P2‐279: Affinity pulldown of gamma‐secretase and associated proteins from human and rat brain

Abstract

The deposition of the Aβ is a pathogenic event in Alzheimer's disease and γ-secretase is responsible for the final cleavage of amyloid precursor protein to generate Aβ. γ-Secretase is a transmembrane (TM) aspartyl protease complex which catalyzes the cleavage of type I TM proteins, including APP and Notch. Four proteins: PS, Nct, Aph-1 and Pen-2 are necessary and sufficient for an active γ-secretase complex, but little is known about how γ-secretase is regulated. Other γ-secretase associated proteins may affect γ-secretase activity, and studies in cell lines have shown that TMP21, CD147 and many other proteins associate with the γ-secretase complex and regulate Aβ production. To investigate whether there are γ-secretase associate proteins in brain, we designed and synthesized a γ-secretase-inhibitor with hydrophilic long linker and a cleavable biotin group (GCB) for effective and specific pulldown of γ-secretase. As a starting material, we used a microsome fraction prepared from rat brain or human brain. The microsomal pellet was dissolved in CHAPSO, the supernatant was incubated with GCB, and streptavidin beads were used for pulldown. The captured proteins from brain samples were digested by trypsin and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The captured proteins were identified by the MASCOT algorithm using the NCBInr data base. The specificity of the pulldown was confirmed by competition using a non-biotinylated inhibitor. Elution by DTT clearly reduced nonspecific binding compared to elution by SDS sample buffer. All the known g-secretase components were identified by LC-MS/MS, as well as the previously reported γ-secretase associated TMP21 and the PS associated syntaxin1 was found to be associated to γ-secretase in rat brain. Finally, we prepared and analyzed membranes from human brain, and identified over 50 proteins potentially associated with γ-secretase. We are currently evaluating their association to γ-secretase and studying their effect on γ-secretase activity. We suggest that the present method can be used for further studies on the composition of the γ-secretase complex.