Two‐step mechanism of inhibition of cathepsin B by cystatin C due to displacement of the proteinase occluding loop

Nycander, Maria; Estrada, Sergio; Mort, John S.; Abrahamson, Magnus; Björk, Ingemar

doi:10.1016/s0014-5793(97)01604-9

Cited by 62 publications

(63 citation statements)

References 30 publications

(80 reference statements)

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“…This concept agrees with higher endopeptidase activity and with the competition between the occluding loop and the propeptide following deprotonation of His 110 changing the pH from 4.0 to 6.0 (Quraishi et al 1999). The flexibility of the loop was further demonstrated by the fact that cystatins A and C were able to displace the loop (Nycander et al 1998;Pavlova et al 2000), and deletion of residues 108-119 abolished the exopeptidase activity of cathepsin B (Illy et al 1997).…”

supporting

confidence: 71%

“…It appears that the occluding loop of cathepsin B can be assisted in assuming its closed or open conformation by particular polypeptides with an induced fit mechanism that promotes either exo-or endoproteolysis. This observation forms the basis of cathepsin B inhibition by its own propeptide (Quraishi et al 1999) and its resistance to cystatin A and C inhibition (Nycander et al 1998;Pavlova et al 2000).…”

Section: Discussionmentioning

confidence: 91%

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A double‐headed cathepsin B inhibitor devoid of warhead

et al. 2008

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Most synthetic inhibitors of peptidases have been targeted to the active site for inhibiting catalysis through reversible competition with the substrate or by covalent modification of catalytic groups. Cathepsin B is unique among the cysteine peptidase for the presence of a flexible segment, known as the occluding loop, which can block the primed subsites of the substrate binding cleft. With the occluding loop in the open conformation cathepsin B acts as an endopeptidase, and it acts as an exopeptidase when the loop is closed. We have targeted the occluding loop of human cathepsin B at its surface, outside the catalytic center, using a high-throughput docking procedure. The aim was to identify inhibitors that would interact with the occluding loop thereby modulating enzyme activity without the help of chemical warheads against catalytic residues. From a large library of compounds, the in silico approach identified [2-[2-(2,4-dioxo-1,3-thiazolidin-3-yl)ethylamino]-2-oxoethyl] 2-(furan-2-carbonylamino) acetate, which fulfills the working hypothesis. This molecule possesses two distinct binding moieties and behaves as a reversible, double-headed competitive inhibitor of cathepsin B by excluding synthetic and protein substrates from the active center. The kinetic mechanism of inhibition suggests that the occluding loop is stabilized in its closed conformation, mainly by hydrogen bonds with the inhibitor, thus decreasing endoproteolytic activity of the enzyme. Furthermore, the dioxothiazolidine head of the compound sterically hinders binding of the C-terminal residue of substrates resulting in inhibition of the exopeptidase activity of cathepsin B in a physiopathologically relevant pH range.

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supporting

confidence: 71%

Section: Discussionmentioning

confidence: 91%

A double‐headed cathepsin B inhibitor devoid of warhead

et al. 2008

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“…52), again using the complex between cystatin B and papain as starting point for the model. Contrary to the inhibition of papain by cystatins, cathepsin B is inhibited in a two-step kinetic reaction (45). As proposed by Nycander et al (45), the first step is regulated by the anchoring of the N-terminal part of the cystatin in the non-primed S pockets.…”

Section: Fig 2 Alignment Of Cystatins With Determined Structuresmentioning

confidence: 97%

Crystal Structure of Human Cystatin D, a Cysteine Peptidase Inhibitor with Restricted Inhibition Profile

Alvarez-Fernandez

Liang

Abrahamson

et al. 2005

Journal of Biological Chemistry

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Cystatins are natural inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases. Cystatin D is a type 2 cystatin, a secreted inhibitor found in human saliva and tear fluid. Compared with its homologues, cystatin D presents an unusual inhibition profile with a preferential inhibition cathepsin S > cathepsin H > cathepsin L and no inhibition of cathepsin B or pig legumain. To elucidate the structural reasons for this specificity, we have crystallized recombinant human Arg 26 -cystatin D and solved its structures at room temperature and at cryo conditions to 2.5-and 1.8-Å resolution, respectively. Human cystatin D presents the typical cystatin fold, with a five-stranded antiparallel ␤-sheet wrapped around a five-turn ␣-helix. The structures reveal differences in the peptidase-interacting regions when compared with other cystatins, providing plausible explanations for the restricted inhibitory specificity of cystatin D for some papain-like peptidases and its lack of reactivity toward legumainrelated enzymes.

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“…The assumption that this interaction acts to constrain the occluding loop into a functional conformation is supported by several findings. Binding of the cysteine protease inhibitor cystatin C to cathepsin B has been shown kinetically to require a two-step mechanism [37], whereas only a single-step mechanism was required for the H110A mutant [38]. Similarly, the binding of the cathepsin B propeptide, which requires relocation of the occluding loop [10], was dramatically enhanced in the case of this mutation [15].…”

Section: Discussionmentioning

confidence: 99%

S′2 substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B

Krupa

Hasnain

Nägler

et al. 2002

Biochemical Journal

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The ability of the lysosomal cysteine protease cathepsin B to function as a peptidyldipeptidase (removing C-terminal dipeptides) has been attributed to the presence of two histidine residues (His110 and His111) present in the occluding loop, an extra peptide segment located in the primed side of the active-site cleft. Whereas His111 is unpaired, His110 is present as an ion pair with Asp22 on the main body of the protease. This ion pair appears to act as a latch to hold the loop in a closed position. The exopeptidase activity of cathepsin B, examined using quenched fluorescence substrates, was shown to have a 20-fold preference for aromatic side chains in the P′3 position relative to glutamic acid as the least favourable residue. Site-directed mutagenesis demonstrated that His111 makes a positive 10-fold contribution to the exopeptidase activity, whereas His110 is critical for this action with the Asp22—His110 ion pair stabilizing the electrostatic interaction by a maximum of 13.9kJ/mol (3.3kcal/mol). These studies showed that cathepsin B is optimized to act as an exopeptidase, cleaving dipeptides from protein substrates in a successive manner, because of its relaxed specificity in P′3 and its other subsites.

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Two‐step mechanism of inhibition of cathepsin B by cystatin C due to displacement of the proteinase occluding loop

Cited by 62 publications

References 30 publications

A double‐headed cathepsin B inhibitor devoid of warhead

A double‐headed cathepsin B inhibitor devoid of warhead

Crystal Structure of Human Cystatin D, a Cysteine Peptidase Inhibitor with Restricted Inhibition Profile

S′2 substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B

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