Proteolytic Activity of Human Osteoclast Cathepsin K

Bossard, Mary J.; Tomaszek, Thaddeus A.; Thompson, Scott K.; Amegadzie, Bernard Y.; Hanning, Charles R.; Jones, Christopher S.; Kurdyla, Jeffrey T.; McNulty, Dean E.; Drake, Fred H.; Gowen, Maxine; Levy, Mark A.

doi:10.1074/jbc.271.21.12517

Cited by 452 publications

(191 citation statements)

References 29 publications

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“…We previously demonstrated that the protease activity responsible for generating p40 was a cysteine protease (15). Cysteine proteases present within the lysosome include cathepsins B, L, H, and S (24) as well as the more recently described O2 and O (25)(26)(27)(28)(29)(30)(31). We considered the possibilities that the MCE could be one of these known proteases or a novel protease.…”

Section: Resultsmentioning

confidence: 99%

Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

Spizz¹,

Blackshear

1997

Journal of Biological Chemistry

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The importance of regulating the cellular concentrations of the myristoylated alanine-rich C kinase substrate (MARCKS), a major cellular substrate of protein kinase C, is indicated by the fact that mice lacking MARCKS exhibit gross abnormalities of central nervous system development and die shortly after birth. We previously identified a novel means of regulating cellular MARCKS concentrations that involved a specific proteolytic cleavage of the protein and implicated a cysteine protease in this process (Spizz, G., and Blackshear, P. J. (1996) J. Biol. Chem. 271, 553-562). Here we show that p40, the carboxyl-terminal fragment resulting from this cleavage of MARCKS, was associated with the mitochondrial/lysosomal pellet fraction of human diploid fibroblasts and that its generation in cells was sensitive to treatment with NH 4 Cl. These data suggest the involvement of lysosomes in the generation and/or stability of p40. The MARCKS-cleaving enzyme (MCE) activity was peripherally associated with a 10,000 ؋ g pellet fraction from bovine liver, and it co-purified with the activity and immunoreactivity of a lysosomal protease, cathepsin B. Cathepsin B catalyzed the generation of p40 from MARCKS in a cell-free system and behaved similarly to the MCE with respect to mutants of MARCKS previously shown to be poor substrates for the MCE. Treatment of fibroblasts with a cell-permeable, specific inhibitor of cathepsin B, CA074-Me, resulted in parallel time-and concentration-dependent inhibition of cathepsin B and MCE activity. Incubation of a synthetic MARCKS phosphorylation site domain peptide with purified cathepsin B resulted in cleavage of the peptide at sites consistent with preferred cathepsin B substrate sites. These data provide evidence for the identity of the MCE as cathepsin B and suggest that this cleavage most likely takes place within lysosomes, perhaps as a result of specific lysosomal targeting sequences within the MARCKS primary sequence. The data also suggest a direct interaction between MARCKS and cathepsin B in cells and leave open the possibility that MARCKS may in some way regulate the protease for which it is a substrate.The myristoylated alanine-rich C kinase substrate (MARCKS) 1 is a prominent cellular substrate for protein kinase C (PKC) (1, 2). Expression of this heat-stable, acidic protein is essential for life, as demonstrated by the perinatal death of mice that are completely deficient in MARCKS (3). Complete lack of expression leads to gross abnormalities of central nervous system development; however, heterozygous mice, which express MARCKS at 50% wild-type levels, appear to be normal.Cellular levels of MARCKS are regulated by both transcriptional and translational mechanisms (4 -14). In addition, we recently demonstrated that the cellular concentrations of MARCKS can also be regulated by a proteolytic event. This proteolytic cleavage results in amino-and carboxyl-terminal fragments of MARCKS that co-exist in cells with the full-length protein. This cleavage of MARCKS was inhibited in intact fi...

