1. Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondria1 pellet, gel filtration of the lysosomal extract and chromatography on CM-Sephadex.2. Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram. By polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, its molecular weight was found to be 23000-24000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8 -6.1 ascertained by analytical isoelectric focusing.3. Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6 -7. In contrast to cathepsin B1, its capability of hydrolyzing N-substituted derivatives of arginine is low and it does not split esters. 4. Greatest activity is obtained close to pH 5.0 with 70-90% of maximal activity at pH 4.0 and pH 6.0 and 30 -40 0 4 at pH 7.0.5. The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl-lysine. Leupeptin acts as a pseudo-irreversible inhibitor.6. The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep-frozen state.Knowledge of the proteolytic enzymes in the cell is one of the preconditions in studying the molecular mechanism of intracellular protein breakdown. All organelles contain proteolytic activity in various amounts [l]. The lysosomes, in particular, are rich in proteinases. A cell fraction enriched in lysosomes showed at pH 3-4 as well as at pH 6-7 the highest activity hydrolyzing cell-derived proteins in comparison to all other cell organelles [l -61. From this finding it was to be concluded, that the lysosomes certainly play an important role in the overall process of intracellular proteolysis also at physiological pH. A wide variety of lysosomal proteinases has been investigated : cathepsin A (lysosomal carboxypeptidase A) [7-131, cathepsin B1 [7,12-201, cathepsin B2 (lysosomal carboxypeptidase B) [7,12 -14,20-221, cathepsin C (dipeptidyl aminopeptidase I) [7,12,13, Dedicated to Professor Horst Hanson on the occasion of his 65th birthday.Ahbveviation. Leu-CH2C1, 1 -chloro-3-amino-5-methyl-~-hexan-2-one; Tos-Lys-CH2C1, 7-amino-l-chloro-3-tosylamido-~-heptan-2-one ; Tos-Phe-CHzC1, l-chlor0-4-phenyl-3-tosylamido-~-butan-2-one; Bz-L-Arg-NHZ, or-N-benzoyl-L-arginine amide.Enzymes. Cathepsin L (EC 3.4.22.-); cathepsin B1 and B2 (EC 3.4.22.1); cathepsinc (EC 3.4.14.1); cathespin D (EC 3.4.23.5). 18,23,24],cathepsin D [6,7,12,13,23,25-311, cathepsin E [7,13] In studies on the proteolytic activity in a lysosomal extract we succeeded in separating cathepsins BI, C and D by gel filtration on Sephadex G-75. With cytosol proteins as well as with azocasein as substrates at pH 6 and 7, the main part of proteolytic activity was shown to be present in the protein fraction with molecular weights between 20000 and 30000 [2,43]. We could show that in addition to cathepsin B1, the...

Regulation of proteolytic enzyme activity is an essential requirement for cells and tissues because proteolysis at the wrong time and location may be lethal. Two principal mechanisms to control the activity of proteases have been developed during evolution. The first is the co-evolution of endogenous inhibitors, typically occurring in cellular compartments separated from those containing active enzymes. The second is the fact that proteases are synthesized as inactive or less active precursor molecules. They are activated, in some cases, upon an appropriate signal like acidification, Ca(++) -binding or, in other cases, by limited intra- or intermolecular proteolysis cleaving off an inhibitory peptide. These regulatory proenzyme regions have attracted much attention during the last decade, since it became obvious that they harbour much more information than just triggering activation. In this review we summarize experimental data concerning three functions of propeptides of clan CA family C1 cysteine peptidases (papain family), namely the selectivity of their inhibitory potency, the participation in correct intracellular targeting and assistance in folding of the mature enzyme. Cysteine peptidases of the CA-C1 family include members from the plant kingdom like papain as well as from the animal kingdom like the lysosomal cathepsins L and B. As it will be shown, the functions are determined by certain structural motifs conserved over millions of years after the evolutionary trails have diverged. The function of propeptides of two other important classes of cysteine peptidases - the calpains, clan CA family C4, and the caspases, clan CD family C 14 - are not considered in this review.

