Papain‐like Cysteine Proteases

Dieter Brömme1

1 Mount Sinai School of Medicine, New York
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.2
DOI:  10.1002/0471140864.ps2102s21
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The name “cysteine protease“ refers to the protease's nucleophilic cysteine residue that forms a covalent bond with the carbonyl group of the scissile peptide bond in substrates. The papain‐like cysteine proteases, classified as the “C1 family” are the most predominant cysteine proteases. These proteases are found in viruses, plants, primitive parasites, invertebrates, and vertebrates alike. Mammalian papain‐like cysteine proteases are also known as cathepsins. This unit discusses cathepsins, and their subcellular and tissue localization, catalytic mechanism, and substrate specificity. Several tables illustrate the properties of the various cathepsins.

PDF or HTML at Wiley Online Library

Table of Contents

  • Sequence Homologies and Evolutionary Relatedness of Cathepsins
  • Catalytic Mechanism of Cathepsins
  • Subcellular and Tissue Localization of Cathepsins
  • Biological and Intrinsic Substrate Specificity
  • Cathepsins and Diseases
  • Recombinant Expression, Purification, and Assays of Cathepsins
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Adachi, W., Kawamoto, S., Ohno, I., Nishida, K., Kinoshita, S., Matsubara, K., and Okubo, K. 1998. Isolation and characterization of human cathepsin V: A major proteinase in corneal epithelium. Investig. Ophthalmol. Vis. Sci. 39:1789‐1796.
   Aibe, K., Yazawa, H., Abe, K., Teramura, K., Kumegawa, M., Kawashima, H., and Honda, K. 1996. Substrate specificity of recombinant osteoclast‐specific cathepsin K from rabbits. Biol. Pharm. Bull. 19:1026‐1031.
   Aronson, N.N.J. and Barrett, A.J. 1978. The specificity of cathepsin B. Hydrolysis of glucagon at the C‐terminus by a peptidyldipeptidase mechanism. Biochem. J. 171:759‐765.
   Brocklehurst, K., Kowlessur, D., O'Driscoll, M., Patel, G., Quenby, S., Salih, E., and Templeton, W., Thomas, E.W., and Willenbrock, F. 1987. Substrate‐derived two‐protonic‐state electrophiles as sensitive kinetic specificity probes for cysteine proteinases. Activation of 2‐pyridyl disulphides by hydrogen‐bonding. Biochem. J. 244:173‐81.
   Brömme, D. and McGrath, M.E. 1996. High level expression and crystallization of recombinant human cathepsin S. Protein Sci. 5:787‐791.
   Brömme, D. and Okamoto, K. 1995. Human cathepsin O2, a novel cysteine protease highly expressed in osteoclastomas and ovary. Molecular cloning, sequencing and tissue distribution. Biol. Chem. Hoppe‐Seyler 376:379‐384.
   Brömme, D. and Schmidt, B.F. 1999. Functional expression of recombinant proteases. In Proteolytic Enzymes—Tools and Targets (E.E. Sterchi and W. Stöcker, eds.) Springer‐Verlag, New York.
   Brömme, D., Steinert, A., Friebe, S., Fittkau, S., Wiederanders, B., and Kirschke, H. 1989. The specificity of bovine spleen cathepsin S. A comparison with liver cathepsins L and B. Biochem. J. 264:475‐481.
   Brömme, D., Bonneau, P.R., Lachance, P., Wiederanders, B., Kirschke, H., Peters, C., Thomas, D.Y., Storer, A.C., and Vernet, T. 1993. Functional expression of human cathepsin S in Saccharomyces cerevisiae. Purification and characterization of the recombinant enzyme. J. Biol. Chem. 268:4832‐4838.
   Brömme, D., Bonneau, P.R., Lachance, P., and Storer, A.C. 1994. Engineering the S2 subsite specificity of human cathepsin S to a cathepsin L‐and cathepsin B‐like specificity. J. Biol. Chem. 269:30238‐30242.
   Brömme, D., Okamoto, K., Wang, B.B., and Biroc, S. 1996. Human cathepsin O2, a matrix protein‐degrading cysteine protease expressed in osteoclasts. Functional expression of human cathepsin O2 in Spodoptera frugiperda and characterization of the enzyme. J. Biol. Chem. 271:2126‐2132.
   Brömme, D., Li, Z., Barnes, M., and Mehler, E. 1999. Human cathepsin V functional expression, tissue distribution, electrostatic surface potential, enzymatic characterization, and chromosomal localization. Biochemistry 38:2377‐2385.
   Brown, J., Matutes, E., Singleton, A., Price, C., Molgaard, H., Buttle, D., and Enver, T. 1998. Lymphopain, a cytotoxic T and natural killer cell‐associated cysteine proteinase. Leukemia. 12:1771‐1781.
   Bühling, F., Gerber, A., Häckel, C., Krüger, S., Köhnlein, T., Brömme, D., Reinhold, D., Ansorge, F., and Welte, T. 1999. Expression of cathepsin K in lung epithelial cells. Am. J. Respir. Crit. Care Med. 20:612‐619.
   Chapman, H.A., Riese, R.J., and Shi, G.P. 1997. Emerging roles for cysteine proteases in human biology. Annu. Rev. Physiol. 59:63‐88.
   D'Alessio, K.J., McQueney, M.S., Brun, K.A., Orsini, M.J., and Debouck, C.M. 1999. Expression in Escherichia coli, refolding, and purification of human procathepsin K, an osteoclast‐specific protease. Protein Expr. Purif. 15:213‐220.
   De Stefanis, D., Demoz, M., Dragonetti, A., Houri, J.J., Ogier‐Denis, E., Codogno, P., Baccino, F.M., and Isidoro, C. 1997. Differentiation‐induced changes in the content, secretion, and subcellular distribution of lysosomal cathepsins in the human colon cancer HT‐29 cell line. Cell Tissue Res. 289:109‐117.
   Deussing, J.R.W., Saftig, P, Peters, C., Ploegh, H.L., and Villadangos, J.A. 1998. Cathepsins B and D are dispensable for major histocompatibility complex class II‐mediated antigen presentation. Proc. Natl. Acad. Sci. U.S.A. 95:4516‐4521.
   Dolenc, I., Turk, B., Pungercic, G., Ritonja, A., and Turk, V. 1995. Oligomeric structure and substrate induced inhibition of human cathepsin C. J. Biol. Chem. 270:21626‐21631.
   Drake, F.H., Dodds, R.A., James, I.E., Connor, J.R., Debouck, C., Richardson, S., Lee‐Rykaczewski, E., Coleman, L., Rieman, D., Barthlow, R., Hastings, G., and Gowen, M. 1996. Cathepsin K, but not cathepsins B, L, or S, is abundantly expressed in human osteoclasts. J. Biol. Chem. 271:12511‐12516.
   Everts, V., Aronson, D.C., and Beertsen, W. 1985. Phagocytosis of bone collagen by osteoclasts in two cases of pycnodysostosis. Calcif. Tissue Int. 37:25‐31.
   Garnero, P., Borel, O., Byrjalsen, I., Ferreras, M., Drake, F.H., McQueney, M.S., Foged, N.T., Delmas, P.D., and Delaisse, J.M. 1998. The collagenolytic activity of cathepsin K is unique among mammalian proteinases. J. Biol. Chem. 273:32347‐32352.
   Gelb, B.D., Shi, G.P., Chapman, H.A., and Desnick, R.J. 1996. Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency. Science 273:1236‐1238.
   Gowen, M., Lazner, F., Dodds, R., Kapadia, R., Feild, J., Tavaria, M., Bertoncello, I., Drake, F., Zavarselk, S., Tellis, I., Hertzog, P., Debouck, C., and Kola, I. 1999. Cathepsin K knockout mice develop osteopetrosis due to a deficit in matrix degradation but not demineralization. J. Bone Miner. Res. 14:1654‐63.
