Structural basis for specificity of papain-like cysteine protease proregions toward their cognate enzymes

Groves, Matthew R.; Coulombe, René; Jenkins, John A.; Cygler, Mirosław

doi:10.1002/(sici)1097-0134(19980901)32:4<504::aid-prot8>3.0.co;2-f

Cited by 72 publications

(53 citation statements)

References 34 publications

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“…Prosegments appear to adopt stably folded structures when bound to the protease domain in their cognate proenzymes and have regions of multiple interfacial contacts with catalytic domains (12), which govern their affinities and specificities (27). Although the majority of isolated prosegments of proteases are either unstructured (23) or possess a loosely packed structure (28), the strongest inhibitory properties demonstrate those isolated prosegments, which reveal a folded tertiary structure (25).…”

Section: Discussionmentioning

confidence: 99%

Prosegment of Tripeptidyl Peptidase I Is a Potent, Slow-binding Inhibitor of Its Cognate Enzyme

Golabek

Dolzhanskaya

Walus

et al. 2008

Journal of Biological Chemistry

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Tripeptidyl peptidase I (TPP I) is the first mammalian representative of a family of pepstatin-insensitive serine-carboxyl proteases, or sedolisins. The enzyme acts in lysosomes, where it sequentially removes tripeptides from the unmodified N terminus of small, unstructured polypeptides. Naturally occurring mutations in TPP I underlie a neurodegenerative disorder of childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2). Generation of mature TPP I is associated with removal of a long prosegment of 176 amino acid residues from the zymogen. Here we investigated the inhibitory properties of TPP I prosegment expressed and isolated from Escherichia coli toward its cognate protease. We show that the TPP I prosegment is a potent, slow-binding inhibitor of its parent enzyme, with an overall inhibition constant in the low nanomolar range. We also demonstrate the protective effect of the prosegment on alkaline pH-induced inactivation of the enzyme. Interestingly, the inhibitory properties of TPP I prosegment with the introduced classic late infantile neuronal ceroid lipofuscinosis disease-associated mutation, G77R, significantly differed from those revealed by wild-type prosegment in both the mechanism of interaction and the inhibitory rate. This is the first characterization of the inhibitory action of the sedolisin prosegment. Tripeptidyl peptidase I (TPP I)2 is an acidic lysosomal hydrolase sequentially removing tripeptides en bloc from the N terminus of small, unstructured polypeptides (for a review, see Ref.1). The natural substrate(s) of the enzyme still remains elusive. TPP I is widely distributed in the human body, with a high expression level in brain tissue (2, 3). Naturally occurring mutations in TPP I underlie a devastating neurodegenerative disorder of early childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2) (4).On the basis of significant sequence homology and inhibitory studies, TPP I was assigned to a family of pepstatin-insensitive serine-carboxyl proteases recently renamed sedolisins (for a Like most proteolytic enzymes, TPP I is synthesized as an inactive precursor, zymogen (8, 9). The prepro-TPP I consists of a 19-amino acid (aa) signal peptide cleaved off when the newly forming polypeptide chain is inserted into the endoplasmic reticulum lumen, the 176-aa prosegment (or prodomain), and a 368-aa catalytic domain. As we demonstrated earlier, the removal of the prosegment from a purified TPP I proenzyme (pro-TPP I) occurs via an autocatalytic, intramolecular mechanism, whereby the mature enzyme does not significantly participate in its own generation (10). Efficient in vitro processing and autoactivation of the proenzyme is triggered by lowering the pH of the proenzyme solution to below 4.5. When pro-TPP I is activated at pH 4.5 and above, it is processed slowly and generates additional 6-and 14-aa N-terminal extensions in the newly formed polypeptide, rendering it enzymatically inactive (10). However, as we showed earlier, the presence of polyanionic compounds, such...

