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Cysteine Proteases and Regulators


Introduction of Cysteine Proteases

In the lysosomal system, proteins will be degraded due to a randomly combined and limited action of many proteases. In order to maintain the effective degradation of biomacromolecules within the lysosomes, there is a variety number of different hydrolases, such as proteases, amylases, lipases and nucleases. Among these are the cysteine cathepsins which belong to the family of papain-like cysteine proteases. More specifically, they are members of the C1 family of papain-like enzymes, the largest and the best characterized family of cysteine peptidases. Cysteine cathepsins have an important role in lysosomal proteases and are widely found in lives. They have special reactive-site properties and their expression pattern presents a tissue-specific fashion. In living bodies, their activity function as maintaining a delicate balance between expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. Researchers can take advantage of the specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures for study and development. All in all, due to their unique reactive-site properties, researchers can regulate the targets simply by the use of appropriate reactive groups.

Classification of Cysteine Proteases

Based on sequence homology, cysteine proteases are divided into different clans, and these clans consist of one or more families (Table 1). The papain family and the calpain family belong to clan CA which is the most abundant among all cysteine proteases. The caspase family is in the second abundant clan CD. Cysteine cathepsins are papain-like cysteine proteinases belonging to clan CA, family C1. There are 11 human cathepsins known as B, H, L, S, C, K, O, F, V, X and W. These proteinases are widely distributed in a range of diverse cells and tissues besides cathepsins S, V, K and W. Caspases are short for cysteinedependent aspartate specific proteases. They have relationships with the human interleukin-1b converting enzyme and are the product of the C. elegans cell death gene CED-3.

Table 1. Families of cysteine proteases

Cysteine Proteases and Regulators

Catalytic mechanism

Generally there are 3 steps for cysteine proteases to catalyze the hydrolysis of peptide bonds (Figure 1). Firstly, a thiol will be deprotonated at the enzyme's active site by an adjacent amino acid with a basic side chain, usually a histidine residue. Secondly, there will be a nucleophilic attack on the substrate carbonyl carbon by the deprotonated cysteine's anionic sulfur. In this step, substrate fragment will be released as well as an amine terminus, then the histidine residue will restore to its deprotonated form, forming a thioester intermediate link between the new carboxy-terminus of the substrate and the cysteine thiol. Finally, on the remaining substrate fragment, there will be a carboxylic acid moiety generated by the hydrolyzation of the thioester bond. Free enzyme is regenerated here too.

Schematic reaction mechanism of the cysteine protease mediated cleavage of a peptide bond.

Figure 1. Schematic reaction mechanism of the cysteine protease mediated cleavage of a peptide bond.

Function of Cysteine Proteases

Cysteine proteases involve in many biological procedures, especially the cysteine cathepsins and caspases. Cysteine cathepsins were thought to exclusively take part in degrading the terminal proteins when cells are dying or autophagy. Besides, an increase in the level of cathepsin expression and activity appears to be implicated in the development of various pathological conditions, such as neurological disorders, cardiovascular diseases, obesity, inflammatory diseases and cancer. For example, researchers recently find that cathepsin K deficiency will result in structural and metabolic changes associated with learning and memory deficits. Unverricht-Lundborg Disease (EPM1) is an autosomal recessive neurodegenerative disorder. The mutations on stefin B gene are found to be related to the progressive myoclonus epilepsy of this disease. According to the genetic studies, when cathepsin C (DPPI) gene mutates resulting loss of function, there will be early-onset periodontitis and palmoplantar keratosis, as well as characteristics of Haim-Munk and Papillon-Lefevre syndromes. Mammals contain two biologically-distinct caspase sub-families: one of these participates in the processing of pro-inflammatory cytokines, while the other is required to elicit and execute the apoptotic response during programmed cell death. For example, caspase-1, -4, -5, and -12 comprise the inflammatory subset in humans, whereas caspase-1, -11, and -12 serve the same function in mice. Caspase-1 or -11 knockout animals, although without any overt apoptotic phenotype, will have trouble in cytokine processing. However the phenotypes of other knockouts are lethal, the early embryonic lethality in caspase-8 knockout mice and the perinatal lethality in caspases-3 and -9 knockout mice are relatively high. It's worth mentioning that caspase-14 involves in keratinocyte differentiation.

