digitonin and Alzheimer-Disease

The four-component intramembrane protease γ-secretase is intricately linked to the development of Alzheimer's disease. Despite recent structural advances, the transmembrane segments (TMs) of γ-secretase remain to be specifically assigned. Here we report a 3D structure of human γ-secretase at 4.32-Å resolution, determined by single-particle, electron cryomicroscopy in the presence of digitonin and with a T4 lysozyme fused to the amino terminus of presenilin 1 (PS1). The overall structure of this human γ-secretase is very similar to that of wild-type γ-secretase determined in the presence of amphipols. The 20 TMs are unambiguously assigned to the four components, revealing principles of subunit assembly. Within the transmembrane region, PS1 is centrally located, with its amino-terminal fragment (NTF) packing against Pen-2 and its carboxyl-terminal fragment (CTF) interacting with Aph-1. The only TM of nicastrin associates with Aph-1 at the thick end of the TM horseshoe, and the extracellular domain of nicastrin directly binds Pen-2 at the thin end. TM6 and TM7 in PS1, which harbor the catalytic aspartate residues, are located on the convex side of the TM horseshoe. This structure serves as an important framework for understanding the function and mechanism of γ-secretase.

Topics: Alzheimer Disease; Amyloid Precursor Protein Secretases; Catalysis; Cell Membrane; Cryoelectron Microscopy; Detergents; Digitonin; Endopeptidases; HEK293 Cells; Humans; Image Processing, Computer-Assisted; Membrane Glycoproteins; Membrane Proteins; Peptide Hydrolases; Presenilin-1; Protein Binding; Protein Structure, Secondary

GAPDH is a redox-sensitive glycolytic enzyme that also promotes apoptosis when translocated to the nucleus and associates with aggregate-prone proteins involved in neurodegenerative disorders. Recent evidence indicates that polymorphic variation within GAPDH genes is associated with an elevated risk of developing Alzheimer's disease (AD). We previously demonstrated that GAPDH readily undergoes disulfide bonding following oxidant exposure, although the consequence of disulfide bonding on GAPDH activity or function is unknown. Here we show that increased GAPDH disulfide bonding is observed in detergent-insoluble extracts from AD patient and transgenic AD mouse brain tissue compared with age-matched controls. Exposure of primary rat cortical neurons to the pro-oxidant amyloid beta peptide promotes nuclear accumulation of a disulfide-linked form of GAPDH, which becomes detergent-insoluble. Disulfide bonding leads to a reduction in GAPDH enzymatic activity and correlates with the appearance of punctate aggregate-like GAPDH staining within the cytoplasm of both oxidant-treated HT22 cells and amyloid beta-treated primary cortical neurons. Our findings suggest that disulfide bonding of GAPDH and subsequent protein aggregate formation may have relevance to the pathophysiology of AD.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Animals; Cell Line; Cell Nucleus; Cysteine; Cytoplasm; Cytosol; Detergents; Digitonin; Disulfides; Dithiothreitol; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Female; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); Glycolysis; Humans; Hydrogen Peroxide; Immunoblotting; Male; Mice; Mice, Transgenic; Models, Biological; Neurodegenerative Diseases; Neurons; Oxidants; Oxidation-Reduction; Polymorphism, Genetic; Protein Isoforms; Rats; Reactive Oxygen Species; Risk; Time Factors