Journal: PLoS ONE

Article Title: Human FGF-21 Is a Substrate of Fibroblast Activation Protein

doi: 10.1371/journal.pone.0151269

Figure Lengend Snippet: (A) Human FGF-21 is cleaved by FAP. Recombinant human FGF-21 was digested by recombinant human FAP and visualized by Coomassie staining of SDS-Page gel. (B) Time course of FGF-21 digestion by FAP quantified by LC/MS extracted ion integration of peaks corresponding to intact (1–181) and cleaved (1–171) forms of FGF-21 (n = 3 per time point per group). Values are mean ± SEM with one phase decay curve fit superimposed. (C) FAP cleavage of FGF-21 is prevented by ARI-3099. ARI-3099 was pre-incubated with recombinant FAP for 30 minutes prior to addition of FGF-21. Reaction products were visualized by Coomassie staining of SDS-Page gel. (D) Recombinant PREP does not cleave FGF-21. Recombinant human PREP was added to recombinant FGF-21 and visualized by Coomassie staining of SDS-Page gel.

Article Snippet: Reactions were carried out at a final concentration of 20 μM FGF-21, 200 nM recombinant human FAP (R&D systems) or PREP (R&D systems) and 16 μM ARI-3099.

Techniques: Recombinant, Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: PLoS ONE

Article Title: Human FGF-21 Is a Substrate of Fibroblast Activation Protein

doi: 10.1371/journal.pone.0151269

Figure Lengend Snippet: (A) FAP cleaves human FGF-21 in mouse, monkey and human plasma. Recombinant FGF-21 was added to plasma to a final concentration of 1 μM in the presence or absence of 16 μM ARI-3099 followed by assessment of intact FGF-21 by sandwich ELISA (n = 3 per group). Values are mean ± SEM. *P < .05 ***P < .001 by ANOVA . (B) FAP activity of mouse, monkey and human plasma as assessed by the FAP-specific fluorescent substrate ARI-3144.

Article Snippet: Reactions were carried out at a final concentration of 20 μM FGF-21, 200 nM recombinant human FAP (R&D systems) or PREP (R&D systems) and 16 μM ARI-3099.

Techniques: Clinical Proteomics, Recombinant, Concentration Assay, Sandwich ELISA, Activity Assay

Journal: Molecular Cancer

Article Title: CREB3L1 promotes tumor growth and metastasis of anaplastic thyroid carcinoma by remodeling the tumor microenvironment

doi: 10.1186/s12943-022-01658-x

Figure Lengend Snippet: CREB3L1 is associated with ECM signaling in thyroid cancer. A GSEA determined the enrichment differences of biological processes depending on the CREB3L1 expression. B-C The evaluated activity scores of the extracellular matrix (ECM) and collagen signaling in NT, PTC and ATC samples. D-E The Pearson correlation analysis of CREB3L1 and ECM or collagen signaling. F Immunofluorescence staining of CREB3L1 and fibroblast marker FAP in thyroid cancer samples. G RT - PCR was used to detect the expression of collagen signals after CREB3L1 knockdown. H The expression of COL5A1 was detected, after CREB3L1 knockdown or overexpression in thyroid cancer cell lines. I-J After CREB3L1 knockdown, Masson trichrome staining and IHC staining were used to detect changes in collagen fibril abundance and the expression of α - SMA after CREB3L1 knockdown, respectively. K Immunofluorescence staining of COL5A1 in ATC cell-derived xenografts after CREB3L1 knockdown. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 NC versus CREB3L1 - KD

Article Snippet: To analyze the α - SMA - , FAP - , or PDGFRα - positive CAFs, found in the 8505C - derived sphere or co - culture with 8505C, the samples were digested and resuspended in PBS, containing 1.5% BSA and incubated with the primary antibodies anti - α - SMA (Cat#14,395–1 - AP, Proteintech), anti - FAP (FAB3715A, R&D Systems, Minnesota, USA), and anti - PDGFRα (#567,950, BD biosciences), respectively.

Techniques: Expressing, Activity Assay, Immunofluorescence, Staining, Marker, Reverse Transcription Polymerase Chain Reaction, Knockdown, Over Expression, Immunohistochemistry, Derivative Assay

Journal: Journal of Medicinal Chemistry

Article Title: Development of Zalfermin, a Long-Acting Proteolytically Stabilized FGF21 Analog

doi: 10.1021/acs.jmedchem.4c00391

Figure Lengend Snippet: Structural model of the zalfermin ( 15 ) FGF21 analog and illustration of interactions with albumin and KLB. Numbering of amino acids corresponds to human FGF21. (A) Amino acid sequence with rendition of the fatty-diacid side chain. The naturally occurring disulfide bridge between Cys75 and Cys93 is indicated by a line. Red font represents a mutation relative to human FGF21. The N-terminal is elongated by an alanine residue (−1Ala) and in-sequence mutations include Asn121Gln, Met168Leu, and Ala180Cys. A C18 diacid gGlu-OEG-OEG-C2DA-Ac side chain is attached at position 180. (B) Representative AlphaFold model of 15 shown in blue and potential interaction with albumin (orange) based on albumin−somapacitan cocrystal structure (PDB entry 6QIO ). The FAP enzyme is colored gray, with the catalytic site and surrounding residues in magenta. FGF21 positions mutated in 15 as well as position 71 (modification site in compound 5 ) and 171 (part of the FAP cleavage Pro171/Ser172 site) are indicated. (C) Crystal structure of FGF21 C-terminal region residues (158-Ser181, in colors) in complex with binding to KLB (in gold) (PDB entry 5VAQ ). The structure predicts introduction of a fatty-diacid side chain on some (red color) but not other (green color) FGF21 amino acid residues to cause steric clash with KLB. For clarity, some FGF21 residues (168, 169, 171, 173, 175–178) are not annotated and red/green color-coded since they are largely hidden from the chosen point of view. FAP: fibroblast activation protein; KLB: beta-klotho. Figure B,C were prepared using PyMOL.

Article Snippet: Five μM of FGF21 analog was incubated in duplicate with FAP (R&D Systems, 3715-SE-010) at pH 7.4 (50 mM Tris, pH 7.4, 100 mM NaCl, 0.05% Tween20) at 37 °C.

Techniques: Sequencing, Mutagenesis, Residue, Modification, Binding Assay, Activation Assay