Identification And Characterization Of Cysteine Proteases (CysPs)

Cysteine proteases (CysPs) are ubiquitous proteolytic proteins found in organisms ranging from viruses. bacteria, fungi, to plants and animals. In plants, CysPs are the most abundant protease type that can be grouped in 10 clans subdivided into 15 families. Among these, the CD clan is comprised by legumains and metacaspases. Legumains (family C13) are lysosomal/vacuolar CysPs, also called Vacuolar Processing Enzymes (VPEs), and were first described in plants where they contribute to the processing and maturation of seed storage proteins. They are synthesized as prolegumain and when they become active, in the correct pH conditions and cellular compartment, they recognize and cleave asparagine (Asn) or aspartic acid (Asp) residues in polypeptides. Metacaspases (family C14) are plant CysPs that recognize residues of arginine (R) or Lysine (K) at the substrate cleavage site instead of Aspartic Acid (D) residues used in animal caspases. They are mainly involved in the regulation of programmed cell death.

The CF clan has pyroglutamyl-peptidase I as its unique peptidase member. It is also known as pyrrolidonecarboxylate peptidase, 5-oxoprolyl peptidase, pyrrolidonecarboxylyl peptidase, pyroglutamate aminopeptidase, pyroglutamyl peptide hydrolase, PYRase, and pyroglutamyl aminopeptidase. They hydrolytically remove the pyroglutamate (pGlu) residue from the amino terminus of pGlu peptides and proteins and have been observed in the tissues of mammals, birds, fish, plants, and bacteria. In the CA clan, we have calpains and papain-like CysPs. Plant genomes contain only one calpain (family C02), known as phytocalpain, that comprise an interesting group of non-classical calpains called as DEK1 subfamily. They carry the active-site residues of cysteine (Cys) on sub-domain PC1, and histidine (His) and asparagine (Asn) on sub-domain PC2, but the maize DEK1 (defective kernel 1) molecule does not depend on Ca2+ for in vitro proteolytic activity. This cysteine-protease is found in other plants including Oryza sativa and Arabidopsis thaliana. Calpains are essential for plant epidermis development. Papain-like cysteine proteases (family C1) (PLCPs) are very stable enzymes and are often found in proteolytically harsh environments such as the apoplast, vacuole, and lysosomes. They are synthetized as pre-proteins and carry targeting signals that ensure they enter the correct endomembrane system. Some PLCPs have an autoinhibitory prodomain that prevents premature activation of the protease. Others PLCPs also carry a C-terminal granulin-like domain that share homology to granulins in animals, which are growth hormones released upon wounding.

PLCPs also play key roles in programmed cell death (PCD) in the development process, as well as that induced by external stimulus, as previously mentioned CysPs. The PLCP family consists of papain, chymopapain, caricain, bromelain, actinidin, ficin, aleurain, etc. Bromelain is the collective name of related CysPs found in species belonging to the Bromeliaceae family, of which pineapple (Ananas comosus) is the most studied. Nevertheless, in recent years, an increased number of identification and characterization studies of CysPs have appeared in other fruits of Bromelia antiacantha, Bromelia pinguin, Pseudonanas macrodontes and Bromelia hieronymi Mez.