Abstract
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.
Highlights
Presenilins (PSs)1 were first identified through genetic studies demonstrating that mutations in them caused Alzheimer’s disease [1, 2]
Both of these antibodies recognized a band of ϳ95 kDa in human brain and in human cell lines indicating that this band is likely to contain fulllength signal peptide peptidase (SPP) (Fig. 1a)
With SPP, we found no evidence for any effect of the epitope tags on the characteristics of the protein that we analyzed in this study
Summary
Presenilins (PSs) were first identified through genetic studies demonstrating that mutations in them caused Alzheimer’s disease [1, 2]. Mutation of either of two conserved aspartates present in adjacent transmembrane domain results in dominant negative PSs that inhibit ␥-secretase activity [14] Despite these recent advances in the understanding of ␥-secretase, the multiprotein complex mediating this activity poses significant problems for studies that would inevitably lead to a more definitive structural and mechanistic understanding of this protease. After its initial in silico identification, one of the presenilin homologs/SPP was identified as an aspartyl I-CliP [17] This protein has been termed signal peptide peptidase as it has been shown to carry out the intramembrane cleavage of signal peptides of major histocompatibility complex class I molecules and hepatitis C virus polyprotein following the initial cleavage of these type II membrane proteins by signal peptidase.
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