Signal peptide peptidase forms a homodimer that is labeled by an active site-directed gamma-secretase inhibitor.
Presenilin (PS) is the presumptive catalytic component of the intramembrane aspartyl protease gamma-secretase complex. Recently a family of presenilin homologs was identified. One member of this family, signal peptide peptidase (SPP), has been shown to be a protease, which supports the hypothesis that PS and presenilin homologs are related intramembrane-cleaving aspartyl proteases. SPP has been reported as a glycoprotein of approximately 45 kDa. Our initial characterization of SPP isolated from human brain and cell lines demonstrated that SPP is primarily present as an SDS-stable approximately 95-kDa protein on Western blots. Upon heating or treatment of this approximately 95-kDa SPP band with acid, a approximately 45-kDa band could be resolved. Co-purification of two different epitope-tagged forms of SPP from a stably transfected cell line expressing both tagged versions demonstrated that the approximately 95-kDa band is a homodimer of SPP. Pulse-chase metabolic labeling studies demonstrated that the SPP homodimer assembles rapidly and is metabolically stable. In a glycerol velocity gradient, SPP sedimented from approximately 100-200 kDa. Significantly the SPP homodimer was specifically labeled by an active site-directed photoaffinity probe (III-63) for PS, indicating that the active sites of SPP and PS/gamma-secretase are similar and providing strong evidence that the homodimer is functionally active. Collectively these data suggest that SPP exists in vivo as a functional dimer.
Nyborg
Andrew C
AC
Department of Neuroscience, Mayo Clinic Jacksonville, Jacksonville, Florida 32224, USA.
Kornilova
Anna Y
AY
Jansen
Karen
K
Ladd
Thomas B
TB
Wolfe
Michael S
MS
Golde
Todd E
TE
D053829:Amyloid Precursor Protein Secretases; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D019281:Dimerization; D010450:Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D000939:Epitopes / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism; D011233:Precipitin Tests; D053764:Presenilin-1; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013347:Subcellular Fractions; D014162:Transfection; D014238:Trichloroacetic Acid / Q000737:chemistry
D004791:Enzyme Inhibitors; D000939:Epitopes; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D053764:Presenilin-1; D014238:Trichloroacetic Acid; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C460085:signal peptide peptidase; C509280:BACE1 protein, human; D005990:Glycerol