Crystal Structure of an Activation Intermediate of Cathepsin E

Ostermann, Nils; Gerhartz, Bernd; Worpenberg, Susanne; Trappe, Jörg; Eder, Jörg

doi:10.1016/j.jmb.2004.07.073

Cited by 29 publications

(34 citation statements)

References 40 publications

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“…The substitution of Tyr-389 with cysteine, which is shorter than tyrosine and not as good a proton donor, could then affect the catalysis by altering the protonation state of the catalytic residue. Similar interactions underlying the aspartyl-mediated catalytic mecha- nism have been reported for cathepsin E (A1 family of aspartic proteases), with Tyr-20 forming a hydrogen bond with Asp-43 (53) and Trichoderma reesei cellobiohydrolase II (family B of glycoside hydrolases), where Tyr-179 interacts with deprotonated Asp-175, maintaining it in a charged state (54,55).…”

Section: Discussionsupporting

confidence: 53%

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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Section: Discussionsupporting

confidence: 53%

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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“…His residues are deprotonated and thereby uncharged at neutral pH but are protonated and positively charged below pH ~6.5. To search for potential heparin-binding regions and, in particular, to search for surfaceexposed His residues that might be involved in pH-dependent interaction with heparin, we constructed a three-dimensional model of murine cathepsin E based on the refined 2.35 Å X-ray structure for human cathepsin E (Ostermann et al, 2004). Because heparin binding was not dependent on dimerization and dimerization does not appear to involve an extensive subunit-subunit interface (Ostermann et al, 2004), only the monomer was modelled.…”

Section: Discussionmentioning

confidence: 99%

“…Three-dimensional modelling of mouse cathepsin E A homology model was constructed using the coordinates for human cathepsin E (PDB code 1TZS) (Ostermann et al, 2004). The sequences of mouse and human cathepsin E were aligned using ClustalW.…”

Section: Affinity Chromatographymentioning

confidence: 99%

A role for cathepsin E in the processing of mast-cell carboxypeptidase A

Henningsson

Yamamoto

Säftig

et al. 2005

Journal of Cell Science

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Mast-cell carboxypeptidase A is stored in the secretory granule and is released, together with a range of other inflammatory mediators, upon mast-cell degranulation. Carboxypeptidase A, like all mast-cell proteases, is stored in the granule as an active enzyme (i.e. with its propeptide removed). Although the processing mechanisms for the other classes of mast-cell proteases (in particular the chymases) have been clarified to some extent, the processing of procarboxypeptidase A is poorly characterized. Here, we show that mast cells from mice lacking the aspartic protease cathepsin E display an accumulation of procarboxypeptidase A, indicating a defect in carboxypeptidase-A processing. By contrast, mast cells lacking cathepsins B, L or D have normal carboxypeptidase-A processing. Furthermore, recombinant cathepsin E was found to process recombinant procarboxypeptidase A in vitro, under conditions resembling those found in mast-cell granules. Immunohistochemical analysis revealed staining for cathepsin E in mast cells from normal mice but not in mast cells from mice lacking heparin, indicating that cathepsin E is bound to heparin proteoglycan within mast-cell granules. In accordance with this notion, affinity chromatography showed that recombinant cathepsin E bound strongly to heparin under acidic conditions (the conditions prevailing in mast-cell granules) but not at neutral pH. Moreover, mast-cell degranulation resulted in the release of cathepsin E. Taken together, our results indicate that cathepsin E is located in mast-cell secretory granules in complex with heparin proteoglycans, and that it has a role in the processing of procarboxypeptidase A into active protease.

