Cysteine Protease Regulator Proteins

 Creative BioMart Cysteine Protease Regulator Proteins Product List
 Cysteine Protease Regulator Proteins Background

Cysteine proteases have a cysteine residue in their active side and therefore employ the sulfhydryl group of the side chain of a cysteine residue as the catalytic nucleophile. The first step of the peptide hydrolysis mechanism involves the deprotonation of the sulfhydryl group in the enzyme's active site by the histidine residue. A catalytic dyad is formed with the imidazole side chain of histidine residue and the sulfhydryl group of the active site cysteine. Some of the cysteine proteases use an extra amino acid to provide additional stabilization via hydrogen bonds, thus forming a catalytic triad. The next step is the nucleophilic attack of the thiolate anion to the carbonyl carbon of the peptide bond to be cleaved and a tetrahedral intermediate is formed. The tetrahedral intermediate is stabilized by the oxyanion hole via hydrogen bonds to the backbone amides. Collapse of the tetrahedral intermediate releases the amino terminus of the substrate and results in the formation of an acyl intermediate. Abstraction of a proton from a water molecule by the catalytic histidine forms an activated water molecule. The thioester bond of the acyl intermediate is then hydrolyzed by the activated water molecule and the free enzyme is regenerated.

Cysteine proteases currently consist of 10 clans. We will focus on clan CA and clan CD cysteine proteases since the inhibitors reported in this thesis are designed for the enzymes belong to these two clans. Clan CA and clan CD cysteine proteases differ slightly in their substrate hydrolysis mechanism. A catalytic dyad of Cys and His is employed by clan CD whereas a catalytic triad of Cys, His, and Asn is used by clan CA cysteine proteases. Stabilization of the tetrahedral intermediate occurs by H bonding to the backbone NH of active site Cys and to the NH2 group of the Gln side chain in clan CA. The oxyanion hole consists of only backbone NH groups in clan CD cysteine proteases.


Clan CA Cysteine Proteases

The majority of cysteine proteases belong to clan CA and these cysteine proteases have been found in viruses, bacteria, protozoa, plants and mammals. Clan CA contains all the families of peptidases that have similar structures to papain.

Papain is a cysteine protease extracted from the tropical papaya fruit. It is the first cysteine protease to be discovered and has been the subject of mechanism and structural studies for many years and it is also the first enzyme to have its crystal structure determined. The substrate specificity of clan CA enzymes is primarily controlled by the S2 subsite. Papain has a very broad specificity compared to other clan CA proteases. It prefers bulky hydrophobic residues in the P2 position, while the S1 subsite is not as selective as the S2 subsite, but has some preference for Arg and Lys over other residues such as Val.

Cathepsin B is a lysosomal cysteine protease with both endopeptidase and dipeptidyl carboxypeptidase activities. The substrate specificity of cathepsin B is similar to papain family and the primary determinant of specificity is the S2 subsite. Cathepsin B can accommodate hydrophobic residues together with arginine in the P2 position due to the location of Glu245 in the S2 subsite. Cathepsin B is implicated in diseases such as arthritis, muscular dystrophy, gingivitis and cancer.

Calpains are a unique family of cysteine proteases since they require calcium to become activated. The major isoforms of calpain (calpain I and calpain II) are nearly identical and differ mostly in the amount calcium they require for activation. Calpain activation is utilized in many cellular processes such as apoptosis, cell differentiation and protein turnover. Calpains prefer hydrophobic residues like Val or Leu in the P2 position but are less selective in the P1 position. Overactivation of calpain has been observed in a variety of disorders, including cataracts, muscular dystrophy, cancer and neurodegenerative diseases.

Cruzain is the only parasitic cysteine protease with a crystal structure. Cruzain is expressed in all life cycles of Trypanasoma cruzi, which is the causative agent of Chagas’ disease in Central America. Cruzain can accommodate hydrophobic residues like phenylalanine together with basic residues such as arginine in the S2 pocket. Cruzain is an excellent target for the development of irreversible inhibitors due to the presence of the enzyme in all stages in the life cycle of the parasite.

Rhodesain is the major cysteine protease of T. brucei rhodesiense, the causative agent of African sleeping sickness. Rhodesain is an important target for the development of antiparasitic chemotherapy due to the involvement of the enzyme in regulating the replication of the parasite. Therefore, inhibition of the enzyme will block the life cycle of the parasite in infected mammalian cells.

TbCatB has recently been identified in T. brucei as a key enzyme in host protein turnover and iron acquisition. TbcatB, a cathepsin B-like enzyme, is an ideal target for the development of new anti-trypanosomal chemotheraphy.


Clan CD Cysteine Proteases

Clan CD is a smaller class of cysteine proteases compared to clan CA cysteine proteases. Clostripain (C11), legumains (C13), caspases (C14), gingipain (C25), separase (C50) and self-cleaving toxin A (C80) belong to clan CD cysteine proteases. The substrate specificity of the members of this class is determined by the residue in the P1 position, such as Asp for caspases, Arg for clostripain, Asn for legumain family, Arg or Lys for gingipain family, Arg for separase family and Leu for self-cleaving toxin A family. Unlike clan CA cysteine proteases, clan CD enzymes employ a catalytic dyad (His-Cys) for substrate hydrolysis.

Asparagine endopeptidases (legumains) cleave their peptide substrates specifically after an asparagine residue. Asparagine endopeptidases (AEs) have been linked to osteoclast formation and bone resorption,19 processing of bacterial antigens20 and elevated levels of human AE was found in many tumors, including carcinomas of the breast, colon, and prostate. AE of the bloodfluke Schistosoma mansoni and AE of the hard tick Ixodes ricinus have an indirect role in host hemoglobin digestion and therefore represent important targets for the development of inhibitors.