Cathepsin S was detected in bovine kidney, spleen, lymph nodes and lung by immunochemical methods. The immunostaining of cathepsin S in kidney was concentrated to the cells of the proximal tubule, where the enzyme was present in cytoplasmic granules. The purification method for cathepsin S from bovine spleen involved (NH4)2SO4 fractionation, chromatography on CM-Sephadex C-50, gel filtration on Sephacryl S-200 and chromatofocusing (pH 8.0-6.0). The enzyme was partially destroyed by autolysis of the homogenate at pH 4.2. The isoelectric point of cathepsin S was 7.0. Cathepsin S was found to hydrolyse proteins at a similar rate to cathepsin L below pH 7.0. At pH values of 7.0-7.5 cathepsin S retained most of its activity, whereas cathepsin L was completely inactive.

Signal transducer and activator of transcription 3 (STAT3) is aberrantly activated in colorectal carcinomas (CRCs). Here, we define the relationship between STAT3 function and the malignant properties of colon carcinoma cells. Elevated activation of STAT3 enhances invasive growth of the CRC cell lines. To address mechanisms through which STAT3 influences invasiveness, the protease mRNA expression pattern of CRC biopsies was analyzed and correlated with the STAT3 activity status. These studies revealed a striking coincidence of STAT3 activation and strong expression of matrix metalloproteinases MMP-1, -3, -7, and -9. Immunohistological examination of CRC tumor specimens showed a clear colocalization of MMP-1 and activated STAT3. Experimentally induced STAT3 activity in CRC cell lines enhanced both the level of MMP-1 mRNA and secreted MMP-1 enzymatic activity. A direct connection of STAT3 activity and transcription from the MMP-1 promoter was shown by reporter gene experiments. Moreover, high-affinity binding of STAT3 to STAT recognition elements in both the MMP-1 and MMP-3 promoter was demonstrated. Xenograft tumors arising from implantation of CRC cells into nude mice showed simultaneous appearance and colocalization of p-Y-STAT3 and MMP-1 expression. Our results link aberrant activity of STAT3 in CRC to malignant tumor progression through upregulated expression of MMPs.

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The peptide-bond-specificity of bovine spleen cathepsin S in the cleavage of the oxidized insulin B-chain and peptide methylcoumarylamide substrates was investigated and the results are compared with those obtained with rat liver cathepsins L and B. Major cleavage sites in the oxidized insulin B-chain generated by cathepsin S are the bonds Glu13-Ala14, Leu17-Val18 and Phe23-Tyr26; minor cleavage sites are the bonds Asn3-Gln4, Ser9-His10 and Leu15-Tyr16. The bond-specificity of this proteinase is in part similar to the specificities of cathepsin L and cathepsin N. Larger differences are discernible in the reaction with synthetic peptide substrates. Cathepsin S prefers smaller neutral amino acid residues in the subsites S2 and S3, whereas cathepsin L efficiently hydrolyses substrates with bulky hydrophobic residues in the P2 and P3 positions. The results obtained from inhibitor studies differ somewhat from those based on substrates. Z-Phe-Ala-CH2F (where Z- represents benzyloxycarbonyl-) is a very potent time-dependent inhibitor for cathepsin S, and inhibits this proteinase 30 times more efficiently than it does cathepsin L and about 300 times better than it does cathepsin B. By contrast, the peptidylmethanes Z-Val-Phe-CH3 and Z-Phe-Lys(Z)-CH3 inhibit competitively both cathepsin S and cathepsin L in the micromolar range.

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The interaction of human recombinant full-length cathepsin S propeptide (amino acids 16-114) with mature cysteine proteinases was studied with respect to selectivity and pH dependence. The inhibitory capacity was tested towards mature human recombinant cathepsin S, purified cathepsin L from rat and Paramecium tetraurelia, rat cathepsin B, human cathepsin H, and papain. The propeptide of cathepsin S strongly inhibited cathepsin S (Ki = 0.27 nM) and the two cathepsin L species (Ki = 0.36 nM) at neutral pH. Papain, and to a minor extent cathepsin H, hydrolyzed the propeptide of cathepsin S, leading to competition with the hydrolysis of the fluorogenic substrates in the respective assays. Cathepsin B activity was nearly unaffected up to micromolar propeptide concentrations in the assay. The inhibition of cathepsin-L-like peptidases was diminished with decreasing pH, probably due to dramatic changes in the conformation of the propeptide. This assumption was supported by far-ultraviolet CD spectroscopy and by the finding of rapid hydrolysis of the cathepsin S propeptide by cathepsin L at pH values less than 5.5.