Year-end Sale % OFF 
Abstract
Intramembrane proteolysis is now firmly established as a prominent biological process, and structure elucidation is emerging as the new frontier in the understanding of these novel membrane-embedded enzymes. Reproducing this unusual hydrolysis within otherwise water-excluding transmembrane regions with purified proteins is a challenging prerequisite for such structural studies. Here we show the bacterial expression, purification, and reconstitution of proteolytically active signal peptide peptidase (SPP), a membrane-embedded enzyme in the presenilin family of aspartyl proteases. This finding formally proves that, unlike presenilin, SPP does not require any additional proteins for proteolysis. Surprisingly, the conserved C-terminal half of SPP is sufficient for proteolytic activity; purification and reconstitution of this engineered fragment of several SPP orthologues revealed that this region defines a functional domain for an intramembrane aspartyl protease. The discovery of minimal requirements for intramembrane proteolysis should facilitate mechanistic and structural analysis and help define general biochemical principles of hydrolysis in a hydrophobic environment.
Highlights
Tremendous efforts have focused on presenilin and the ␥-secretase complex, a putative membrane-embedded aspartyl protease that produces the amyloid- protein implicated in Alzheimer disease and that plays a key role in Notch receptor signaling during cell differentiation [5]
Four different variants were expressed in E. coli strain C43: signal peptide peptidase (SPP) from Homo sapiens, SPP from S. pombe, and SPP and SPP-like protein 3 (SPPL3) from Drosophila melanogaster, each as an N-terminal MBP fusion protein containing the PelB leader sequence for the proper insertion
The inhibition by (ZLL)2 ketone was concentration-dependent, with an IC50 of ϳ15 M compared with 2 M full-length SPP. These findings suggest that the chemistry of proteolysis is the same for both the full-length protein and the functional domain and that the four C-terminal transmembrane domains may be sufficient for SPP proteolytic activity
Summary
In each case the activity was blocked by the specific SPP transition-state analogue inhibitor, (ZLL)2 ketone, and the cleaved product formed from the bacterially expressed SPP co-migrated with product formed from solubilized membranes from mammalian cells (Fig. 1, b and f). D, proteolytic activity of DDM-solubilized human SPP functional domain and its inhibition by (ZLL)2 ketone.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