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Section: Discussionmentioning

confidence: 99%

Prosegment of Tripeptidyl Peptidase I Is a Potent, Slow-binding Inhibitor of Its Cognate Enzyme

Golabek

Dolzhanskaya

Walus

et al. 2008

Journal of Biological Chemistry

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“…The primary role of this motif is thought to be stabilization of the structural arrangement of helixes ␣1-␣3 and strand ␤1 of the pro-peptide into a discrete globular domain. In procaricain/procathepsin L, the packing of helices ␣1 and ␣2 is determined by the interactions of the three aromatic residues F/W22p, W25p, and F/W46p (1PCI numbering), among which the last residue is part of the ERFNIN motif (44). The residues that occupy the corresponding positions in the rproDer p 1-CN structure are F8, Y11, and F32 (Fig.…”

Section: The Folding Of the Pro-peptidementioning

confidence: 99%

“…However, neither E38p nor R42p, which are part of the ERFNIN motif, are structurally conserved in rproDer p 1-CN, and this part of the network is thus not present in the mite enzyme. The relative orientation of helix ␣2 and strand ␤1 is similar in procaricain and procathepsin L, and the two residues that stabilize this fold are I53p and N57p through side chain hydrophobic contacts and hydrogen bonds, respectively (44). Both residues are part of the ERFNIN motif and are again not conserved in rproDer p 1-CN because they are located C terminally in helix ␣2, which is truncated in rproDer p 1-CN.…”

Section: The Folding Of the Pro-peptidementioning

confidence: 99%

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The Crystal Structure of Recombinant proDer p 1, a Major House Dust Mite Proteolytic Allergen

Meno

Thorsted

Ipsen

et al. 2005

The Journal of Immunology

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Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant molecule was produced. The sequence of the proDer p 1 allergen was modified to reduce glycosylation and to abolish enzymatic activity. The resulting rproDer p 1 preparation was homogenous and stable and yielded crystals diffracting to a resolution of 1.61 Å. The active site is located in a large cleft on the surface of the molecule. The 80-aa pro-peptide adopts a unique fold that interacts with the active site cleft and a substantial adjacent area on the mature region, excluding access to the cleft and the active site. Studies performed using crossed-line immunoelectrophoresis and IgE inhibition experiments indicated that several epitopes are covered by the pro-peptide and that the epitopes on the recombinant mature molecule are indistinguishable from those on the natural one. The structure confirms previous results suggesting a preference for aliphatic residues in the important P2 position in substrates. Sequence variations in related species are concentrated on the surface, which explains the existence of cross-reacting and species-specific antibodies. This study describes the first crystal structure of one of the clinically most important house dust mite allergens, the cysteine protease Der p 1.

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“…This mechanism prevents premature proteolysis and targets enzymic activities to the compartment where their function is required. In many cases, activation occurs by the removal of an inhibitory segment, usually located in the N-terminus of the peptidase [2,4,5].…”

Section: Introductionmentioning

confidence: 99%

Identification of furin pro-region determinants involved in folding and activation

Bissonnette

Charest

Longpré

et al. 2004

Biochemical Journal

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The pro-region of the subtilisin-like convertase furin acts early in the biosynthetic pathway as an intramolecular chaperone to enable proper folding of the zymogen, and later on as an inhibitor to constrain the activity of the enzyme until it reaches the transGolgi network. To identify residues that are important for proregion function, we initially identified amino acids that are conserved among the pro-regions of various mammalian convertases. Site-directed mutagenesis of 17 selected amino acids within the 89-residue pro-region and biosynthetic labelling revealed that I60A-furin and H66A-furin were rapidly degraded in a proteasome-dependent manner, while W34A-furin and F67A-furin did not show any autocatalytic activation. Intriguingly, the latter mutants proteolytically cleaved pro-von Willebrand factor precursor to the mature polypeptide, suggesting that the mutations permitted proper folding, but did not allow the pro-region to exercise its role in inhibiting the enzyme. Homology modelling of furin's pro-region revealed that residues Ile-60 and His-66 might be crucial in forming the binding interface with the catalytic domain, while residues Trp-34 and Phe-67 might be involved in maintaining a hydrophobic core within the pro-region itself. These results provide structural insights into the dual role of furin's proregion.

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Structural basis for specificity of papain-like cysteine protease proregions toward their cognate enzymes

Cited by 72 publications

References 34 publications

Prosegment of Tripeptidyl Peptidase I Is a Potent, Slow-binding Inhibitor of Its Cognate Enzyme

Prosegment of Tripeptidyl Peptidase I Is a Potent, Slow-binding Inhibitor of Its Cognate Enzyme

The Crystal Structure of Recombinant proDer p 1, a Major House Dust Mite Proteolytic Allergen

Identification of furin pro-region determinants involved in folding and activation

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