Affinity pulldown of ?-secretase and associated proteins from human and rat brain.

ABSTRACT: ?-Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins, including amyloid precursor protein (APP) and Notch. A functional complex is dependent on the assembly of four proteins: presenilin (PS), nicastrin, Aph-1 and Pen-2. Little is known about how the substrates are selected by ?-secretase, but it has been suggested that ?-secretase associated proteins (GSAPs) could be of importance. For instance, it was recently reported from studies in cell lines that TMP21, a transmembrane protein involved in trafficking, binds to ?-secretase and regulates the processing of APP-derived substrates without affecting Notch cleavage. Here, we present an efficient and selective method for purification and analysis of ?-secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a ?-secretase inhibitor coupled to biotin via a long linker and a S-S bridge. After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to LC-MS/MS analysis, and proteins were identified by sequence data from MS/MS spectra. All of the known ?-secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to ?-secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the ?-secretase complex.

SUBMITTER: Teranishi Y 

PROVIDER: S-EPMC4373488 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature