Cysteine cathepsins are a family of proteases identified in cancer, atherosclerosis, osteoporosis, arthritis, and a number of other diseases. As this number continues to rise, so does the need for low cost, broad use quantitative assays to detect their activity and can be translated to the clinic in the hospital or in low resource settings. Multiplex cathepsin zymography is one such assay that detects subnanomolar levels of active cathepsins K, L, S, and V in cell or tissue preparations observed as clear bands of proteolytic activity after gelatin substrate SDS-PAGE with conditions optimal for cathepsin renaturing and activity. Densitometric analysis of the zymogram provides quantitative information from this low cost assay. After systematic modifications to optimize cathepsin zymography, we describe reduced electrophoresis time from 2 h to 10 min, incubation assay time from overnight to 4 h, and reduced minimal tissue protein necessary while maintaining sensitive detection limits; an evaluation of the pros and cons of each modification is also included. We further describe image acquisition by Smartphone camera, export to Matlab, and densitometric analysis code to quantify and report cathepsin activity, adding portability and replacing large scale, darkbox imaging equipment that could be cost prohibitive in limited resource settings.
Cathepsins Zymography Detection Cysteine protease Cancer
This is a preview of subscription content, log in to check access.
This study was funded by the Georgia Cancer Coalition (M.O.P.) and the Institutional Research and Academic Career Development Awards (IRACDA Grant Number K12 GM000680, NIH/NIGMS) (J.E.D.).
Chapman, H. A., Riese, R. J., & Shi, G. P. (1997). Emerging roles for cysteine proteases in human biology. Annual Review of Physiology,59, 63–88.CrossRefGoogle Scholar
Sukhova, G. K., Zhang, Y., Pan, J. H., Wada, Y., Yamamoto, T., Naito, M., et al. (2003). Deficiency of cathepsin S reduces atherosclerosis in LDL receptor-deficient mice. The Journal Clinical Investigation,111(6), 897–906.Google Scholar
Lecaille, F., Bromme, D., & Lalmanach, G. (2008). Biochemical properties and regulation of cathepsin K activity. Biochimie,90(2), 208–226.CrossRefGoogle Scholar
Lutgens, E., Lutgens, S. P., Faber, B. C., Heeneman, S., Gijbels, M. M., de Winther, M. P., et al. (2006). Disruption of the cathepsin K gene reduces atherosclerosis progression and induces plaque fibrosis but accelerates macrophage foam cell formation. Circulation,113(1), 98–107.CrossRefGoogle Scholar
Platt, M. O., Ankeny, R. F., Shi, G. P., Weiss, D., Vega, J. D., Taylor, W. R., et al. (2007). Expression of cathepsin K is regulated by shear stress in cultured endothelial cells and is increased in endothelium in human atherosclerosis. American Journal of Physiology. Heart and Circulatory Physiology,292(3), H1479–H1486.CrossRefGoogle Scholar
Littlewood-Evans, A. J., Bilbe, G., Bowler, W. B., Farley, D., Wlodarski, B., Kokubo, T., et al. (1997). The osteoclast-associated protease cathepsin K is expressed in human breast carcinoma. Cancer Research,57(23), 5386–5390.Google Scholar
Platt, M. O., Ankeny, R. F., & Jo, H. (2006). Laminar shear stress inhibits cathepsin L activity in endothelial cells. Arteriosclerosis, Thrombosis, and Vascular Biology,26(8), 1784–1790.CrossRefGoogle Scholar
Sukhova, G. K., Shi, G. P., Simon, D. I., Chapman, H. A., & Libby, P. (1998). Expression of the elastolytic cathepsins S and K in human atheroma and regulation of their production in smooth muscle cells. The Journal of Clinical Investigation,102(3), 576–583.CrossRefGoogle Scholar
Shi, G. P., Sukhova, G. K., Grubb, A., Ducharme, A., Rhode, L. H., Lee, R. T., et al. (1999). Cystatin C deficiency in human atherosclerosis and aortic aneurysms. The Journal of Clinical Investigation,104(9), 1191–1197.CrossRefGoogle Scholar
Hansen, L., Parker, I., Sutliff, R. L., Platt, M. O., & Gleason, R. L., Jr. (2012). Endothelial dysfunction, arterial stiffening, and intima-media thickening in large arteries from HIV-1 transgenic mice. Annals of Biomedical Engineering,22, 22.Google Scholar
Pang, M., Martinez, A. F., Fernandez, I., Balkan, W., & Troen, B. R. (2007). AP-1 stimulates the cathepsin K promoter in RAW 264.7 cells. Gene,403(1–2), 151–158.CrossRefGoogle Scholar
Yasuda, Y., Li, Z., Greenbaum, D., Bogyo, M., Weber, E., & Bromme, D. (2004). Cathepsin V, a novel and potent elastolytic activity expressed in activated macrophages. Journal of Biological Chemistry,279(35), 36761–36770.CrossRefGoogle Scholar
Park, K. Y., Li, W. A., & Platt, M. O. (2012). Patient specific proteolytic activity of monocyte-derived macrophages and osteoclasts predicted with temporal kinase activation states during differentiation. Integrative Biology: Quantitative Biosciences from Nano to Macro,4(12), 1459–1469.Google Scholar
Tournu, C., Obled, A., Roux, M. P., Deval, C., Ferrara, M., & Bechet, D. M. (1998). Glucose controls cathepsin expression in Ras-transformed fibroblasts. Archives of Biochemistry and Biophysics,360(1), 15–24.CrossRefGoogle Scholar
Mohamed, M. M., & Sloane, B. F. (2006). Cysteine cathepsins: Multifunctional enzymes in cancer. Nature Reviews Cancer,6(10), 764–775.CrossRefGoogle Scholar
Brubaker, K. D., Vessella, R. L., True, L. D., Thomas, R., & Corey, E. (2003). Cathepsin K mRNA and protein expression in prostate cancer progression. Journal of Bone and Mineral Research,18(2), 222–230.CrossRefGoogle Scholar
Vasiljeva, O., Reinheckel, T., Peters, C., Turk, D., Turk, V., & Turk, B. (2007). Emerging roles of cysteine cathepsins in disease and their potential as drug targets. Current Pharmaceutical Design,13(4), 387–403.CrossRefGoogle Scholar
Wijkmans, J., & Gossen, J. (2011). Inhibitors of cathepsin K: A patent review (2004–2010). Expert Opinion on Therapeutic Patents,21(10), 1611–1629.CrossRefGoogle Scholar
Bromme, D., & Lecaille, F. (2009). Cathepsin K inhibitors for osteoporosis and potential off-target effects. Expert Opinion on Investigational Drugs,18(5), 585–600.CrossRefGoogle Scholar
Palermo, C., & Joyce, J. A. (2008). Cysteine cathepsin proteases as pharmacological targets in cancer. Trends in Pharmacological Sciences,29(1), 22–28.CrossRefGoogle Scholar
Blum, G., Mullins, S. R., Keren, K., Fonovic, M., Jedeszko, C., Rice, M. J., et al. (2005). Dynamic imaging of protease activity with fluorescently quenched activity-based probes. Nature Chemical Biology,1(4), 203–209.CrossRefGoogle Scholar
Joyce, J. A., Baruch, A., Chehade, K., Meyer-Morse, N., Giraudo, E., Tsai, F. Y., et al. (2004). Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis. Cancer Cell,5(5), 443–453.CrossRefGoogle Scholar
Li, W. A., Barry, Z. T., Cohen, J. D., Wilder, C. L., Deeds, R. J., Keegan, P. M., et al. (2010). Detection of femtomole quantities of mature cathepsin K with zymography. Analytical Biochemistry,401(1), 91–98.CrossRefGoogle Scholar
Chen, B., & Platt, M. O. (2011). Multiplex zymography captures stage-specific activity profiles of cathepsins K, L, and S in human breast, lung, and cervical cancer. Journal of Translational Medicine,9, 109.CrossRefGoogle Scholar
Wilder, C. L., Park, K. Y., Keegan, P. M., & Platt, M. O. (2011). Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues. Archives of Biochemistry and Biophysics,516(1), 52–57.CrossRefGoogle Scholar
Edge, S. B., & Compton, C. C. (2010). The American Joint Committee on Cancer: The 7th edition of the AJCC cancer staging manual and the future of TNM. Annals of Surgical Oncology,17(6), 1471–1474.CrossRefGoogle Scholar
Keegan, P. M., Wilder, C. L., & Platt, M. O. (2012). Tumor necrosis factor alpha stimulates cathepsin K and V activity via juxtacrine monocyte-endothelial cell signaling and JNK activation. Molecular and Cellular Biochemistry,367(1–2), 65–72. doi:10.1007/s11010-012-1320-0.CrossRefGoogle Scholar
Barry, Z. T., & Platt, M. O. (2012). Cathepsin S cannibalism of cathepsin K as a mechanism to reduce type I collagen degradation. Journal of Biological Chemistry,287(33), 27723–27730. doi:10.1074/jbc.M111.332684.CrossRefGoogle Scholar
Gallagher, S.R. (2001). One-dimensional SDS gel electrophoresis of proteins. Current Protocols in Protein Science. doi:10.1002/0471140864.ps1001s00.