Three-dimensional Structure of the Signal Peptide Peptidase

Miyashita, Hiroyuki; Maruyama, Yasushi; Isshiki, Hayato; Osawa, Satoko; Ogura, Toshihiko; Mio, Kazuhiro; Sato, Chikara; Tomita, Taisuke; Iwatsubo, Takeshi

doi:10.1074/jbc.m111.260273

Cited by 22 publications

(25 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, a detailed structural analysis of the whole ␥-secretase complex is still required. Intriguingly, PSH forms a tetrameric assembly with its N-ter- minal half, which is reminiscent of the single particle analysis data on human SPP (96). The majority of the catalytic site architecture in PSH is formed by its C-terminal half, which is in accordance with the result that the C-terminal portion is the minimal proteolytic core domain of SPP (64).…”

Section: Detailed Structure Of Gxgd Proteasessupporting

confidence: 73%

“…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. We have purified the human SPP protein using the baculovirus/Sf9 cell system and analyzed its structure by single particle analysis (96). Enzymatically active SPP forms a bullet-shaped homotetramer with dimensions of 85 ϫ 85 ϫ 130 Å at 22 Å resolution.…”

Section: Signal Peptide Peptidasementioning

confidence: 99%

See 1 more Smart Citation

Structural Biology of Presenilins and Signal Peptide Peptidases

Tomita

Iwatsubo

2013

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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 ...

show abstract

Section: Detailed Structure Of Gxgd Proteasessupporting

confidence: 73%

Section: Signal Peptide Peptidasementioning

confidence: 99%

Structural Biology of Presenilins and Signal Peptide Peptidases

Tomita

Iwatsubo

2013

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…33,34 Additionally, endogenous DDM-solubilized human and drosophila SPP formed higher molecular weight complexes around 180−200 kDa. 21 We utilized a photolabeling approach to elucidate whether endogenous active SPP is a homodimer or monomer. We synthesized JC10, a photoprobe structurally similar to JC8 except with the addition of a disulfide bond in the biotin linker that can be eluted from the streptavidin matrix with the reducing agent TCEP (Figure 3A).…”

Section: Resultsmentioning

confidence: 99%

γ-Secretase Inhibitors and Modulators Induce Distinct Conformational Changes in the Active Sites of γ-Secretase and Signal Peptide Peptidase

Gertsik

Chau

2015

ACS Chem. Biol.

View full text Add to dashboard Cite

γ-Secretase inhibitors (GSIs) and modulators (GSMs) are at the frontline of cancer and Alzheimer’s disease research, respectively. While both are therapeutically promising, not much is known about their interactions with proteins other than γ-secretase. Signal peptide peptidase (SPP), like γ-secretase, is a multispan transmembrane aspartyl protease that catalyzes regulated intramembrane proteolysis. We used active site-directed photophore walking probes to study the effects of different GSIs and GSMs on the active sites of γ-secretase and SPP and found that nontransition state GSIs inhibit labeling of γ-secretase by activity-based probes but enhance labeling of SPP. The opposite is true of GSMs, which have little effect on the labeling of γ-secretase but diminish labeling of SPP. These results demonstrate that GSIs and GSMs are altering the structure of not only γ-secretase but also SPP, leading to potential changes in enzyme activity and specificity that may impact the clinical outcomes of these molecules.

show abstract

“…AtSPP-GFP migrated at a size smaller than 65 kDa, whereas HsSPP-GFP migrated at a size more than 72 kDa. While the reasons underlying this anomalous migration remain to be elucidated, previous Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) studies have shown that HsSPP forms a high molecular mass complex under DDM-solubilized conditions [23]. HsSPP and AtSPP may assemble into complexes of different molecular masses.…”

Section: Resultsmentioning

confidence: 99%

Experimental detection of proteolytic activity in a signal peptide peptidase of Arabidopsis thaliana

et al. 2013

Self Cite

View full text Add to dashboard Cite

BackgroundSignal peptide peptidase (SPP) is a multi-transmembrane aspartic protease involved in intramembrane-regulated proteolysis (RIP). RIP proteases mediate various key life events by releasing bioactive peptides from the plane of the membrane region. We have previously isolated Arabidopsis SPP (AtSPP) and found that this protein is expressed in the ER. An AtSPP-knockout plant was found to be lethal because of abnormal pollen formation; however, there is negligible information describing the physiological function of AtSPP. In this study, we have investigated the proteolytic activity of AtSPP to define the function of SPPs in plants.ResultsWe found that an n-dodecyl-ß-maltoside (DDM)-solubilized membrane fraction from Arabidopsis cells digested the myc-Prolactin-PP-Flag peptide, a human SPP substrate, and this activity was inhibited by (Z-LL)2-ketone, an SPP-specific inhibitor. The proteolytic activities from the membrane fractions solubilized by other detergents were not inhibited by (Z-LL)2-ketone. To confirm the proteolytic activity of AtSPP, the protein was expressed as either a GFP fusion protein or solely AtSPP in yeast. SDS-PAGE analysis showed that migration of the fragments that were cleaved by AtSPP were identical in size to the fragments produced by human SPP using the same substrate. These membrane-expressed proteins digested the substrate in a manner similar to that in Arabidopsis cells.ConclusionsThe data from the in vitro cell-free assay indicated that the membrane fraction of both Arabidopsis cells and AtSPP recombinantly expressed in yeast actually possessed proteolytic activity for a human SPP substrate. We concluded that plant SPP possesses proteolytic activity and may be involved in RIP.

show abstract

Three-dimensional Structure of the Signal Peptide Peptidase

Cited by 22 publications

References 53 publications

Structural Biology of Presenilins and Signal Peptide Peptidases

Structural Biology of Presenilins and Signal Peptide Peptidases

γ-Secretase Inhibitors and Modulators Induce Distinct Conformational Changes in the Active Sites of γ-Secretase and Signal Peptide Peptidase

Experimental detection of proteolytic activity in a signal peptide peptidase of Arabidopsis thaliana

Contact Info

Product

Resources

About