Cysteine cathepsins: from structure, function and regulation to new frontiers

Abstract

It is more than 50 years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive-site properties and an uneven tissue-specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive-site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate "warhead". The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation. Together with the growing number of non-endosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Fig. 1

3D schematic representation of the substrate-binding sites of papain-like proteases along the active-site cleft. The representation is based on the proposed revised definition of substrate-binding sites based on the crystal structures of substrate-mimicking inhibitors bound to the protease's active sites . The substrate-binding sites of the papain-like proteases are located on the left (S1, S3, S2′) and right (S2, S1′) side of the active-site cleft in accordance with the standard view orientation. According to papain numbering, L-domain loops include residues Gln19-Cys25 and Arg59-Tyr67 whereas R-domain loops contain residues Leu134-His159 and Asn175-Ser205, respectively. The active-site residues Cys25 and His159 and the disulphide Cys22-Cys63 are indicated.

Fig. 2

3D-based sequence alignment of the mature form of cysteine cathepsins. (A) The structural alignment of eight human cysteine cathepsins, porcine cathepsin H and papain, as a representative member of the papain-family, was done with the program MAIN . The PDB codes of the structures are as follows: cathepsin B (1huc) , cathepsin C (1k3b) , cathepsin F (1m6d) , cathepsin H (8pch) , cathepsin K (1mem) , cathepsin L (1icf) , cathepsin S (1glo) , cathepsin V (1fh0) , cathepsin X (1ef7) , and papain (9pap) . According to cathepsin L numbering, the residues that form the substrate-binding site, within the L-domain loops (Q19-C25, Q60-L69) and R-domain loops (I136-H163, N187-A214), are boxed and highlighted in light gray. (B) The structure-based sequence alignment of human cathepsins O and W was guided using the known 3D structure of human cathepsin L (1icf) using the Expresso (3D-Coffee) program . In both cases the leading sequence was human cathepsin L (1icf) and it is shown in uppercase letters whereas the non-conserved residues are indicated in lowercase letters. Identical residues are shown as dots and the gaps are marked by the dash symbol. Active site residues Cys25 and His163 are indicated by an asterisk.

Fig. 3

Fold of the mature form of cysteine cathepsins–endopeptidases. The fold of the two-chain form of native cathepsin L (1icf) is shown in the green ribbon representation. The parts corresponding to the secondary structure elements, α-helices and β-sheets, are shown in blue and red color, respectively. The side chains of the reactive-site cysteine (Cys25) and histidine (His163) are indicated in a ball-and-stick representation.

Fig. 4

Additional features of cysteine cathepsins–exopeptidases. Chain traces of exopeptidases cathepsins B, X, H and C are indicated in blue, yellow, green and red color, respectively, over the surface of the endopeptidase cathepsin L (1icf) .

Fig. 5

Procathepsin B fold. The propeptide part of human procathepsin B (2pbh) is shown as a chain trace over the surface of the mature part of the enzyme. The α-helices are shown in red color. The surfaces of the reactive-site residues, cysteine and histidine, are marked with yellow and green color, respectively.

Fig. 6

Mechanisms of the cysteine cathepsins involvement in cancer progression. Extracellular cysteine cathepsins promote tumor progression by initiating a proteolytic cascade that results in the activation of a urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs) and plasminogen. Collectively, active proteases, including cysteine cathepsins themselves, can degrade all of the components of the extracellular matrix, promoting tumor progression and metastasis. The degradation of cell-adhesion proteins (e.g., E-cadherin) would additionally increase the disseminative ability of tumor cells. Alternatively, translocated into the cytosol cysteine cathepsins are shown to act as initiator proteases in lysosome-mediated cell death through the degradation of antiapoptotic Bcl-2 family members and the proteolytic activation of Bid, engaging the mitochondrial pathway of apoptosis.