Fluorometric Microassays for the Determination of Cathepsin L and Cathepsin S Activities in Tissue Extracts

Werle, Bernd; Staib, A; Jülke, Britta; Ébert, W.; Zladoidsky, Pavel; Sekirnik, Andreja; Kos, Janko; Spieß, Eberhard

doi:10.1515/bc.1999.138

Cited by 30 publications

(25 citation statements)

References 25 publications

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“…showed that the replacement of a 50 mmol/L citrate assay buffer by a 50 mmol/L MES buffer led to the total abolition of the inhibition of cathepsins L, S, and K by the rCatKPP (9). In these experiments, the fluorimetric assays were done in a 100 mmol/L sodium acetate buffer (pH 5.5), as has been published previously (20,21), and is suitable for use with the each of the cathepsin L -like cysteine cathepsins.…”

Section: Expression and Purification Of Rcatsppmentioning

confidence: 80%

Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L–like proteases in tumor microenvironment

Burden¹,

Snoddy

Buick

et al. 2008

Molecular Cancer Therapeutics

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Human cathepsin L along with cathepsin S, K, and V are collectively known as cathepsin L -like proteases due to their high homology. The overexpression and aberrant activity of each of these proteases has been implicated in tumorigenesis. These proteases contain propeptide domains that can potently inhibit both their cognate protease and other proteases within the cathepsin L -like subfamily. In this investigation, we have produced the cathepsin S propeptide recombinantly and have shown that it is a potent inhibitor of the peptidolytic, elastinolytic, and gelatinolytic activities of the cathepsin L -like proteases. In addition, we show that this peptide is capable of significantly attenuating tumor cell invasion in a panel of human cancer cell lines. Furthermore, fusion of an IgG Fc-domain to the COOH terminus of the propeptide resulted in a chimeric protein with significantly enhanced ability to block tumor cell invasion. This Fc fusion protein exhibited enhanced stability in cell-based assays in comparison with the unmodified propeptide species. This approach for the combined inhibition of the cathepsin L -like proteases may prove useful for the further study in cancer and other conditions where their aberrant activity has been implicated. Furthermore, this strategy for simultaneous inhibition of multiple cysteine cathepsins may represent the basis for novel therapeutics to attenuate tumorigenesis. [Mol Cancer Ther 2008;7(3):538 -47]

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Section: Expression and Purification Of Rcatsppmentioning

confidence: 80%

Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L–like proteases in tumor microenvironment

Burden¹,

Snoddy

Buick

et al. 2008

Molecular Cancer Therapeutics

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“…Assays used 20 M benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin (ZPhe-Arg-AMC; Anaspec) at pH 7.5 in 100 mM Na-Acetate with 2.5 mM EDTA, 0.01% Triton X-100, and 2.5 mM DTT in a microtiter plate [24] with ~0.3 g of extract at 37 °C. The amount of product was determined by excitation at 355 nm and emission at 460 nm using a kinetic reading for a microtiter plate and comparison with 7-amino-4-methylcoumarin standards from Anaspec.…”

Section: Methodsmentioning

confidence: 99%

Upregulation of elastase proteins results in aortic dilatation in mucopolysaccharidosis I mice

Tittiger

Knutsen

et al. 2008

Molecular Genetics and Metabolism

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Mucopolysaccharidosis I (MPS I), known as Hurler syndrome in the severe form, is a lysosomal storage disease due to -L-iduronidase (IDUA) deficiency. It results in fragmentation of elastin fibers in the aorta and heart valves via mechanisms that are unclear, but may result from the accumulation of the glycosaminoglycans heparan and dermatan sulfate. Elastin fragmentation causes aortic dilatation and valvular insufficiency, which can result in cardiovascular disease. The pathophysiology of aortic disease was evaluated in MPS I mice. MPS I mice have normal elastic fiber structure and aortic compliance at early ages, which suggests that elastin assembly is normal. Elastin fragmentation and aortic dilatation are severe at 6 months, which is temporally associated with marked increases in mRNA and enzyme activity for two elastin-degrading proteins, matrix metalloproteinase-12 (MMP-12) and cathepsin S. Upregulation of these genes likely involves activation of STAT proteins, which may be induced by structural stress to smooth muscle cells from accumulation of glycosaminoglycans in lysosomes. Neonatal intravenous injection of a retroviral vector normalized MMP-12 and cathepsin S mRNA levels and prevented aortic disease. We conclude that aortic dilatation in MPS I mice is likely due to degradation of elastin by MMP-12 and/or cathepsin S. This aspect of disease might be ameliorated by inhibition of the signal transduction pathways that upregulate expression of elastase proteins, or by inhibition of elastase activity. This could result in a treatment for patients with MPS I, and might reduce aortic aneurism formation in other disorders.

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“…Cathepsin L -type activity in cell lysates was measured using the previously described method 22 with modifications. Briefly, an aliquot of 15 g of total protein diluted in 100 L of double distilled water was incubated in 870 L of 0.34 M sodium acetate buffer, pH 5.5, containing 2 mM EDTA -Na 2 ( Sigma).…”

Section: Cathepsin L -Type Activity Assaymentioning

confidence: 99%

Selective suppression of cathepsin L by antisense cDNA impairs human brain tumor cell invasion in vitro and promotes apoptosis

et al. 2003

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Invasion and metastasis of certain tumors are accompanied by increased mRNA protein levels and enzymatic activity of cathepsin L. Cathepsin L has also been suggested to play a role in the proteolytic cascades associated with apoptosis. To investigate the role of cathepsin L in brain tumor invasion and apoptosis, the human glioma cell line, IPTP, was stably transfected with full -length antisense and sense cDNA of cathepsin L. Down -regulation of cathepsin L by antisense cDNA significantly impaired ( up to 70% ) glioma cell invasion in vitro and markedly increased glioma cell apoptosis induced by staurosporine. Compared to control and parental cell lines, antisense down -regulation of cathepsin L was associated with an earlier induction of caspase -3 activity. Up -regulation of cathepsin L activity by sense cDNA was associated with reduced apoptosis and later induction of caspase -3 activity. Moreover, down -regulation of cathepsin L lowered the expression of antiapoptotic protein Bcl -2, whereas up -regulation increased the expression of Bcl -2, indicating that cathepsin L acts upstream of caspase -3. These data show that cathepsin L is an important protein mediating the malignancy of gliomas and its inhibition may diminish their invasion and lead to increased tumor cell apoptosis by reducing apoptotic threshold.

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Fluorometric Microassays for the Determination of Cathepsin L and Cathepsin S Activities in Tissue Extracts

Cited by 30 publications

References 25 publications

Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L–like proteases in tumor microenvironment

Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L–like proteases in tumor microenvironment

Upregulation of elastase proteins results in aortic dilatation in mucopolysaccharidosis I mice

Selective suppression of cathepsin L by antisense cDNA impairs human brain tumor cell invasion in vitro and promotes apoptosis

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