   Guncar, G., Podobnik, M., Pungercar, J., Strukelj, B., Turk, V., and Turk, D. 1998. Crystal structure of porcine cathepsin H determined at 2.1 A resolution: Location of the mini‐chain C‐terminal carboxyl group defines cathepsin H aminopeptidase function. Structure 6:51‐61.
   Haeckel, C., Krueger, S., Buehling, F., Brömme, D., Franke, K., Schuetze, A., Roese, I., and Roessner, A. 1999. Expression of cathepsin K in the human embryo and fetus. Dev. Dyn. 216:89‐95.
   Hasnain, S., Hirama, T., Huber, C.P., Mason, P., and Mort, J.S. 1993. Characterization of cathepsin B specificity by site‐directed mutagenesis. Importance of Glu245 in the S2‐P2 specificity for arginine and its role in transition state stabilization. J. Biol. Chem. 268:235‐240.
   Kafienah, W., Brömme, D., Buttle, D.J., Croucher, L.J., and Hollander, A.P. 1998. Human cathepsin K cleaves native type I and II collagens at the N‐terminal end of the triple helix. Biochem. J. 331:727‐732.
   Katunuma, N., Matsunaga, Y., and Saibara, T. 1994. Mechanism and regulation of antigen processing by cathepsin B. Adv. Enzyme Regul. 34:145‐158.
   Kirschke, H., Langner, J., Wiederanders, B., Ansorge, S., and Bohley, P. 1977. Cathepsin L. A new proteinase from rat‐liver lysosomes. Eur. J. Biochem. 74:293‐301.
   Kirschke, H., Schmidt, I., and Wiederanders, B. 1986. Cathepsin S. The cysteine proteinase from bovine lymphoid tissue is distinct from cathepsin L (EC Biochem. J. 240:455‐459.
   Kirschke, H., Barrett, A.J., and Rawlings, N.D. 1995. Proteinases 1: Lysosomal cysteine proteinases. In Protein Profiles 2(14):1588‐1643, Academic Press.
   Knight, C.G. 1980. Human cathepsin B. Application of the substrate N‐benzyloxycarbonyl‐L‐arginyl‐L‐arginine2‐naphthylamide to a study of the inhibition by leupeptin. Biochem. J. 189(3):447‐453.
   Koblinski, J.E., Ahram, M., and Sloane, B.F. 2000. Unraveling the role of proteases in cancer. Clin. Chim. Acta 291:113‐135.
   Kopitar, G., Dolinar, M., Strukelj, B., Pungercar, J., and Turk, V. 1996. Folding and activation of human procathepsin S from inclusion bodies produced in Escherichia coli. Eur. J. Biochem 236:558‐562.
   Lindley, H. 1972. The specificity of dipeptidyl aminopeptidase I (cathepsin C) and its use in peptide sequence studies. Biochem. J. 126:683‐685.
   Linnevers, C., Smeekens, S.P., and Brömme, D. 1997a. Human cathepsin W, a putative cysteine protease predominantly expressed in CD8+ T‐lymphocytes. FEBS Lett. 405:253‐259.
   Linnevers, C.J., McGrath, M.E., Armstrong, R., Mistry, F.R., Barnes, M., Klaus, J.L., Palmer, J.T., Katz, B.A., and Brömme, D. 1997b. Expression of human cathepsin K in Pichia pastoris and preliminary crystallographic studies of an inhibitor complex. Protein Sci. 6:919‐921.
   Mai, J., Waisman, D.M., and Sloane, B.F. 2000. Cell surface complex of cathepsin B/annexin II tetramer in malignant progression. Biochim. Biophys. Acta 1477:215‐230.
   McGrath, M.E., Klaus, J.L., Barnes, M.G., and Brömme, D. 1997. Crystal structure of human cathepsin K complexed with a potent inhibitor. Nature Struct. Biol. 4:105‐109.
   McKerrow, J.H. 1999. Development of cysteine protease inhibitors as chemotherapy for parasitic diseases: Insights on safety, target validation, and mechanism of action. Int. J. Parasitol. 29:833‐937.
   McKerrow, J.H., Engel, J.C., and Caffrey, C.R. 1999. Cysteine protease inhibitors as chemotherapy for parasitic infections. Bioorg. Med. Chem. 7:639‐644.
   Musil, D., Zucic, D., Turk, D., Engh, R.A., Mayr, I., Huber, R., Popovic, T., Turk, V., Towatari, T., Katunuma, N., and Bode, W. 1991. The refined 2.15 Å X‐ray crystal structure of human liver cathepsin B: The structural basis for its specificity. EMBO J. 10:2321‐2330.
   Nagler, D.K., Zhang, R., Tam, W., Sulea, T., Purisima, E.O., and Menard, R. 1999. Human cathepsin X: A cysteine protease with unique carboxypeptidase activity. Biochemistry 38:12648‐12654.
   Nakagawa, T., Roth, W., Wong, P., Nelson, A., Farr, A., Deussing, J., Villadangos, J.A., Ploegh, H., Peters, C., and Rudensky, A.Y. 1998. Cathepsin L: Critical role in Ii degradation and CD4 T cell selection in the thymus. Science 280:450‐453.
   Nakagawa, T.Y., Brissette, W.H., Lira, P.D., Griffiths, R.J., Petrushova, N., Stock, J., McNeish, J.D., Eastman, S.E., Howard, E.D., Clarke, S.R., Rosloniec, E.F., Elliott, E.A., and Rudensky, A.Y. 1999. Impaired invariant chain degradation and antigen presentation and diminished collagen‐induced arthritis in cathepsin S null mice. Immunity 10:207‐217.
   Pham, C.T. and Ley, T.J. 1999. Dipeptidyl peptidase I is required for the processing and activation of granzymes A and B in vivo. Proc. Natl. Acad. Sci. U.S.A. 96:8627‐8632.
   Polgar, L. and Halasz, P. 1982. Current problems in mechanistic studies of serine and cysteine proteinases. Biochem. J. 207:1‐10.
   Rao, N.V., Rao, G.V., and Hoidal, J.R. 1997. Human dipeptidyl‐peptidase I. Gene characterization, localization, and expression. J. Biol. Chem. 272:10260‐10265.
   Rawlings, N.D. and Barrett, A.J. 1994. Families of cysteine peptidases. Methods Enzymol. 244:461‐86.
   Ren, W.P., Fridman, R., Zabrecky, J.R., Morris, L.D., Day, N.A., and Sloane, B.F. 1996. Expression of functional recombinant human procathepsin B in mammalian cells. Biochem. J. 319:793‐800.
   Rich, D.H., Brown, M.A., and Barrett, A.J. 1986. Purification of cathepsin B by a new form of affinity chromatography. Biochem. J. 235:731‐734.
   Riese, R.J., Wolf, P., Brömme, D., Natkin, L.R., Villadangos, J.A., Ploegh, H.L., and Chapman, H.A. 1996. Essential role for cathepsin S in MHC class II‐associated invariant chain processing and peptide loading. Immunity 4:357‐366.
   Rozhin, J., Sameni, M., Ziegler, G., and Sloane, B.F. 1994. Pericellular pH affects distribution and secretion of cathepsin B in malignant cells. Cancer Res. 54:6517‐6525.
   Saftig, P., Wehmeyer, O., Hunziker, E., Jones, S., Boyde, A., Rommerskirch, W., and von Figura, K. 1998. Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin K‐deficient mice. Proc. Natl. Acad. Sci. U.S.A. 95:13453‐13458.
   Santamaria, I., Velasco, G., Pendas, A.M., Fueyo, A., and Lopez‐Otin, C. 1998. Cathepsin Z, a novel human cysteine proteinase with a short propeptide domain and a unique chromosomal location. J. Biol. Chem. 273:16816‐16823.
   Schechter, I. and Berger, A. 1967. On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 27:157‐62.
   Shi, G.P., Villadangos, J.A., Dranoff, G., Small, C., Gu, L., Haley, K.J., Riese, R., Ploegh, H.L., and Chapman, H.A. 1999. Cathepsin S required for normal MHC class II peptide loading and germinal center development. Immunity 10:197‐206.
