Proteomic Profiling of γ-Secretase Substrates and Mapping of Substrate Requirements

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Abstract
The presenilin/γ-secretase complex, an unusual intramembrane aspartyl protease, plays an essential role in cellular signaling and membrane protein turnover. Its ability to liberate numerous intracellular signaling proteins from the membrane and also mediate the secretion of amyloid-β protein (Aβ) has made modulation of γ-secretase activity a therapeutic goal for cancer and Alzheimer disease. Although the proteolysis of the prototypical substrates Notch and β-amyloid precursor protein (APP) has been intensely studied, the full spectrum of substrates and the determinants that make a transmembrane protein a substrate remain unclear. Using an unbiased approach to substrate identification, we surveyed the proteome of a human cell line for targets of γ-secretase and found a relatively small population of new substrates, all of which are type I transmembrane proteins but have diverse biological roles. By comparing these substrates to type I proteins not regulated by γ-secretase, we determined that besides a short ectodomain, γ-secretase requires permissive transmembrane and cytoplasmic domains to bind and cleave its substrates. In addition, we provide evidence for at least two mechanisms that can target a substrate for γ cleavage: one in which a substrate with a short ectodomain is directly cleaved independent of sheddase association, and a second where a substrate requires ectodomain shedding to instruct subsequent γ-secretase processing. These findings expand our understanding of the mechanisms of substrate selection as well as the diverse cellular processes to which γ-secretase contributes. All cells face the challenge of removing transmembrane proteins from the lipid bilayer for the purpose of signaling or degradation. One molecular solution to this problem is the multiprotein enzyme complex γ-secretase, which is able to hydrolyze several known transmembrane proteins within the hydrophobic lipid environment. Due to its central role in the pathogenesis of Alzheimer disease, modulation of γ-secretase activity has become a therapeutic goal. However, the number and diversity of proteins that can be cleaved by this protease remain unknown, and the attributes that target these proteins to γ-secretase are unclear. In this study, we used an unbiased approach to substrate identification and surveyed the proteome for targets of γ-secretase. Of the thousands of proteins detectable, only a relative few were substrates of γ-secretase, all of which were type I transmembrane proteins. In addition to validating several of these novel substrates, we compared them to other proteins that we identified as nonsubstrates and determined that there are specific domains that can activate or inhibit γ-secretase processing. These findings should advance our understanding of the many cellular processes regulated by γ-secretase and may offer insights into how γ-secretase can be exploited for therapeutic purposes.

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