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“…Our model of CDR1 foresees no obvious partners for intramolecular disulfide bond formation for Cys 358 and Cys 396 leaving them as prime candidates for intermolecular disulfide bond formation despite their slightly concealed location. This disulfide bridge-mediated homodimerization reminds of human cathepsin E, because this enzyme, to the best of our knowledge, is the only A1 AP that exists as a disulfide bridged homodimer (29,30). However, although cathepsin E monomers are fully catalytically active and the disulfide bond appears to increase mostly the enzyme FIGURE 7.…”

Section: Discussionmentioning

confidence: 87%

“…required for optimal catalytic activity are some of the unusual properties observed for recombinant CDR1 that distinguishes it from the typical plant APs. Even though there are examples of aspartic protease displaying at least one of the above mentioned properties: CND41 is well known for its insensitivity to pepstatin (3), renin, and the human immunodeficiency virus-proteinase are both optimally active at pH around 6.0 (25,26), BACE and renin show significant protease activity without irreversible removal of their prosegment (27,28), and cathepsin E is active as a homodimer (29,30), CDR1 is unique in displaying all these features. This reflects a pattern of unprecedented functional complexity and is consistent with a specific biological function.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Recombinant CDR1, an Arabidopsis Aspartic Proteinase Involved in Disease Resistance

Simões

Faro

Bur

et al. 2007

Journal of Biological Chemistry

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The Arabidopsis thaliana constitutive disease resistance 1 (CDR1) gene product is an aspartic proteinase that has been implicated in disease resistance signaling (Xia, Y., Suzuki, H., Borevitz, J., Blount, J., Guo, Z., Patel, K., Dixon, R. A., and Lamb, C. (2004) EMBO J. 23, 980 -988). This apoplastic enzyme is a member of the group of "atypical" plant aspartic proteinases. As for other enzymes of this subtype, CDR1 has remained elusive until recently as a result of its unusual properties and localization. Here we report on the heterologous expression and characterization of recombinant CDR1, which displays unique enzymatic properties among plant aspartic proteinases. The highly restricted specificity requirements, insensitivity toward the typical aspartic proteinase inhibitor pepstatin A, an unusually high optimal pH of 6.0 -6.5, proteinase activity without irreversible prosegment removal, and dependence of catalytic activity on formation of a homo-dimer are some of the unusual properties observed for recombinant CDR1. These findings unveil a pattern of unprecedented functional complexity for Arabidopsis CDR1 and are consistent with a highly specific and regulated biological function. Plant aspartic proteinases (APs)3 are widely distributed in the plant kingdom and have been detected or purified from monocot and dicot species as well as gymnosperms (1). APs are synthesized as single chain preproenzymes and, upon activation, converted to mature two-chain enzymes. A characteristic feature of "typical" plant AP precursors is the presence of a protein domain of ϳ100 amino acids known as the plant-specific insert, which is highly similar to saposin-like proteins and is removed from the precursors upon activation into the mature form of the enzymes (1).Recently published results on plant APs with unexpected localizations, unusual sequences, and novel structural arrangements (2-10) as well as the identification of a wide variety of AP-like proteins in the Arabidopsis genome (11, 12) have triggered a redefinition of the classification of plant APs. Faro and Gal have recently proposed a new classification that takes into account the diversity of plant APs by grouping them into typical, nucellin-like, and atypical members, with the latter group comprising the majority of the newly identified Arabidopsis APs (11). Despite having low sequence identity, these atypical and nucellin-like APs share several common features such as 1) the absence of the internal segment plant-specific insert in their sequence, 2) an unusually high number of cysteines, 3) the type of amino acids preceding the first catalytic triad, and 4) unexpected localizations, which clearly differentiate them from the well studied typical plant APs. Thus far, only a rather small number of atypical and nucellin-like APs have been studied (2-10). However, the proposed roles in highly regulated processes like plastid homeostasis (tobacco CND41) (9, 10), disease resistance (Arabidopsis CDR1) (7), or programmed cell death (Barley nucellin and Arabidopsis PCS1) (2,...

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Crystal Structure of an Activation Intermediate of Cathepsin E

Cited by 29 publications

References 40 publications

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

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

A role for cathepsin E in the processing of mast-cell carboxypeptidase A

Characterization of Recombinant CDR1, an Arabidopsis Aspartic Proteinase Involved in Disease Resistance

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