Deducing the Transmembrane Domain Organization of Presenilin-1 in γ-Secretase by Cysteine Disulfide Cross-Linking

Kornilova, Anna Y.; Kim, Jennifer; Laudon, Hanna; Wolfe, Michael S.

doi:10.1021/bi060107k

Cited by 28 publications

(23 citation statements)

References 46 publications

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“…Thus, we speculate that the C-terminal region of TMD1 is an essential domain for maintaining the integrity of PS1 structure via hydrophobic interactions with other TMDs. The observation that C92 in TMD1 was cross-linked with C410 or C419 in TMD8, the latter being totally embedded within the lipid bilayer (Sato et al, 2008), by oxidation-mediated crosslinking (Kornilova et al, 2006), supports this view.…”

Section: Discussionmentioning

confidence: 63%

Participation of Transmembrane Domain 1 of Presenilin 1 in the Catalytic Pore Structure of the γ-Secretase

Takagi

Tominaga²,

Sato³

et al. 2010

J. Neurosci.

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␥-Secretase is an intramembrane-cleaving protease that is responsible for the generation of amyloid-␤ peptides linked to the pathogenesis of Alzheimer's disease. Using a substituted cysteine accessibility method, we have previously shown that the hydrophilic "catalytic pore" structure of ␥-secretase is formed by the transmembrane domains (TMDs) 6, 7, and 9 of presenilin 1 (PS1), the catalytic subunit of ␥-secretase, within the membrane. Here, we analyzed the structure in and around the first hydrophobic region, the putative TMD1, of PS1, of which the precise function as well as three-dimensional location within ␥-secretase remained unknown. We found that TMD1 is located in proximity to the catalytic GxGD and PAL motifs within the C-terminal fragment of PS1, facing directly the catalytic pore. Competition experiments using known ␥-secretase inhibitors suggested that the N-terminal region of TMD1 functions as a subsite during proteolytic action of the ␥-secretase. Intriguingly, binding of inhibitors affected water accessibility of residues at the membrane border of TMD1, suggesting the possibility of a dynamic motion of TMD1 during the catalytic process. Our results provide mechanistic insights into the functional role of TMD1 of PS1 in the intramembrane-cleaving activity of the ␥-secretase.

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Section: Discussionmentioning

confidence: 63%

Participation of Transmembrane Domain 1 of Presenilin 1 in the Catalytic Pore Structure of the γ-Secretase

Takagi

Tominaga²,

Sato³

et al. 2010

J. Neurosci.

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“…Thus, these mutations seem to be well tolerated and do not affect significantly the structure of PS1, as assessed in our cell biological experiments. Recently, Kornilova et al (42) have suggested that cysteines Cys-92, -410, and -419 may contribute to the active site of PS1. However, they did not check to what extent their replacement with serine (or alanine) interferes with this function.…”

Section: Discussionmentioning

confidence: 99%

Contribution of Presenilin Transmembrane Domains 6 and 7 to a Water-containing Cavity in the γ-Secretase Complex

Tolia

Chávez‐Gutiérrez

Strooper

2006

Journal of Biological Chemistry

132

150

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␥-Secretase is a multiprotein complex responsible for the intramembranous cleavage of the amyloid precursor protein and other type I transmembrane proteins. Mutations in Presenilin, the catalytic core of this complex, cause Alzheimer disease. Little is known about the structure of the protein and even less about the catalytic mechanism, which involves proteolytic cleavage in the hydrophobic environment of the cell membrane. It is basically unclear how water, needed to perform hydrolysis, is provided to this reaction. Presenilin transmembrane domains 6 and 7 seem critical in this regard, as each bears a critical aspartate contributing to catalytic activity. Current models imply that both aspartyl groups should closely oppose each other and have access to water. This is, however, still to be experimentally verified. Here, we have performed cysteine-scanning mutagenesis of both domains and have demonstrated that several of the introduced residues are exposed to water, providing experimental evidence for the existence of a water-filled cavity in the catalytic core of Presenilin. In addition, we have demonstrated that the two aspartates reside within this cavity and are opposed to each other in the native complex. We have also identified the conserved tyrosine 389 as a critical partner in the catalytic mechanism. Several additional amino acid substitutions affect differentially the processing of ␥-secretase substrates, implying that they contribute to enzyme specificity. Our data suggest the possibility that more selective ␥-secretase inhibitors could be designed. The Presenilin (PS)2 proteins are the prototypic members of a group of aspartic proteases involved in regulated intramembrane proteolysis, a mechanism responsible for cleavage of peptide bonds within the lipid bilayer (1, 2). More than 150 mutations in Presenilin 1 and 10 mutations in Presenilin 2 have been associated with Alzheimer disease (for a list of the mutations, see molgen.ua.ac.be/ADMutations), demonstrating their pivotal role in the pathogenesis of the disease. Presenilins are critical for the ␥-secretase cleavage of the amyloid precursor protein (APP) that generates the amyloid ␤ peptide (A␤) (3). They are also responsible for the intramembrane proteolysis of several other type I transmembrane proteins (reviewed in Ref. 4), including the S3 cleavage of Notch that releases the Notch intracellular domain (NICD), a major regulator of gene transcription (5). Together with Presenilin, three other membrane proteins are necessary and sufficient for processing by ␥-secretase (6 -8), i.e. Nicastrin, APH-1, and PEN-2. Nicastrin appears to recognize the free N terminus of potential substrates (9), whereas the catalytic core resides in Presenilin (reviewed in Ref. 10).Most recent topological studies propose a nine-transmembrane domain model for Presenilins, with the N terminus oriented toward the cytosol and the C terminus toward the extracellular space (11-13). Mutation of two conserved aspartates, Asp-257 and Asp-385, located in transmembrane domains (TMs)...

