DNA Accelerates the Inhibition of Human Cathepsin V by Serpins

Ong, Poh Chee; McGowan, Sheena; Pearce, Mary Catherine; Irving, James A.; Kan, Wan-Ting; Grigoryev, Sergei A.; Turk, Boris; Silverman, Gary A.; Brix, Klaudia; Bottomley, Stephen P.; Whisstock, James C.; Pike, Robert N.

doi:10.1074/jbc.m706991200

Cited by 42 publications

(31 citation statements)

References 43 publications

(40 reference statements)

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“…1b, both proteins showed a mobility shift toward the positive electrode following incubation with heparin. We also examined the ability to bind DNA using agarose gel mobility shift analysis but found no binding for either serpin (data not shown), in agreement with the findings of Ong et al (21) for SCCA-1.…”

Section: Serpins Scca-1 and Scca-2 Bind The Glycosaminoglycansupporting

confidence: 77%

“…This template mechanism is found for most plasma serpin enhancement by heparin, but for SCCA-1 rate enhancement in the presence of DNA, a non-templating saturation effect was seen as a result of protease binding only (21). Using a range of heparin concentrations (Fig.…”

Section: Binding Of Heparin Quenches the Intrinsic Tryptophanmentioning

confidence: 99%

“…For a number of serpins, this activity can be enhanced by the presence of cofactors, most notably the effects of glycosaminoglycans on plasma serpin inhibition. Other ligands can also modulate serpin activity, and Ong et al (21) showed that SCCA-1 activity against cathepsin V can be enhanced in the presence of DNA, but that unlike the nuclear serpin MENT, SCCA-1 does not bind DNA directly, and enhancement appears to be mediated via the protease.…”

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confidence: 99%

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Heparin Enhances Serpin Inhibition of the Cysteine Protease Cathepsin L

Higgins

Fox

Kowalski

et al. 2010

Journal of Biological Chemistry

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The glycosaminoglycan heparin is known to possess antimetastatic activity in experimental models and preclinical studies, but there is still uncertainty over its mechanism of action in this respect. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III, but a similar cofactor role has not been previously investigated for proteases linked to metastasis. The squamous cell carcinoma antigens (serpins B3 and B4) are tumor-associated proteins that can inhibit papainlike cysteine proteases, including cathepsins L, K, and S. In this study, we show that SCCA-1 (B3) and SCCA-2 (B4) can bind heparin as demonstrated by affinity chromatography, native PAGE gel shifts, and intrinsic fluorescence quenching. Binding was specific for heparin and heparan sulfate but not other glycosaminoglycans. The presence of heparin accelerated inhibition of cathepsin L by both serpins, and in the case of SCCA-1, heparin increased the second order inhibition rate constant from 5.4 ؋ 10 5 to >10 8 , indicating a rate enhancement of at least 180-fold. A templating mechanism was shown, consistent with ternary complex formation. Furthermore, SCCA-1 inhibition of cathepsin L-like proteolytic activity secreted from breast and melanoma cancer cell lines was significantly enhanced by heparin. This is the first example of glycosaminoglycan enhancement of B-clade serpin activity and the first report of heparin acting as a cofactor in serpin cross-class inhibition of cysteine proteases. Most importantly, this finding raises the possibility that the anticancer properties of heparin may be due, at least partly, to enhanced inhibition of prometastatic proteases.

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Section: Serpins Scca-1 and Scca-2 Bind The Glycosaminoglycansupporting

confidence: 77%

Section: Binding Of Heparin Quenches the Intrinsic Tryptophanmentioning

confidence: 99%

mentioning

confidence: 99%

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Heparin Enhances Serpin Inhibition of the Cysteine Protease Cathepsin L

Higgins

Fox

Kowalski

et al. 2010

Journal of Biological Chemistry

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“…It was demonstrated that the inhibitory activity of MENT on cathepsin L, rather than DNA binding, is crucial for mediating its effect (29). DNA was reported to accelerate the rate at which MENT inhibited cathepsin V, a human ortholog of mammalian cathepsin L, up to 50-fold (50). Therefore, the possible interaction of stefin B with DNA was analyzed by gel shift assay, as described previously (50).…”

