Role of ADAMs in the Ectodomain Shedding and Conformational Conversion of the Prion Protein

Abstract

The cellular prion protein (PrPC) is essential for the pathogenesis and transmission of prion diseases. PrPC is bound to the plasma membrane via a glycosylphosphatidylinositol anchor, although a secreted, soluble form has also been identified. Previously we reported that PrPC is subject to ectodomain shedding from the membrane by zinc metalloproteinases with a similar inhibition profile to those involved in shedding the amyloid precursor protein. Here we have used gain-of-function (overexpression) and loss-of-function (small interfering RNA knockdown) experiments in cells to identify the ADAMs (a disintegrin and metalloproteinases) involved in the ectodomain shedding of PrPC. These experiments revealed that ADAM9 and ADAM10, but not ADAM17, are involved in the shedding of PrPC and that ADAM9 exerts its effect on PrPC shedding via ADAM10. Using dominant negative, catalytically inactive mutants, we show that the catalytic activity of ADAM9 is required for its effect on ADAM10. Mass spectrometric analysis revealed that ADAM10, but not ADAM9, cleaved PrP between Gly228 and Arg229, three residues away from the site of glycosylphosphatidylinositol anchor attachment. The shedding of another membrane protein, the amyloid precursor protein β-secretase BACE1, by ADAM9 is also mediated via ADAM10. Furthermore, we show that pharmacological inhibition of PrPC shedding or activation of both PrPC and PrPSc shedding by ADAM10 overexpression in scrapie-infected neuroblastoma N2a cells does not alter the formation of proteinase K-resistant PrPSc. Collectively, these data indicate that although PrPC can be shed through the action of ADAM family members, modulation of PrPC or PrPSc ectodomain shedding does not regulate prion conversion.

Prion and TNFα: TAC(E)it agreement between the prion protein and cell signaling

“…However, because regulation of ADAM enzymes could be cell context-dependent, at this stage a direct PrP C -TACE interaction cannot be excluded. In support of this possibility stands the above mentioned enzyme-substrate interaction allowing PrP C shedding by ADAM10, 32 and the notion that-among all ADAM family members-ADAM10 shares with TACE the highest degree of sequence homology. 29 Finally, given that N-terminal octapeptide repeats of PrP C bind metal ions (primarily Cu 2+ but also Zn 2+ and other ions), 38,39 one may tentatively suggest that the interaction serves for PrP C to deliver Zn 2+ to TACE catalytic site (Fig.…”

“…This was achieved by stimulating serotonergic and noradrenergic neurons with antibodymediated PrP C cross-linking that, by triggering Fyn-dependent NADPH oxidase two distinct endo-proteolytic events, termed α-and β-cleavage, of which the α-cleavage implicates ADAM9, ADAM10 and ADAM17 (TACE). [33][34][35] Although the latter report has recently been questioned, 32 the patho-physiologic relevance of PrP C shedding and of the endo-proteolytic processes remains unclear.…”

“…One example of this functional interaction is the shedding of the far C-end of PrP C itself by the coordinated action of ADAM9 and ADAM10. 32 In particular, following activation by ADAM9 by an as yet unknown mechanism, ADAM10 cleaves PrP C three amino acid residues prior to the GPI anchor attachment site. Also the central region of PrP C undergoes Figure 1.…”

The P’s and Q’s of cellular PrP-Aβ interactions

“…The endoproteases involved in PrP cleavage are a little more controversial than those for APP [29][30][31] but irrespective of whether or not PrP C is subject to cleavage by any endoprotease that also acts upon APP, there is nonetheless a curious parallel between the alternative cleavage pathways that can process APP and PrP C . 32 Thus β-like cleavage of PrP C to generate a C2-PrP fragment is potentially pathologic, as C2 PrP can form PrP Sc , and alternatively, an α-like cleavage to generate C1-PrP is protective with regards to prion misfolding since C1-PrP cannot form PrP Sc .…”

PrPCHomodimerization Stimulates the Production of PrPCCleaved Fragments PrPN1 and PrPC1

“…In contrast, its physiological function is still under debate (Roucou et al, 2004;Steele et al, 2007). PrP C undergoes several physiological cleavages, the ␤-and ␣-cleavage, at amino acids 89/90 and 110 -111/112 of human PrP C , respectively; and a cleavage at amino acids 228/229 resulting in ectodomain shedding (Chen et al, 1995;Mangé et al, 2004;Taylor et al, 2009). …”