Beta-endoproteolysis of the cellular prion protein by dipeptidyl peptidase-4 and fibroblast activation protein

Abstract

The cellular prion protein (PrP C ) converts to alternatively folded pathogenic conformations (PrP Sc ) in prion infections and binds neurotoxic oligomers formed by amyloid-β α-synuclein, and tau. β-Endoproteolysis, which splits PrP C into N- and C-terminal fragments (N2 and C2, respectively), is of interest because a protease-resistant, C2-sized fragment (C2 Sc ) accumulates in the brain during prion infections, seemingly comprising the majority of PrP Sc at disease endpoint in mice. However, candidates for the underlying proteolytic mechanism(s) remain unconfirmed in vivo. Here, a cell-based screen of protease inhibitors unexpectedly linked type II membrane proteins of the S9B serine peptidase subfamily to PrP C β-cleavage. Overexpression experiments in cells and assays with recombinant proteins confirmed that fibroblast activation protein (FAP) and its paralog, dipeptidyl peptidase-4 (DPP4), cleave directly at multiple sites within PrP C ’s N-terminal domain. For wild-type mouse and human PrP C substrates expressed in cells, the rank orders of activity were human FAP ~ mouse FAP > mouse DPP4 > human DPP4 and human FAP > mouse FAP > mouse DPP4 >> human DPP4, respectively. C2 levels relative to total PrP C were reduced in several tissues from FAP-null mice, and, while knockout of DPP4 lacked an analogous effect, the combined DPP4/FAP inhibitor linagliptin, but not the FAP-specific inhibitor SP-13786, reduced C2 Sc and total PrP Sc levels in two murine cell-based models of prion infections. Thus, the net activity of the S9B peptidases FAP and DPP4 and their cognate inhibitors/modulators affect the physiology and pathogenic potential of PrP C .

Application of N-Terminal Labeling Methods Provide Novel Insights into Endoproteolysis of the Prion Protein in Vivo

“…We then focused on PrP fragmentation and used a combination of immunoprecipitation and in vitro labeling of the N-termini; by these means, we identified fragments corresponding to α- and β-cleavage of mouse PrP C and novel fragments commencing at residues 74, 81, 92. In addition to these findings, fragments commencing at residues 71, 79, and 87 from ex vivo samples corroborate recent data indicating endoproteolysis of PrP C in vitro and in vivo by S9B peptidase subfamily …”

“…DPP6, a catalytically dead S9B protein, has been described as an interactor of PrP C . , Additionally, DPP2 was ranked among the top proteases predicted from the cleavage site information generated by subtiligase-based N-terminomics. Intriguingly, overexpression and in vitro cleavage assays with some members of the S9B protease family, namely, DPP4 and fibroblast activation protein (but not DPP6 or the cytoplasmically expressed S9B family members DPP8 and DPP9), proved adept at β-cleavage of S3 PrP, and WT PrP C to a lesser extent, cleaving directly at multiple sites within the sequence . Noting that DPP4 is also implicated in generating N-terminally truncated pathogenic forms of Alzheimer’s disease Aβ peptide, a deeper exploration of the S9B proteases (which may have complex patterns of regulation exhibiting changes in localization independent of catalytic activity) may be warranted.…”

“…In addition to these findings, fragments commencing at residues 71, 79, and 87 from ex vivo samples corroborate recent data indicating endoproteolysis of PrP C in vitro and in vivo by S9B peptidase subfamily. 25 ■…”

Cleavage site-directed antibodies reveal the prion protein in humans is shed by ADAM10 at Y226 and associates with misfolded protein deposits in neurodegenerative diseases

“…The interest in endogenous proteolytically generated PrP fragments is steadily increasing 19,20,106–109 with more and more functions and pathological implications being suggested, particularly for ‘sPrP’ 45–51 . Yet due to the lack of appropriate tools, most studies either did not sufficiently discriminate between sPrP and other released PrP forms (e.g., on EVs) or drew their conclusions from experiments using synthetic PrP, considering the latter to be a suitable analogue for physiological sPrP.…”

“…Some conserved endogenous cleavage events within PrP have been identified over the years, yet their biological relevance is just starting to be understood as more systematic studies are being conducted 5,17–20 . This also applies to the constitutive, membrane-proximate shedding by the metalloprotease ADAM10 21–23 , which is of particular interest as it releases nearly full-length PrP, i.e., shed PrP (sPrP), into the extracellular space while leaving only the GPI anchor and a few amino acids behind at the plasma membrane.…”