   Sol‐Church, K., Frenck, J., Troeber, D., and Mason, R.W. 1999. Cathepsin P, a novel protease in mouse placenta. Biochem. J. 343:207‐209.
   Sol‐Church, K., Frenck, J., and Mason, R.W. 2000. Cathepsin Q, a novel lysosomal cysteine protease highly expressed in placenta. Biochem. Biophys. Res. Commun. 267:791‐795.
   Takahashi, T., Dehdarani, A.H., and Tang, J. 1988. Porcine spleen cathepsin H hydrolyzes oligopeptides solely by aminopeptidase activity. J. Biol. Chem. 263:10952‐10957.
   Thompson, S.K., Halbert, S.M., Bossard, M.J., Tomaszek, T.A., Levy, M.A., Zhao, B., Smith, W.W., Abdel‐Meguid, S.S., Janson, C.A., D'Alessio, K.J., McQueney, M.S., Amegadzie, B.Y., Hanning, C.R., DesJarlais, R.L., Briand, J., Sarkar, S.K., Huddleston, M.J., Ijames, C.F., Carr, S.A., Garnes, K.T., Shu, A., Heys, J.R., Bradbeer, J., Zembryski, D., and Lee‐Rykaczewski, L. 1997. Design of potent and selective human cathepsin K inhibitors that span the active site. Proc. Natl. Acad. Sci. U.S.A. 94:14249‐14254.
   Toomes, C., James, J., Wood, A.J., Wu, C.L., McCormick, D., Lench, N., Hewitt, C., Moynihan, L., Roberts, E., Woods, C.G., Markham, A., Wong, M., Widmer, R., Ghaffar, K.A., Pemberton, M., Hussein, I.R., Temtamy, S.A., Davies, R., Read, A.P., Sloan, P., Dixon, M.J., and Thakker, N.S. 1999. Loss‐of‐function mutations in the cathepsin C gene result in periodontal disease and palmoplantar keratosis. Nature Genet. 23:421‐424.
   van Noorden, C.J.F., Vogels, I.M., Everts, V., and Beertsen, W. 1987. Localization of cathepsin B activity in fibroblasts and chondrocytes by continuous monitoring of the formation of a final fluorescent reaction product using 5‐nitrosalicylaldehyde. Histochem. J. 19:483‐487.
   Velasco, G., Ferrando, A.A., Puente, X.S., Sanchez, L.M., and Lopez‐Otin, C. 1994. Human cathepsin O. Molecular cloning from a breast carcinoma, production of the active enzyme in Escherichia coli, and expression analysis in human tissues. J. Biol. Chem. 269:27136‐27142.
   Wang, B., Shi, G.P., Yao, P.M., Li, Z., Chapman, H.A., and Brömme, D. 1998. Human cathepsin F. J. Biol. Chem. 273:32000‐32008.
   Weber, E., Gunther, D., Laube, F., Wiederanders, B., and Kirschke, H. 1994. Hybridoma cells producing antibodies to cathepsin L have greatly reduced potential for tumour growth. Cancer Res. Clin. Oncol. 120:564‐567.
   Wex, T., Levy, B., Smeekens, S.P., Ansorge, S., Desnick, R.J., and Brömme, D. 1998. Genomic structure, chromosomal localization, and expression of human cathepsin W. Biochem. Biophys. Res. Commun. 248:255‐261.
   Willstädter, R. and Bamann, E. 1929. Über die Proteasen der Magenschleimhaut. Hoppe‐Seylers Z. Physiol. Chem. 180:127‐143.
   Xia, L., Kilb, J., Wex, H., Lipyansky, A., Breuil, V., Stein, L., Palmer, J.T., Dempster, D.W., and Brömme, D. 1999. Localization of rat cathepsin K in osteoclasts and resorption pits: Inhibition of bone resorption cathepsin K‐activity by peptidyl vinyl sulfones. Biol. Chem. 380:679‐687.
PDF or HTML at Wiley Online Library