Etsuko Murata

Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivo

Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer‐graphic modeling based on the stereo‐structure. The common fragment, N‐(L‐trans‐carbamoyloxyrane‐2‐carbonyl)‐phenylalanine‐dimethylamide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10−7 M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10−7 M in vitro.

New functions of lactoferrin and β-casein in mammalian milk as cysteine protease inhibitors

We found new inhibitory function of lactoferrin and beta-casein in milk against cysteine proteases using reverse zymography. The inhibition of cathepsin L by lactoferrin was strongest and the inhibition kinetics were of a non-competitive type. Heat denatured lactoferrin lost the inhibitory activity completely, therefore the tertiary structure is essential to show the inhibition. Native lactoferrin was not degraded by papain during the assay condition. The intramolecular peptide, Y(679)-K(695), of lactoferrin is an active domain and the synthesized peptide inhibited cysteine proteases. The Y(679)-K(695) peptide showed 90% homology with the sequences of a common active site of cystatin family. beta-Casein and the active domain, synthesized L(133)-Q(151), peptide inhibited cysteine proteases. Lactoferrin and beta-casein in milk might play a role in antiseptic and antiinfectious functions due to cysteine protease inhibition of bacteria and viruses.

Catechin derivatives: Specific inhibitor for caspases-3, 7 and 2, and the prevention of apoptosis at the cell and animal levels

Tea‐catechin derivatives are shown to inhibit activities of caspases‐3, 2 and 7 in vitro, and prevented experimental apoptosis at the cell and animal levels. Epigallo‐catechin‐gallate showed the strongest inhibition at 1 × 10−7 M to these caspases, but cysteine cathepsins and caspase‐8 were not inhibited. Caspase‐3 inhibition showed a 2nd‐order allosteric‐type, but the inhibition of caspases‐2 and 7 showed a non‐competitive‐type. The apoptosis‐test using cultured HeLa cells was inhibited by these catechins. In rat hepatocytes, apoptosis was induced by d‐galactosamine in vivo. In this case, caspase‐3 activity in the cytoplasm, the serum aminotransferases and dUTP nick formation detected by TUNNEL‐staining were effects, and these elevations were suppressed by administration of catechin.

Study of the functional share of lysosomal cathepsins by the development of specific inhibitors.

To analyze the functional share of individual cathepsins, we developed powerful and specific inhibitors for individual cathepsins using computer graphics of substrate binding pockets based on X-ray crystallography. These new inhibitors were named CLIK group. Epoxy succinate peptide derivatives, CLIK-066, 088, 112, 121, 148, 181, 185 and 187, are typical specific inhibitors for cathepsin L. Aldehyde derivatives CLIK-060 and CLIK-164 showed specific inhibition against cathepsin S and cathepsin K, respectively. We found that pyridoxal phosphate (PLP), a coenzyme form of vitamin B6, inhibits all cathepsins and also new artificially synthesized pyridoxal derivatives, CLIK-071 and -072, in which the phosphate esters of PLP were replaced by propionic acid, exhibited strong inhibition for cathepsins. Furthermore, CLIK-071 was easy to incorporate into cells and showed powerful inhibition for intracellular cathepsins. Using these selective inhibitors, the allotment of individual cathepsin functions in cells has been studied as follows. Cathepsin L and/or K participate in bone resorption based on bone type-1 collagen degradation and the L-type protease inhibitors suppressed the bone resorption. Cathepsins B and S participate in antigen presentations based on antigen processing and invariant chain degradation, respectively. Also cathepsin L participates in cell apoptosis mediated by caspase III activation.

Chondroitin sulfate proteoglycan is a potent enhancer in the processing of procathepsin L

Abstract The acceleration effect of chondroitin-4-sulfate(CS) proteoglycan on the processing of procathepsin L in vitro was investigated using enzyme purified from the culture medium of MLC cells. Procathepsin L was slightly processed even when it was incubated without CSproteoglycan for 60 min in 50 mM acetate buffer, pH 5.5, and trace amounts of the 31 kDa mature form and 35 38 kDa intermediates of cathepsin L were formed. On the other hand, in the presence of CSproteoglycan, procathepsin L was completely converted to the mature form within the same 60 minute time period. Moreover, ZPheArgMCA hydrolyzing activity was increased significantly by the incubation with CSproteoglycan, while no considerable increase in the activity was observed during the incubation without CSproteoglycan. Since the specific cathepsin L inhibitor, CLIK-195, inhibited the processing of procathepsin L accelerated by CSproteoglycan, the trace amount of cathepsin L activity may participate in the processing. These results suggest that CSproteoglycan may play a role in accelerating the processing of procathepsin L as an endogenous enhancer in the extracellular environment in vivo.

A novel apoptosis cascade mediated by lysosomal lactoferrin and its participation in hepatocyte apoptosis induced by d‐galactosamine

A new apoptosis cascade mediated by lysosomal lactoferrin was found in apoptotic liver induced by d‐galactosamine. Caspase‐3 and lactoferrin were increased in the apoptotic liver cytoplasm and serum transaminases were elevated. Recombinant lactoferrin stimulated procaspase‐3 processing at 10−6–10−7 M to an extent similar to that by granzyme B in vitro. Lactoferrin changed procaspase‐3 structure susceptible to the processing. Synthetic peptide Y679‐K695 in lactoferrin molecule inhibited the processing of procaspase‐3 by lactoferrin. Lactoferrin in lysosomes was decreased and lactoferrin released into cytoplasm was increased quantitatively in d‐galactosamine induced apoptotic liver, and procaspase‐3 in cytoplasm was processed to caspase‐3.

New cysteine protease inhibitors in physiological secretory fluids and their medical significance

New cysteine protease inhibitors in human tears and milk were found and their medical significance was studied. As the protective components against bacterial infection in eyes, we detected four kinds of biologically active proteins in normal human tears including three kinds of cysteine protease inhibitors. Using our reverse zymography of normal tears, the three kinds of cysteine protease inhibitors were found to be 78, 20 and 15 kDa and were determined to be lactoferrin, VEG protein and cystatin S, respectively. The C-terminus area 17 mer peptide, Y679-K695 of lactoferrin molecule showed strong homology with a common active domain of cystatin family and the synthesized peptide itself showed considerable inhibition of cysteine proteases. Not only disease-specific changes of these inhibitor contents, but disease-specific new inhibitors were also found in patient tears in special autoimmune diseases. The characteristic 35 kDa inhibitor band which was detected specifically in the cases of Behcets disease tears, an autoimmune disease, was determined to be a lachrymal acidic proline-rich protein family based on the Nterminus sequence analysis. The 65 kDa inhibitor of tears in Haradas autoimmune-disease was determined to be a human Ig heavy chain V-III region. Also lactoferrin content in Haradas disease was very low compared with that of normal tears. Also we found two cathepsin inhibitors, lactoferrin and β-casein, in milk of human and bovine using reverse zymography. They may also play a role in bacterio-cidal and viro-cidal functions in milk. The L133-Q151 in human β-casein molecule is the active inhibitory domain. It is most important to know from biological aspects that the concentration of these inhibitors in natural milk can inhibit cysteine proteases of bacteria. Surprisingly, the 50 times diluted milk inhibited papain completely, because lactoferrin and casein contents in milk are very high. We want to emphasize that these inhibitors in milk play a sufficient role in the protection of bacteria. Index