Proteolytically generated soluble Tweak Receptor Fn14 is a blood biomarker for g-secretase activity (2022)
Figure 1

Figure 1: Fn14 is processed by the protease γ-secretase

A. Domain structure of Fn14. Fn14 has a short, compact N-terminal extracellular domain. It consists of 53 amino acids that contains three disulfide bridges (dashed lines) that form the cysteine-rich domain, CRD (red). Fn14 has a single transmembrane domain (blue) and a short C-terminal cytosolic tail with a TRAF binding site (green) important for its signaling function. Protein N- and C-termini (N, C) are indicated.

B. Detection of sFn14 in conditioned media. HEK293E cells were transfected with either empty vector or a plasmid encoding human Fn14 that bears an N-terminal HA-tag and a C-terminal double FLAG-tag. Conditioned media and lysates of the transfected cells were collected and analyzed by immunoblotting with the indicated antibodies. Calnexin served as a loading control. Shown are representative blots from N=3 experiments.

C. Generation of sFn14 is sensitive to the γ-secretase inhibitor DAPT. HEK293E cells were transfected with either Fn14 or C99 (C-terminal fragment of APP), both containing a N-terminal HA-tag and C-terminal double FLAG-tag. One day after transfection, cells were treated with γ-secretase inhibitor DAPT (1 μM), broad-spectrum metalloprotease inhibitor TAPI-1 (50 μM), or the corresponding amount of vehicle DMSO as indicated. The conditioned media and the lysates were blotted with anti-HA antibody. Shown are representative blots from N=4 experiments.

D. Schematic representation of Fn14 shedding by the γ-secretase complex. γ-Secretase is a hetero-tetrameric complex consisting of the indicated subunits, with presenilin being the catalytic subunit and the ectodomain of nicastrin forming a lid-like structure on top of the γ-secretase complex. Proteolysis takes place at the catalytic core (indicated by black arrow head), within the lipid bilayer. Proteolysis results in one fragment released into the extracellular space (sFn14), and one fragment into the cytosol (Fn14 ICD, intracellular domain).

E. In vitro γ-secretase cleavage assay. HEK293E cells were transfected with epitope-tagged Fn14 or C99. Cellular membranes were collected and incubated under indicated conditions for the γ-secretase activity assay. Reactions were terminated and ultracentrifuged. Supernatant (containing γ-secretase cleavage products) was used for detecting the ICD fragment while the pellet was used to blot for full length proteins Fn14 or C99. Blotting is done by anti-Flag antibody. Shown are representative blots from N=3 experiments.

F. sFn14 production requires the proteolytic presenilin subunit of γ-secretase. HEK293 cells stably transfected with APP carrying the Swedish double mutation (K595N/M596L) and with a CRISPR/CAS9-mediated knockout of presenilin 1 (PS1 KO) or of both PS1 and PS2 (PS1/2 dKO), were transiently transfected with epitope-tagged Fn14. Conditioned media and lysates of the transfected cells were blotted with the indicated antibodies. C83 is a small C-terminal fragment of APP (containing the C-terminal 83 amino acids), which is also subjected to γ-secretase processing. Upon longer exposure, the C99 (generated by BACE1 from APP) is also visible in the PS1/2 dKO condition. Shown are representative blots from N=4 experiments.

G. Quantification of results from panel F. sFn14 levels in the conditioned media quantified and normalized to WT sFn14 signal.

Data Information: Panel (G) shows mean ± SEM, along with p-values calculated by ordinary one-way ANOVA test with Tukey's multiple comparison test. Number of biological replicates performed is indicated in the corresponding panel legend.