The Structure of Pepstatin

Morishima, Hazime; Takita, Tomohisa; Aoyagi, Takaaki; Takeuchi, Tomio; Umezawa, Hamao

doi:10.7164/antibiotics.23.263

Cited by 104 publications

(29 citation statements)

References 5 publications

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“…Recombinant Sap9 and Sap10 exhibit an unusually broad pH spectrum, with activities above neutral pH and an incomplete inhibition by pepstatin A. Secreted Sap proteases display typical features of aspartic proteases, including acidic pH optima ("acid proteases"), a bell-shaped (Gaussian) pH dependence of activity (55), and sensitivity to the inhibitor pepstatin A (43). To test whether Sap9 and Sap10 follow this prototypic scheme, we measured the proteolytic activities of recombinant Sap9 and Sap10 (rSap9 and rSap10) against the fluorescencequenched substrate casein and compared these to Sap2 and Sap6 (representing the two main subfamilies of Sap1 to 3 and Sap4 to 6).…”

Section: Resultsmentioning

confidence: 99%

Proteolytic Cleavage of Covalently Linked Cell Wall Proteins by Candida albicans Sap9 and Sap10

et al. 2011

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The cell wall of the human-pathogenic fungus Candida albicans is a robust but also dynamic structure which mediates adaptation to changing environmental conditions during infection. Sap9 and Sap10 are cell surfaceassociated proteases which function in C. albicans cell wall integrity and interaction with human epithelial cells and neutrophils. In this study, we have analyzed the enzymatic properties of Sap9 and Sap10 and investigated whether these proteases cleave proteins on the fungal cell surface. We show that Sap9 and Sap10, in contrast to other aspartic proteases, exhibit a near-neutral pH optimum of proteolytic activity and prefer the processing of peptides containing basic or dibasic residues. However, both proteases also cleaved at nonbasic sites, and not all tested peptides with dibasic residues were processed. By digesting isolated cell walls with Sap9 or Sap10, we identified the covalently linked cell wall proteins (CWPs) Cht2, Ywp1, Als2, Rhd3, Rbt5, Ecm33, and Pga4 as in vitro protease substrates. Proteolytic cleavage of the chitinase Cht2 and the glucan-cross-linking protein Pir1 by Sap9 was verified using hemagglutinin (HA) epitope-tagged versions of both proteins. Deletion of the SAP9 and SAP10 genes resulted in a reduction of cell-associated chitinase activity similar to that upon deletion of CHT2, suggesting a direct influence of Sap9 and Sap10 on Cht2 function. In contrast, cell surface changes elicited by SAP9 and SAP10 deletion had no major impact on the phagocytosis and killing of C. albicans by human macrophages. We propose that Sap9 and Sap10 influence distinct cell wall functions by proteolytic cleavage of covalently linked cell wall proteins.

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Section: Resultsmentioning

confidence: 99%

Proteolytic Cleavage of Covalently Linked Cell Wall Proteins by Candida albicans Sap9 and Sap10

et al. 2011

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“…The preparation used for Fig. 1 still had residual activity at pH 7. It is known that pepstatin, a microbial inhibitor of pepsin (31), is also effective in inhibiting cathepsin D (32). The inhibitor also binds to cathepsin D at pH 7. When this inhibitor was tested with cartilage extract (Table I), it was found to inhibit PGS digestion at pH 5 quite strongly, but to have only a small effect on the digestion at pH 7.…”

Section: Methodsmentioning

confidence: 99%

Neutral Proteases and Cathepsin D in Human Articular Cartilage

Sapolsky¹,

Howell²,

Woessner³

1974

J. Clin. Invest.

100

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A B S T R A C T Proteolytic enzymes have been studied in extracts of human articular cartilage by the use of micromethods. The digestion of hemoglobin at pH 3.2 and of cartilage proteoglycan at pH 5 was shown to be due chiefly to cathepsin D. Cathepsin D was purified 900-fold from human patellar cartilage. Its identity was established by its specific cleavage of the B chain of insulin. At least six multiple forms of cathepsin D are present in cartilage; these corresponded to bovine forms 4-9. Cathepsin D had no action on proteins at pH 7.4. However, cartilage extracts digested proteoglycan, casein, and histone at this pH. The proteolytic activities against these three substrates were purified about 170-, 160-, and 70-fold, respectively. Each activity appeared in multiple forms on DEAE-Sephadex chromatography. The three activities appear to be different since cysteine inhibited casein digestion, aurothiomalate inhibited histone digestion, and neither inhibited proteoglycan digestion. Tests with a wide range of inhibitors and activators suggest that these three activities differ from other neutral proteases described in the literature.

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“…18,19 Compared with other structurally related cyanobacterial secondary metabolites (Figure 2), compounds 1–3 are hybrids between the grassystatins A–C 23 and the tasiamides. 24–26, 30,31 Compounds 1–3 share the Leu-statine unit of grassystatins A–C 23 and have closely related residues flanking the statine moiety to those of the tasiamides.…”

Section: Resultsmentioning

confidence: 99%

Grassystatins D–F, Potent Aspartic Protease Inhibitors from Marine Cyanobacteria as Potential Antimetastatic Agents Targeting Invasive Breast Cancer

Al-Awadhi¹,

Law

Paul

et al. 2017

J. Nat. Prod.

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Three new modified peptides named grassystatins D–F (1–3) were discovered from a marine cyanobacterium from Guam. Their structures were elucidated using NMR spectroscopy and mass spectrometry (MS). The hallmark structural feature in the peptides is a statine unit which contributes to their aspartic protease inhibitory activity preferentially targeting cathepsins D and E. Grassystatin F (3) was the most potent analogue with IC50 values of 50 and 0.5 nM against cathepsins D and E, respectively. The acidic tumor microenvironment is known to increase the activation of some of the lysosomal proteases associated with tumor metastasis such as cathepsins. Because cathepsin D is a biomarker in aggressive forms of breast cancer and linked to poor prognosis, the effects of cathepsin D inhibition by 1 and 3 on the downstream cellular substrates, cystatin C and PAI-1, were investigated. Furthermore, the functional relevance of targeting cathepsin D substrates was evaluated by examining the effect of 1 and 3 on the migration of MDA-MD-231 cells. Grassystatin F (3) inhibited the cleavage of cystatin C and PAI-1, the activities of their downstream targets cysteine cathepsins and tPA, as well as the migration of the highly aggressive triple negative breast cancer cells, phenocopying the effect of siRNA mediated knockdown of cathepsin D.

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The Structure of Pepstatin

Cited by 104 publications

References 5 publications

Proteolytic Cleavage of Covalently Linked Cell Wall Proteins by Candida albicans Sap9 and Sap10

Proteolytic Cleavage of Covalently Linked Cell Wall Proteins by Candida albicans Sap9 and Sap10

Neutral Proteases and Cathepsin D in Human Articular Cartilage

Grassystatins D–F, Potent Aspartic Protease Inhibitors from Marine Cyanobacteria as Potential Antimetastatic Agents Targeting Invasive Breast Cancer

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