A C-terminal Region of Signal Peptide Peptidase Defines a Functional Domain for Intramembrane Aspartic Protease Catalysis

Signal peptide peptidase (SPP) is an atypical aspartic protease that hydrolyzes peptide bonds within the transmembrane domain of substrates and is implicated in several biological and pathological functions. Here, we analyzed the structure of human SPP by electron microscopy and reconstructed the three-dimensional structure at a resolution of 22 Å . Enzymatically active SPP forms a slender, bullet-shaped homotetramer with dimensions of 85 ؋ 85 ؋ 130 Å . The SPP complex has four concaves on the rhombus-like sides, connected to a large chamber inside the molecule. Intriguingly, the N-terminal region of SPP is sufficient for the tetrameric assembly. Moreover, overexpression of the N-terminal region inhibited the formation of the endogenous SPP tetramer and the proteolytic activity within cells. These data suggest that the homotetramer is the functional unit of SPP and that its N-terminal region, which works as the structural scaffold, has a novel modulatory function for the intramembrane-cleaving activity of SPP.The intramembrane-cleaving proteases (I-CLiPs) 5 that sever the transmembrane domains of their substrates have been identified in a range of organisms and play a variety of roles in

Section: Discussionmentioning

confidence: 92%

Section: N-terminal Region Of Spp Is Responsible For Tetramericmentioning

confidence: 99%

Section: N-terminal Region Of Spp Is Responsible For Tetramericmentioning

confidence: 99%

See 1 more Smart Citation

Three-dimensional Structure of the Signal Peptide Peptidase

Miyashita

Maruyama

Isshiki

et al. 2011

“…SPP shares a series of inhibitors with ␥-secretase (25,26), suggesting that SPP and PS have similar catalytic sites. Although they are similar in protease activity, SPP does not require additional subunit(s) for activity (27). Thus, ␥-secretase subunits, NCT, Aph-1, and Pen2 may not be required for proteolysis itself.…”

mentioning

confidence: 99%

Nicastrin Is Dispensable for γ-Secretase Protease Activity in the Presence of Specific Presenilin Mutations

Futai

Yagishita

Ishiura

2009

␥-Secretase is a multisubunit membrane protein complex consisting of presenilin (PS1), nicastrin (NCT), anterior pharynx-1, and presenilin enhancer 2. To analyze the activity of familial Alzheimer disease mutants and to understand the roles of the subunits, we established a yeast transcriptional activator Gal4p system with artificial ␥-secretase substrates containing amyloid precursor protein or Notch fragments. The ␥-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4p release from the membrane-bound substrates, i.e. growth of yeast on histidine and adenine, or ␤-galactosidase assay. We screened and evaluated ␥-secretase mutants using this reconstitution system in yeast, which does not possess endogenous ␥-secretase activity. When we introduced familial Alzheimer mutants of PS1 in this system, their activities were shown to be loss of function. Although the protease activity of wild type PS1 depends on the other three subunits introduced, we obtained 15 new PS1 mutants, which are active in the absence of NCT. They possessed a S438P mutation at the ninth transmembrane domain (TM9) together with one missense mutation distributed through transmembrane and loop regions. These mutations were not related to familial Alzheimer mutations of PS1 as identified so far. The S438P mutant was partially active but required other mutations for full activation. Results of the ␤-galactosidase assay suggested that they have wild type protease activities, which were further confirmed by the endoproteolysis of PS1, amyloid ␤ peptides, and Notch intracellular domain production in mammalian cells. These results suggest that NCT is dispensable for the protease activity of ␥-secretase.

“…Thus, the structure of the catalytic site of SPP should resemble that of PS, although the water accessibility of residues around the active site in SPP remains unknown. Moreover, expression of human SPP in yeast and in bacteria reconstitutes the proteolytic activity (21,64), suggesting that the SPP protein has activity on its own and does not require other cofactors. Intriguingly, the recombinant C-terminal half of SPP is sufficient for proteolytic activity in vitro, indicating that this region is the minimal catalytic domain of SPP.…”

Section: Signal Peptide Peptidasementioning

confidence: 99%

Structural Biology of Presenilins and Signal Peptide Peptidases

Tomita

Iwatsubo

2013

Presenilin and signal peptide peptidase are multispanning intramembrane-cleaving proteases with a conserved catalytic GxGD motif. Presenilin comprises the catalytic subunit of ␥-secretase, a protease responsible for the generation of amyloid-␤ peptides causative of Alzheimer disease. Signal peptide peptidase proteins are implicated in the regulation of the immune system. Both protease family proteins have been recognized as druggable targets for several human diseases, but their detailed structure still remains unknown. Recently, the x-ray structures of some archaeal GxGD proteases have been determined. We review the recent progress in biochemical and biophysical probing of the structure of these atypical proteases.Intramembrane proteolysis is an atypical hydrolysis of peptide bonds within the lipid bilayer. Several lines of evidence suggest that this unusual cleavage is involved in numerous biological processes encompassing all branches of life. So far, three families of intramembrane-cleaving proteases have been discovered: rhomboid, site-2 protease, and GxGD proteases, including ␥-secretase and signal peptide peptidase (SPP). 2␥-Secretase is a key enzyme in the production of amyloid-␤ peptide (A␤), a major component of senile plaques in the brains of patients with Alzheimer disease, from amyloid-␤ precursor protein (APP) ( Fig. 1) (1). In particular, ␥-secretase cleavage determines the level of A␤42, the most aggregation-prone species of A␤ predominantly deposited in the brains of Alzheimer disease patients (2, 3). Moreover, ␥-secretase mediates the proteolysis-dependent signaling of several type I membrane proteins, including the Notch receptor, which is involved in the maintenance of stem cells and in the development of cancer (4).Thus, rational design of ␥-secretase inhibitors (GSIs) and modulators (GSMs) based on the molecular mechanism of ␥-secretase would pave the way toward development of novel drugs (5,6). The identity of the ␥-secretase had been a long-time mystery. PSEN genes were identified as familial Alzheimer disease (FAD)-linked genes encoding novel proteins, the presenilins (PSEN), lacking the traditional conserved aspartic protease motif (i.e. D(S/T)G) (7-9). Functional analyses in vitro and in vivo showed that presenilin (PS) is required for and modulates ␥-secretase-mediated cleavage of APP (10 -15). However, simple overexpression of PS does not significantly alter the ␥-secretase activity in cells. Soon after the biochemical analysis of PS, it was recognized that PS forms a large membrane protein complex, and only overexpressed PS protein that is incorporated into the complex affects the APP cleavage. Crucial evidence of the enzymatic function of PS was obtained by chemical biology; transition state analog-type GSIs directly target PS (16,17). In addition, the identification of membrane-embedded aspartate residues critical for enzymatic activity (18), as well as inhibitor binding experiments, revealed a novel conserved GxGD motif among species (19). In fact, the GxGD motif is conserved in ...