Regulators of Cysteine Proteases

Inhibitors of papain-like cysteine proteases

Cystatins are the best studied endogenous inhibitors of papain-like cysteine proteases. They can be found both in animals and plants. There is a superfamily of cystatin homologous proteins in which can be sub-grouped into four families, namely the stefins, the cystatins, the kininogens and noninhibitory homologs of cystatins. In fact, the first three inhibitors are truly inhibitory, the last ones are some homologs like fetuins and histidine-rich glycoprotein containing two cystatin-like domains. Their major function is to protect organisms from degrading by endogenous proteases accidentally released from lysosomes. Besides, they are with antiviral properties and can prevent the invasion by microorganisms and parasites. That's why cystatins are considered as emergency inhibitors. Cystatins, on the other hand, were shown to efficiently block the activity of these proteases at neutral conditions which is their optimum pH for catalyzing, and this has been demonstrated by the stefin B- cathepsin S interaction.

Caspase inhibitors

Researchers have found three protein families having ability to ablate caspase activity both in vitro and in vivo. One is the inhibitors of apoptosis protein (IAP) family which function as regulating cellular apoptosis by direct caspase inhibition. Besides these endogenous regulators, there are inhibitors like the virally encoded cowpox virus CrmA and baculovirus p35 proteins. They are generated early in infection to suppress caspase-mediated host defenses. According to the in vitro data, each of the inhibitors has a characteristic specificity profile against human caspases and these profiles fit the biologic function of the inhibitors well.

Zymogen activation

The serine protease precursors trypsinogen and chymotrypsinogen, and the aspartic protease precursor pepsinogen are called zymogens which are the large precursor proteins of various proteases. This mechanism can protect cells from damaging by proteases produced by itself. Once delivered to its specific compartment, processing of the enzyme starts, which include cleavage of the pro-domain and activation of the mature enzyme. In processing of cysteine protease, pH change has great importance. In lysosomes or food vacuoles, enzymes get activated by controlled proteolysis which involves autocatalysis or trans-activation. Auto-catalysis is a common mode of activation of cysteine proteases itself from their zymogens. The pH has great importance in this procedure. It will destroy the interactions between the prodomain and the mature domain, making the cleavage site within the prodomain loop to the active site. An N-terminal prodomain is cleaved off from the whole enzyme at active site then the enzyme gets activated. Trans-activation involves cleavage by another active molecule of the same enzyme that cleaves within the residues lying at the junction of prodomain and mature domain such as pepsin, Cathepsin D, and legumain/asparaginyl endopeptidase.

References:

  1. Turk V, et al. Cysteine cathepsins: From structure, function and regulation to new frontiers. BBA - Proteins and Proteomics. 2012, 1824(1):68-88.
  2. Brocklehurst K, Philpott M P. Cysteine proteases: mode of action and role in epidermal differentiation. Cell & Tissue Research. 2013, 351(2):237-244.
  3. Kopitar-Jerala N. The Role of Cysteine Proteinases and their Inhibitors in the Host-Pathogen Cross Talk. Current Protein & Peptide Science. 2012, 13(8).
  4. Sonia V, et al. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets. Frontiers in Pharmacology. 2016, 7.
  5. Turk B, et al. Regulating cysteine protease activity: essential role of protease inhibitors as guardians and regulators. Current Pharmaceutical Design. 2002, 8(18).
  6. Mcilwain D R, et al. Caspase Functions in Cell Death and Disease. Cold Spring Harbor Perspectives in Biology. 2013, 5(4):a008656.

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