Cathepsins proteinase activity

Several lines of evidence indicate that neoplastic cells per se are the main source of extracellular thiol proteinase activity. Recent studies (39) have shown that malignant human breast tumors maintained in organ culture secrete high levels of a cathepsin B-like enzyme into the culture medium. Moreover high levels of cathepsin B-like enzyme are present in the serum of patients with a wide variety of cancers, and these levels decrease when the cancer tissue is removed or treated with therapeutic agents (64, 65). Cathepsin B-like enzyme from cultured cells of malignant tumors (39,66) possesses enzymic properties similar to those of cathepsin B with respect to specificity, affinity, and pH optima for synthetic substrates. It hydrolyzes Bz-Arg-Arg-2-naphthylamide and is inhibited by leupeptin. However, the tumor enzyme is much more stable than cathepsin B to inactivation above pH 7. It has a molecular weight of about 33,000-35,000. The distribution of cathepsin B-like activity was determined in fractions of control and neoplastic epithelial cells from human ectocervix (66). The activity is present mainly in the mitochondrial and lysosomal fractions of normal cells but mainly in the plasma membranes and nuclei of neoplastic cells. 

Fig. 4. Time courses of change in activities of cathepsin B (a) and cathepsin L (b) from rat liver treated with E-64 (L) and leupeptin. E-64 (L) and leupeptin were dissolved in 0.9% NaCl. Rats weighing 100 to 120 g were given a single ip injection of 1.0 mg/100 g body weight of proteinase inhibitors. At the times after injection as indicated, rats were killed, the mitochondrial-lysosomal fraction was isolated, and cathepsin B and cathepsin L activities were determined. Without inhibitor (O), with E-64 (L) ( ), with leupeptin ( ). (Revised from Ref 70.)... 

Starvation of animals increases the rate of protein degradation in liver. 81,82) and muscle 83-85) and causes about twofold increase of cathepsin D and A within 24 hours in the liver 55, 56). However, increases of cathepsin B and L are much less than that of catheptsin D (55). In the livers of protein-depleted rats, cathepsins B, L, and D decrease during the course of protein depletion 49,81), their activities being one-third of that of controls (on 20% casein diet) after protein depletion for 10 days. It is, however, not known whether changes of proteinase activity are associated with changes in protein synthesis. 

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Eosinophils may be increased in some patients, particularly during exacerbations. Activated inflammatory cells release a variety of mediators, most notably leukotriene B4, interleukin-8, and tumor necrosis factor-a (TNF-a). Various proteinases, such as elastase, cathepsin G, and proteinase-3, are secreted by activated neutrophils. These mediators and proteinases are capable of sustaining inflammation and damaging lung structures. 

C5a is inactivated by the myeloperoxidase-H202 system, which oxidises a methionine residue (Met 70) on the molecule group A streptococcal endo-proteinases also abolish chemotactic activity of C5a and related compounds. Neutrophil lysosomal enzymes (e.g. elastase and cathepsin G) also destroy C5a chemotactic activity, but as these proteases are inhibited by the serum antiproteinases, a -antiproteinase and a2-macroglobulin, the physiological role of neutrophilic proteases in the inactivation of C5a is questionable. Two chemotactic factor inactivators have been found in human serum an a-globulin that specifically and irreversibly inactivates C5-derived chemotactic factors, and a / -globulin that inactivates bacterial chemotactic factors. These activities are heat labile (destroyed by treatment at 56 °C for 30 min) and are distinct from those attributable to anaphylatoxin inactivator. An apparently specific inhibitor of C5-derived chemotactic activity has also been described in human synovial fluid and peritoneal fluid. This factor (molecular mass of 40 kDa) is heat stable and acts directly on C5a. 

The functional proteins in the cell have to be protected in order to prevent premature degradation. Some of the intracellularly active proteolytic enzymes are therefore enclosed in lysosomes (see p. 234). The proteinases that act there are also known as cathepsins. Another carefully regulated system for protein degradation is located in the cytoplasm. This consists of large protein complexes (mass 2 10 Da), the proteasomes. Proteasomes contain a barrel-shaped core consisting of 28 subunits that has a sedimentation coef cient (see p. 200) of 20 S. Proteolytic activity (shown here by the scissors) is localized in the interior of the 20-S core and is therefore protected. The openings in the barrel are sealed by 19-S particles with a complex structure that control access to the core. 

Milk contains at least two proteinases, plasmin (alkaline milk proteinase) and cathepsin D (acid milk proteinase) and possibly several others, i.e. two thiol proteinases, thrombin and an aminopeptidase. In terms of activity and technological significance, plasmin is the most important of the indigenous proteinases and has been the subject of most attention. The relevant literature has been reviewed by Grufferty and Fox (1988) and Bastian and Brown (1996). 

Cathepsin D (EC3.4.23.5). It has been known for more than 20 years that milk also contains an acid proteinase, (optimum pH ss 4.0) which is now known to be cathepsin D, a lysozomal enzyme. It is relatively heat labile (inactivated by 70°C x 10 min). Its activity in milk has not been studied extensively and its significance is unknown. At least some of the indigenous acid proteinase is incorporated into cheese curd its specificity on asl- and / -caseins is quite similar to that of chymosin but it has very poor milk-clotting activity (McSweeney, Fox and Olson, 1995). It may contribute to proteolysis in cheese but its activity is probably normally overshadowed by chymosin, which is present at a much higher level. 

Other proteinases. The presence of low levels of other proteolytic enzymes in milk has been reported (see Fox and McSweeney, 1996). Most of these originate from somatic cells, and their level increases during mastitic infection. The presence of cathepsin D, a lysozomal enzyme, in milk suggests that all the lysozomal proteinases are present in milk although they may not be active. These minor proteinases are considered to be much less significant than plasmin, but more work on the subject is necessary. 

Serine proteases, released from immune cell granules, process cytokines and growth factors that control multiple cellular process [56], Proteinase 3, cathepsin G, and elastase all cleave membrane-bound TNF-o, IL-1, and IL-18, and activate epidermal growth factor receptor (EGFR) and toll-like receptor-4 (TLR-4). These actions inhibit growth and lead to apoptosis with transcriptional nuclear factor kB (NF-kB) inactivation. Bik suppresses release of TNF-o, IL-1, and IL-18 and prevents EGFR and TLR-4 activation. Activation of NF-kB is a mediator of cell proliferation, whereas inhibition of NF-/. B leads to apoptosis [82]. Overall, Bik inhibition of immune cell serine proteases increases cell proliferation and stability. 

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