, and His 10 -LRP1 ICD (at a 2:2:4 μM ratio). Samples were loaded onto an SDS-PAGE gel to separate the soluble (supernatant, S) and co-sedimented (pellet, P) fractions (upper panel). After Coomassie staining, densitometry-based quantification of the individual bands was performed. The binding of SNX17 and Retriever to liposomes was quantified as the percentage of total protein bound to the pellet under each condition, with VPS35L serving as a representative band of the Retriever complex. The values in the graph (bottom panel) represent the mean ± SD of six biological replicates, derived from three independent liposome preparations and two separate protein purifications of Retriever and His-Sumo3-SNX17. Statistical significance was tested using one-way ANOVA followed by Tukey’s test for multiple comparisons. * p = 0.03, ** p = 0.002, *** p = 0.0002, **** p = 0.0000006, ns not significant. .

Article Snippet: Peptide SNX17 CT-18 (MNVEIGNPTYKMYE) , Genscript , N/A.

Techniques: Fluorescence, Imaging, Incubation, Staining, Membrane, Binding Assay, Sedimentation, Liposomes, SDS Page, Derivative Assay

Journal: EMBO Reports

Article Title: Selective cargo and membrane recognition by SNX17 regulates its interaction with Retriever

doi: 10.1038/s44319-024-00340-1

Figure Lengend Snippet: ( A ) Fluorescent imaging of GUVs stained with Marina Blue DHPE lipid dye (shown in cyan) to study the interaction of Retriever-mKate2 (red) with SNX17 WT or mutants on membranes. Scale bar: 5 μm. ( B – E ) Study of Retriever recruitment onto liposome membranes of various compositions in the presence of His-Sumo3-SNX17 and the cargo His 10 -L2 FBR or His 10 -LRP1 ICD . Liposomes lacking phosphatidylinositol ( C ) or containing PI3P ( B ), PI5P ( D ), or PI(3,5)P 2 ( E ) were analyzed. Supernatant (S) and pellet (P) fractions were separated and visualized via SDS-PAGE followed by Coomassie staining (left). The binding of SNX17 and Retriever to liposomes was quantified as the percentage of total protein bound to the pellet in each condition, with VPS35L serving as a representative band of the Retriever complex (right). Bars represent mean ± SD from three ( B ) or two ( C – E ) biological replicates, derived from independent liposomes preparations and two separate protein purifications of Retriever and His-Sumo3-SNX17. One-way ANOVA followed by Tukey’s test for multiple comparisons was performed for statistical analysis in ( B ). ** p = 0.004, *** p = 0.0003, ns not significant.

Article Snippet: Peptide SNX17 CT-18 (MNVEIGNPTYKMYE) , Genscript , N/A.

Techniques: Imaging, Staining, Liposomes, SDS Page, Binding Assay, Derivative Assay

Journal: EMBO Reports

Article Title: Selective cargo and membrane recognition by SNX17 regulates its interaction with Retriever

doi: 10.1038/s44319-024-00340-1

Figure Lengend Snippet: ( A ) Cargo-mediated activation: SNX17 encounters its cargo, and this interaction through the FERM domain triggers the release of the SNX17 C-terminal region. With the C-terminal residues exposed, SNX17 binds and recruits Retriever. Subsequently, SNX17 binding to PI3P at the membrane through the PX domain promotes the attachment of the complex to the membrane. ( B ) Membrane-mediated activation: SNX17 initially binds to PI3P, leading to its attachment to the membrane and subsequent exposure of the Retriever-binding motif. The movement of the C-terminal residues of SNX17 enables Retriever recruitment and cargo binding. The predicted interaction between VPS26C and SNX17, observed in the AF2-multimer model for the complex SNX17:L2 17mer :VPS26C:VPS35L 110-598 , was used to illustrate the proposed approach of Retriever to the membrane in ( A , B ).

Article Snippet: Peptide SNX17 CT-18 (MNVEIGNPTYKMYE) , Genscript , N/A.

Techniques: Activation Assay, Binding Assay, Membrane

Journal: EMBO Reports

Article Title: Selective cargo and membrane recognition by SNX17 regulates its interaction with Retriever

doi: 10.1038/s44319-024-00340-1

Figure Lengend Snippet:

Article Snippet: Peptide SNX17 CT-18 (MNVEIGNPTYKMYE) , Genscript , N/A.

Techniques: Recombinant, Plasmid Preparation, Construct, Sequencing, Cell Culture, Transfection, Membrane, Blocking Assay, Software, Inverted Microscopy