Affinity pulldown of γ-secretase and associated proteins from human and rat brain.
γ-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 γ-secretase, but it has been suggested that γ-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 γ-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 γ-secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a γ-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 γ-secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ-secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the γ-secretase complex.
Teranishi
Yasuhiro
Y
The Karolinska Institutet (KI) Dainippon Sumitomo Pharma Alzheimer Center (KASPAC), KI-Alzheimer's Disease Research Center, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Novum, Huddinge, Sweden.
Hur
Ji-Yeun
JY
Welander
Hedvig
H
Frånberg
Jenny
J
Aoki
Mikio
M
Winblad
Bengt
B
Frykman
Susanne
S
Tjernberg
Lars O
LO
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D029543:Nucleocytoplasmic Transport Proteins; D010446:Peptide Fragments / Q000378:metabolism*; D053764:Presenilin-1 / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D050827:Syntaxin 1 / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D029543:Nucleocytoplasmic Transport Proteins; D010446:Peptide Fragments; D053764:Presenilin-1; D050827:Syntaxin 1; C484395:TMED10 protein, human; C100672:Tmed10 protein, rat; D053829:Amyloid Precursor Protein Secretases