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“…Tyr389 may also contribute to the catalytic site . The conformation of the active site also depends on more remote sequences within PS1, such as the C-terminal PAL motif and Cys residues in TMD1 and TMD8 (Kornilova et al, 2006). Binding of an active-site-directed inhibitor prevents the disulphide cross-linking between TMD1 and TMD8, implicating their close proximity to or allosteric interaction with the catalytic site.…”

Section: Introductionmentioning

confidence: 99%

“…What the low-resolution 3D-EM does not reveal is how and where NCT, APH1 and PEN2 associate in the complex relative to PS or the contribution of TMDs bordering the translucent inner space. The lateral thin-density regions may for instance represent interfaces between different components, PS heterodimers [TMD1-TMD8 (Kornilova et al, 2006)] [TMD6-TMD7 (Sato et al, 2006;Tolia et al, 2006)] or even two PS molecules. Indeed, some studies indicate that the catalytic core consists of a PS dimer, one PS providing the substrate-binding site and the other the catalytic cleavage site (Cervantes et al, 2004;Schroeter et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Building γ-secretase – the bits and pieces

Spasić

Annaert

2008

Journal of Cell Science

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occurs under tight control of ER-Golgi recycling regulators, which allows defined quantities of complexes to reach postGolgi compartments, where ␥-secretase activity is regulated by multiple other factors. 3D-EM rendering reveals a complex with a translucent inner space, suggesting the presence of a water-filled cavity required for intramembrane proteolysis. Despite huge efforts, we are now only beginning to unravel the assembly, stoichiometry, activation and subcellular location of ␥-secretase. Presenilin at the heart of ␥-secretase Mammalian PSs come in two flavours, PS1 and PS2, which are highly homologous. Both are polytopic membrane proteins and have ten hydrophobic domains, of which nine are proposed to span the membrane (Laudon et al., 2005;Oh and Turner, 2005;Spasic et al., 2006) (Fig. 1). PSs are initially translocated in the ER as fulllength proteins but get converted by endoproteolysis within hydrophobic domain 7 to stable N-and C-terminal fragments (NTFs and CTFs, respectively) that associate to form a heterodimer. Although both fragments are part of the catalytic ␥-secretase, endoproteolysis is not a requirement for activity (Li et al., 2000;Steiner et al., 1999) (reviewed in Dillen and Annaert, 2006).Two highly conserved aspartate residues (Asp257 and Asp385 in human PS1) within transmembrane domain 6 (TMD6) and TMD7 constitute the core of the catalytic site. Mutation of either abolishes ␥-secretase activity Wolfe et al., 1999). Together with surrounding residues, they mark a highly conserved YD/GxGD consensus motif (Steiner et al., 2000). Tyr389 may also contribute to the catalytic site . The conformation of the active site also depends on more remote sequences within PS1, such as the C-terminal PAL motif and Cys residues in TMD1 and TMD8 (Kornilova et al., 2006). Binding of an active-site-directed inhibitor prevents the disulphide cross-linking between TMD1 and TMD8, implicating their close proximity to or allosteric interaction with the catalytic site. Finally, catalytic activity must be sensitive to subtle changes in the overall TMD conformation, given the effects of the scattered FAD-linked mutations.These interactions between remote parts of the molecule fit with the idea that PS1 adopts a ring-like topology (Annaert et al., 2001). This model is based on the identification of APP-binding regions in PS1 -TMD1 (extending to part of the first luminal loop domain) and the C-terminus -and led to the idea that the substrate must first bind to a remote docking site at the heterodimer interface prior to entering the active site. Indeed, subsequent imaging and biochemical studies have demonstrated that transition-state ␥-secretase inhibitors that bind to PS1 do not affect the interaction between PS1 and the APP C-terminal fragment (Berezovska et al., 2003; Esler et al., 2002). Similarly, inhibitors based on the substrate TMD do not prevent labelling of the complex by active-sitedirected inhibitors (Kornilova et al., 2003). Interestingly, these peptide inhibitors label only PS heterodimers and not th...

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Deducing the Transmembrane Domain Organization of Presenilin-1 in γ-Secretase by Cysteine Disulfide Cross-Linking

Cited by 28 publications

References 46 publications

Participation of Transmembrane Domain 1 of Presenilin 1 in the Catalytic Pore Structure of the γ-Secretase

Participation of Transmembrane Domain 1 of Presenilin 1 in the Catalytic Pore Structure of the γ-Secretase

Contribution of Presenilin Transmembrane Domains 6 and 7 to a Water-containing Cavity in the γ-Secretase Complex

Building γ-secretase – the bits and pieces

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