Section: Visualization Of Intracellular Met-75 Cathepsin L-stefin B Imentioning

confidence: 99%

Stefin B Interacts with Histones and Cathepsin L in the Nucleus

Čeru¹,

Konjar²,

Maher³

et al. 2010

Journal of Biological Chemistry

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Cysteine cathepsins are involved in protein degradation (1) and the development and function of the immune system (2). Cathepsin L is an endopeptidase that is able to perform limited proteolysis in the endosomes and lysosomes of specific cell types. Besides its role in hair formation and skin metabolism, it is involved in T-cell selection and NKT cell development (3). It participates in processing the major histocompatibility complex II invariant chain in thymic cortex epithelial cells (4), encephalin in chromaffin granules of neuroendocrine cells (5), and in the degradation and recycling of growth factors and their receptors in epidermal keratinocytes (6). Cathepsin L is also associated with an endosomal processing step during invasion of cells by Ebola virus (7), severe acute respiratory syndrome (SARS) coronavirus (8), and murine hepatitis coronavirus (9). As the result of gene duplication, the human genome encodes for two cathepsin L-like proteases, namely the human cathepsin L and cathepsin V (cathepsin L2), whereas in mouse only cathepsin L is present (10). At the protein level, mouse cathepsin L displays a higher sequence homology to human cathepsin V than to human cathepsin L (11). Cathepsin V shares 80% protein sequence identity with cathepsin L, but in contrast to the ubiquitously expressed cathepsin L, its expression is restricted to thymus and testis (11,12).Recently, the otherwise endosomal proteinase cathepsin L has been reported to be active in the nucleus. It cleaves the CUX1 transcription factor and as a result accelerates progression into the S phase of the cell cycle (13). Cathepsin L deficiency was shown to cause a global rearrangement of chromatin structure and redistribution of specifically modified histones (14). In addition, cathepsin L was found to cleave histone H3.2 in the nucleus during mouse embryonic stem cell differentiation (15).Cathepsin L is inhibited in vitro by a number of proteins as follows: cystatins (16), thyropins (17), and some of the serpins (18,19). Type 1 cystatins, or stefins, are mainly intracellular, whereas type 2 cystatins are predominantly secreted (20,21). Stefin B is localized in the cytosol and nucleus of proliferating cells (22). Loss-of-function mutations in the cystatin B (CTSB, stefin B) gene are found in patients with Unverricht-Lundborg disease (EPM1), but its physiological implication in the pathogenesis of the disease has yet to be defined (23-26). EPM1 is an autosomal recessive inherited disease in which patients suffer from myoclonic jerks, tonic-clonic epileptic seizures, and progressive decline in cognition (26). Histopathological examination of the brain has shown neural degeneration in several areas of the central nervous system, with cerebellar damage and serious alterations of Purkinje cells (27). The most common mutation in EPM1 patients is a dodecamer repeat expansion in the stefin B (CSTB) gene promoter region that leads to reduced mRNA and protein levels (23,25). In addition, four mutations in the coding region were reported in EPM1 (23,28...

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“…Moreover, heparan sulphate has been implicated in the regulation of the conformational plasticity of a cathepsin L homologue from Trypanosoma brucei, possibly by allosteric mechanisms 19 . Similarly, DNA has been shown to augment the inhibition of cathepsin V by serpins 20 .…”

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confidence: 99%

A novel allosteric mechanism in the cysteine peptidase cathepsin K discovered by computational methods

et al. 2014

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Allosteric modifiers have the potential to fine-tune enzyme activity. Therefore, targeting allosteric sites is gaining increasing recognition as a strategy in drug design. Here we report the use of computational methods for the discovery of the first small-molecule allosteric inhibitor of the collagenolytic cysteine peptidase cathepsin K, a major target for the treatment of osteoporosis. The molecule NSC13345 is identified by high-throughput docking of compound libraries to surface sites on the peptidase that are connected to the active site by an evolutionarily conserved network of residues (protein sector). The crystal structure of the complex shows that NSC13345 binds to a novel allosteric site on cathepsin K. The compound acts as a hyperbolic mixed modifier in the presence of a synthetic substrate, it completely inhibits collagen degradation and has good selectivity for cathepsin K over related enzymes. Altogether, these properties qualify our methodology and NSC13345 as promising candidates for allosteric drug design.

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DNA Accelerates the Inhibition of Human Cathepsin V by Serpins

Cited by 42 publications

References 43 publications

Heparin Enhances Serpin Inhibition of the Cysteine Protease Cathepsin L

Heparin Enhances Serpin Inhibition of the Cysteine Protease Cathepsin L

Stefin B Interacts with Histones and Cathepsin L in the Nucleus

A novel allosteric mechanism in the cysteine peptidase cathepsin K discovered by computational methods

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