Proteolytically generated soluble Tweak Receptor Fn14 is a blood biomarker for γ‐secretase activity

Abstract

Fn14 is a cell surface receptor with key functions in tissue homeostasis and injury but is also linked to chronic diseases. Despite its physiological and medical importance, the regulation of Fn14 signaling and turnover is only partly understood. Here, we demonstrate that Fn14 is cleaved within its transmembrane domain by the protease γ‐secretase, resulting in secretion of the soluble Fn14 ectodomain (sFn14). Inhibition of γ‐secretase in tumor cells reduced sFn14 secretion, increased full‐length Fn14 at the cell surface, and enhanced TWEAK ligand‐stimulated Fn14 signaling through the NFκB pathway, which led to enhanced release of the cytokine tumor necrosis factor. γ‐Secretase‐dependent sFn14 release was also detected ex vivo in primary tumor cells from glioblastoma patients, in mouse and human plasma and was strongly reduced in blood from human cancer patients dosed with a γ‐secretase inhibitor prior to chimeric antigen receptor (CAR)‐T‐cell treatment. Taken together, our study demonstrates a novel function for γ‐secretase in attenuating TWEAK/Fn14 signaling and suggests the use of sFn14 as an easily measurable pharmacodynamic biomarker to monitor γ‐secretase activity in vivo.

Cleavage efficiency of the intramembrane protease γ‐secretase is reduced by the palmitoylation of a substrate's transmembrane domain

“…if the proteins were above the hyperbolic FDR curves in the volcano plots (Figure 1A-D and Table S1 for proteomic data). In HeLa cells, this yielded TNFRSF12A (better known as FN14), which is a known γsecretase substrate 23 and validates the screening approach. Additional hits were PTPRCAP and C16orf54 in Jurkat cells, which have been little described to date.…”

“…HEK293 cells were seeded on 12-well plates (Corning) and transfected with a pcDNA3.1 plasmid carrying the different plasma membrane constructs (FN14, FXYD3, FXYD6, PMEPA1 and PTTG1IP full length, LYRIC and SYT12 with a shortened cytoplasmic tail: amino acid 1-104 and AA 1-95, respectively, and ITGB1 with a shortened N-terminus, amino acids AA 703-798 to keep the construct sizes similar) tagged with an N-terminal HA-tag (YPYDVPDYA) and a C-terminal double FLAG tag with a three amino acid linker (GLEDY KDD DDK DYK DDDDK) as described before. 23 Shortly, transfection was performed by Lipofectamine™ 2000, according to the manufacturer's instructions (Thermo Fisher, US). Cells were incubated overnight in 1 mL DMEM (Gibco) with 10% FCS, containing either 1 μM of the γsecretase inhibitor DAPT (Sigma) or DMSO as control.…”

“…As a positive control for a substrate, we included FN14. 23 As a positive control for a nonsubstrate, we included a C-terminal fragment of integrin β1 (ITGB1) carrying 25 amino acids of its natural ectodomain, which was previously established as a nonsubstrate. 24 All proteins carried an N-terminal HA-epitope tag and a C-terminal double FLAG-epitope tag (Figure 2A for a scheme).…”

“…In contrast to the endogenously expressed short proteins, where DAPT increased protein abundance of FN14 in the proteomic experiment (Figure 1A), this increase was not seen upon overexpression of FN14 (Figure 2B), in line with our previous study. 23 Presumably, the transfected protein is much more abundant than the endogenous protein so only a small fraction is processed by γsecretase during the experiment. The full-length proteins were detected with various abundance when the same amounts of total lysate protein were loaded (Figure 2B,C, top two panels).…”

“…16,19,20 As a consequence, most mechanistic studies with γsecretase rely on the use of model substrates with artificially truncated ectodomains. An exception are three recently discovered γsecretase substrates, the B cell maturation antigen (BCMA), the TWEAK receptor FN14 (also known as TWEAK receptor, TNR12, CD266 or Fibroblast growth factor-inducible protein 14) and synaptotagmin-7, [21][22][23] which do not require ectodomain shedding, because they have short ectodomains of 54 (BCMA), 53 (FN14) or 16 (Syt-7) amino acids. The three proteins constitute the emerging subfamily of naturally short γsecretase substrates, which offer the advantage for mechanistic studies that their direct cleavage by γsecretase can be monitored without interference by an initial ectodomain shedding step and that expression of pre-truncated forms of the proteins is not required.…”