The β-secretase β-site APP cleaving enzyme (BACE1) is a central drug target for Alzheimer’s disease. Clinically tested, BACE1-directed inhibitors also block the homologous protease BACE2. Yet little is known about physiological BACE2 substrates and functions in vivo. Here, we identify BACE2 as the protease shedding the lymphangiogenic vascular endothelial growth factor receptor 3 (VEGFR3). Inactivation of BACE2, but not BACE1, inhibited shedding of VEGFR3 from primary human lymphatic endothelial cells (LECs) and reduced release of the shed, soluble VEGFR3 (sVEGFR3) ectodomain into the blood of mice, nonhuman primates, and humans. Functionally, BACE2 inactivation increased full-length VEGFR3 and enhanced VEGFR3 signaling in LECs and also in vivo in zebrafish, where enhanced migration of LECs was observed. Thus, this study identifies BACE2 as a modulator of lymphangiogenic VEGFR3 signaling and demonstrates the utility of sVEGFR3 as a pharmacodynamic plasma marker for BACE2 activity in vivo, a prerequisite for developing BACE1-selective inhibitors for safer prevention of Alzheimer’s disease.

The cell surface receptor TREM2 is a key genetic risk factor and drug target in Alzheimer’s disease (AD). In the brain, TREM2 is expressed in microglia, where it undergoes proteolytic cleavage, linked to AD risk, but the responsible protease in microglia is still unknown. Another microglia-expressed AD risk factor is catalytically inactive rhomboid 2 (iRhom2, RHBDF2), which binds to and acts as a non-catalytic subunit of the metalloprotease ADAM17. A potential role in TREM2 proteolysis is not yet known. Using microglial-like BV2 cells, bone marrow-derived macrophages and primary murine microglia, we identify iRhom2 as a modifier of ADAM17-mediated TREM2 shedding. Loss of iRhom2 increased TREM2 in cell lysates and at the cell surface and enhanced TREM2 signaling and microglial phagocytosis of the amyloid β-peptide (Aβ). This study establishes ADAM17 as a physiological TREM2 protease in microglia, and suggests iRhom2 as a potential drug target for modulating TREM2 proteolysis in AD. * iRhom2 : (inactive Rhomboid protein 2) RHBDF2 : (Rhomboid 5 Homolog 2) TREM2 : (Triggering Receptor Expressed on Myeloid Cells 2) ADAM17 : (A Disintegrin And Metalloproteinase 17) ADAM10 : (A Disintegrin And Metalloproteinase 10) CSF1R : (Colony Stimulating Factor 1 Receptor) SYK : (Spleen Tyrosine Kinase) hiSPECS : (high-performance secretome protein enrichment with click sugars).

The intramembrane protease gamma-secretase has broad physiological functions, but also contributes to Notch-dependent tumors and Alzheimer's disease. While gamma-secretase cleaves numerous membrane proteins, only few nonsubstrates are known. Thus, a fundamental open question is how gamma-secretase distinguishes substrates from nonsubstrates and whether sequence-based features or post-translational modifications of membrane proteins contribute to substrate recognition. Using mass spectrometry-based proteomics, we identified several type I membrane proteins with short ectodomains that were inefficiently or not cleaved by gamma-secretase, including 'pituitary tumor-transforming gene 1-interacting protein' (PTTG1IP). To analyze the mechanism preventing cleavage of these putative nonsubstrates, we used the validated substrate FN14 as a backbone and replaced its transmembrane domain (TMD), where gamma-cleavage occurs, with the one of nonsubstrates. Surprisingly, some nonsubstrate TMDs were efficiently cleaved in the FN14 backbone, demonstrating that a cleavable TMD is necessary, but not sufficient for cleavage by gamma-secretase. Cleavage efficiencies varied by up to 200-fold. Other TMDs, including that of PTTG1IP, were still barely cleaved within the FN14 backbone. Pharmacological and mutational experiments revealed that the PTTG1IP TMD is palmitoylated, which prevented cleavage by gamma-secretase. We conclude that the TMD sequence of a membrane protein and its palmitoylation can be key factors determining substrate recognition and cleavage efficiency by gamma-secretase. The intramembrane protease gamma-secretase has broad physiological functions. However, a fundamental open question is how gamma-secretase distinguishes substrates from nonsubstrates and whether sequence-based features or post-translational modifications of membrane proteins contribute to substrate recognition. Using mass spectrometry-based proteomics and domain swap experiments, this study demonstrates that palmitoylation within the C-terminal half of a substrate's transmembrane domain constitutes a new mechanism that can suppress cleavage by gamma-secretase.image

Intramembrane proteases play a pivotal role in biology and medicine, but how these proteases decode cleavability of a substrate transmembrane (TM) domain remains unclear. Here, we study the role of conformational flexibility of a TM domain, as determined by deuterium/hydrogen exchange, on substrate cleavability by γ-secretase in vitro and in cellulo . By comparing hybrid TMDs based on the natural amyloid precursor protein TM domain and an artificial poly-Leu non-substrate, we find that substrate cleavage requires conformational flexibility within the N-terminal half of the TMD helix (TM-N). Robust cleavability also requires the C-terminal TM sequence (TM-C) containing substrate cleavage sites. Since flexibility of TM-C does not correlate with cleavage efficiency, the role of the TM-C may be defined mainly by its ability to form a cleavage-competent state near the active site, together with parts of presenilin, the enzymatic component of γ-secretase. In sum, cleavability of a γ-secretase substrate appears to depend on cooperating TM domain segments, which deepens our mechanistic understanding of intramembrane proteolysis.

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.