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Section: Resultsmentioning

confidence: 99%

Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

Spizz¹,

Blackshear

1997

Journal of Biological Chemistry

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“…The pycnodysostosis candidate gene cathepsin K, CTSK, contributes significantly to the enzymatic activity that has been ascribed to cysteine class proteases, which are implicated in degradation of the protein components within bone during the resorptive stage of organ remodelling. 17 Kinetic and biochemical studies indicate that cathepsin K is intimately involved in the process of bone resorption. Thus it represents a new molecular target in treatment of diseases associated with excessive bone losssuch as osteoporosis.…”

Section: Discussionmentioning

confidence: 99%

Cathepsin K gene mutations and 1q21 haplotypes in patients with pycnodysostosis in an outbred population

et al. 2000

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The molecular genetics of the autosomal recessive disorder pycnodysostosis was studied in five independent families from an outbred Caucasian population. We found two new mutations and one recently described mutation in the cathepsin K gene by sequencing DNA from eight patients with pycnodysostosis: a one base transition in exon 8, c926T > C, causing a single amino acid substitution leucine ® proline, L309P; A 3' splice site mutation in intron 2, c121-1G > A, causing deletion of all exon 3, 41V-81Mdel; and the exon 3 missense mutation c236G > A leading to residue G79E. In three of the families patients were homozygous for 926T > C. In the remaining two families patients were heterozygous for 926T > C and 121-1G > A in one case, and for 926T > C and 236G > A in the other case. Assays using genomic DNA were developed for all three mutations. We tested 150 healthy control persons and observed the mutation frequencies: 0 to 300 for 121-1G > A and 236G > A and 1 to 150 for 926T > C. One patient from each family was haplotyped with eight microsatellite markers surrounding the cathepsin K gene on chromosome 1q21. A very rare, P = 1.8 ؋ 10 -6 to P = 0.0004, and highly preserved area around the presumed disease locus was common to all the patients. This haplotype was found on seven chromosomes identical by state, IBS, out of the possible eight carrying the 926T > C mutation. Founder effect, locus homogeneity, and allele heterogeneity regarding pycnodysostosis within this population are discussed. Finally, the first pregnancy and delivery described in a patient with pycnodysostosis is reported.

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“…[17][18][19] The time frame of the changes in cathepsin S and K expression and the local- The relative amounts of elastin degradation are presented as the absorbance determined. Elastolytic activity of cathepsin S (100 nmol) was used as a positive control.…”

Section: Discussionmentioning

confidence: 99%

“…16 Despite their lysosomal origin and optimal acidic pH, some of cathepsins including cathespin S and K can be secreted and retain a large portion of their proteolytic activity at neutral pH. [17][18][19] Among the members of the cathepsin family, cathepsin S and K express potent elastolytic as well as collagenolytic activities. 19 -21 Although it has been demonstrated that vascular SMCs have the ability to express these cathespins, 6,22 cathepsins have received much less consideration in the involvement in the neointima formation.…”

mentioning

confidence: 99%

Increased Expression of Elastolytic Cysteine Proteases, Cathepsins S and K, in the Neointima of Balloon-Injured Rat Carotid Arteries

Cheng

Kuzuya

Sasaki

et al. 2004

The American Journal of Pathology

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The matrix-degrading activity of several proteases are involved in the accelerated breakdown of extracellular matrix associated with vascular remodeling during the development of atherosclerosis and vascular injury-induced neointimal formation. Previous studies have shown that the potent elastolytic cysteine proteases, cathepsins S and K, are overexpressed in atherosclerotic lesions in human and animal models. However, the role of these cathepsins in vascular remodeling remains unclear. In the present study, the expressions of cathepsin S and K and their inhibitor cystatin C were examined during arterial remodeling using a rat carotid artery balloon-injury model. The increase in both cathepsin S and K mRNA levels was observed from day 1 and day 3 through day 14 following the induction of balloon injury, respectively. Western blotting analysis revealed that both cathepsin S and K protein levels also increased in the carotid arteries during neointima formation, coinciding with an increase elastolytic activity assayed using Elastin-Congo red, whereas, no significant change in the expressions of cystatin C mRNA and protein was observed during follow-up periods after injury. Immunohistochemistry, Western blot, and in situ hybridization showed that the increase of cathepins S and K and the decrease of cystatin C occurred preferentially in the developing neointima. These findings suggest that cathepsin S and K may participate in the pathological arterial remodeling associated with restenosis.

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Proteolytic Activity of Human Osteoclast Cathepsin K

Cited by 452 publications

References 29 publications

Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

Cathepsin K gene mutations and 1q21 haplotypes in patients with pycnodysostosis in an outbred population

Increased Expression of Elastolytic Cysteine Proteases, Cathepsins S and K, in the Neointima of Balloon-Injured Rat Carotid Arteries

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