Structural study of regulated intramembrane proteolysis of p75NTR by γ-secretase.

Abstract

Programmed cell death (PCD) maintains the tissue size in a multicellular organism. Uncontrolled PCD in neurons has long been implicated in neurodegenerative diseases, e.g. Alzheimer's disease. p75 neurotrophin receptor (p75NTR) plays an important role in regulating neuronal growth and also involves in apoptotic signaling, which leads to morphological changes in cells in PCD. When binding to nerve growth factor (NGF), p75NTR maintains cell survival and proliferation; on the contrary, p75NTR induces apoptosis when the NGF precursor (pro-NGF) binds onto p75NTR. The complex formation of the pro-NGF, p75NTR, and sortilin initiates the apoptotic signaling. The subsequent proteolytic processing on p75NTR by γ-secretase relays the signaling to the neuronal apoptosis. p75NTR, the founding member of tumor necrosis factor (TNF) receptor superfamily, is a type I transmembrane protein, involved in the downstream cell death signaling after the release of intracellular domain (ICD) through regulated intramembrane proteolysis (RIP) executed by γ-secretase. Detailed molecular mechanism of how the γ-secretase cleaves p75NTR and releases its death domain is still unknown. To answer this question, our ultimate goal is to study the molecular mechanism of the p75NTR cleavage by γ-secretase using cryogenic electron microscopy (cryo-EM). We generated four p75NTR mutants with various truncated mutants in Sf9 insect cells to determine the cleavage site for the 𝛾-secretase. We characterized the bindings using size-exclusion chromatography, SDS-PAGE, and negative-stain EM. p75NTR mutants generated and characterized their bindings with γ -secretase. We will stabilize the complex formation and probe the interaction between these proteins using single-particle cryo-EM. The result will gain a fundamental understanding of how p75NTR cleavage triggers the neuronal apoptosis signaling.