Eiki Kominami

Structures and Functions of Lysosomal Thiol Proteinases and Their Endogenous Inhibitor

Publisher Summary This chapter discusses the structures and functions of lysosomal thiol proteinases and their endogenous inhibitor. There is a very high concentration of cathepsin L in liver, which is similar to that of cathepsin B. When lysosomes are broken during homogenization of tissues, the thiol proteinase inhibitor binds to proteinases immediately. Various proteinase inhibitors of microbial origin are useful in elucidating the importance of lysosomal thiol proteinases in protein degradation; however, these inhibitors do not distinguish between cathepsin B, cathepsin L, and other thiol proteinases. Thus, specific substrates for use in the assay of individual thiol proteinases and specific inhibitors of respective proteinases must be developed. Addition of proteinase inhibitors to cells or their injection in vivo inhibits lysosomal proteinase activities, causes formation of autophagic vacuoles, and induces synthesis of hemoglobin-hydrolyzing thiol proteinase. Various blood proteins—such as asialoproteins—are degraded in liver via processes involving receptor-mediated binding, pinocytosis, fusion with primary lysosomes, and subsequent hydrolysis in secondary lysosomes.

Molecular cloning and sequencing of cDNA for rat cathepsin L

A near full‐length cDNA for rat cathepsin L was isolated. The deduced protein comprises 334 amino acid residues (M r 37 685) containing a typical signal sequence (N‐terminal 17 residues), pro‐peptide (96 residues), and the sequence for mature cathepsin L (221 residues). Rat cathepsin L shows 94% amino acid identity with mouse cysteine proteinase. Amino acid sequence homologies of rat cathepsin L with rat cathepsins H and B are 45 and 25%, respectively. These facts indicate that mouse cysteine proteinase is probably mouse cathepsin L and that cathepsin L is more closely related to cathepsin H than cathepsin B.

A new function of kininogens as thiol-proteinase inhibitors: inhibition of papain and cathepsins B, H and L by bovine, rat and human plasma kininogens.

Kininogen Thiol‐proteinase inhibitor Papain Cathepsin B Cathepsin H Cathepsin L

Biosyntheses and processing of lysosomal cysteine proteinases in rat macrophages

The intracellular processing and release of three lysosomal cysteine proteinases, cathepsin B, H and L, by rat peritoneal macrophages were investigated by pulse‐chase experiments. Newly synthesized procathepsins B (39 kDa), H(41 kDa) and L (39 kDa) after 15 min labeling were processed to the mature, single‐chain enzymes within 1 h. The single‐chain forms of cathepsin B, H and L were further processed to two‐chain forms at different rates: conversion of cathepsin L to the two‐chain form was rapid, whereas the conversions cathepsin B and H took at least 6 h. Macrophages released 30% of the procathepsins B and L, and 10% of the procathepsin H.

Effect of proteinase inhibitors on intracellular processing of cathepsin B, H and L in rat macrophages

The effects of various proteinase inhibitors on the processing of lysosomal cathepsins B, H and L were investigated in cultured rat peritoneal macrophages. The processing of newly synthesized pro‐cathepsins B, H and L to the mature single‐chain enzymes was sensitive to a metal chelator,1,10‐phenanthroline, and a synthetic metalloendopeptidase substrate, Z‐Gly‐Leu‐NH2, and insensitive to inhibitors of serine proteinases, aspartic proteinases and cysteine proteinases. Inhibitors of cysteine proteinases, E‐64‐d and leupeptin, inhibited the processing of the single‐chain forms of cathepsins B, H and L to the two‐chain forms. These results suggest that (a) metal endopeptidase(s) is (are) involved in the propeptide processing of cathepsin B, H and L, and that proteolytic cleavages of the mature single‐chain cathepsins are accomplished by cysteine proteinases in lysosomes.

A new enzyme that specifically inactivates apo-protein of pyridoxal enzymes

Abstract We found a new inactivating enzyme in small intestine and skeletal muscles, which specifically reacts with apo-proteins of pyridoxal enzymes. This inactivating enzyme does not react with all the non-pyridoxal enzymes tested. The inactivation by this enzyme is prevented by the addition of PALP. The enzyme splits apo-pyridoxal enzymes into smaller protein and oligopeptides. The activity of inactivating enzyme increases from 10–20 fold only in the case of B 6 deficiency compaired with that of normal rats.

Molecular cloning and sequencing of cDNA for rat cathepsin H. Homology in pro-peptide regions of cysteine proteinases.

A cDNA for rat cathepsin H was isolated and sequenced. The deduced protein comprising 333 amino acid residues is composed of a typical signal sequence (21 residues), a pro‐peptide region (92 residues) and a mature enzyme region (220 residues). The amino acid sequence in the pro‐peptide region, in particular, residues Phe‐(−41) to Ser‐(−29) of cathepsin H, is highly homologous to the pro‐peptide regions of other cysteine proteinases. This homologous region may play a role in the processing of cysteine proteinases.

Inhibitions of degradation of rat liver aldolase and lactic dehydrogenase by N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine or leupeptin in vivo

Abstract When injected into rats, leupeptin and E-64 (N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine), potent thiol protease inhibitors of microbial origin, inhibited cathepsin B (EC 3.4.22.1) and cathepsin L (EC 3.4.22.-) in the lysosomal fraction of liver. Both compounds strongly inhibited cathepsin B, but E-64 had more effect than leupeptin on cathepsin L. Neither compound inhibited cathepsin D (EC 3.4.23. 5). E-64 reduced the apparent turnover rate of aldolase (EC 4. 1.2.13) markedly and the turnover rates of lactic dehydrogenase (EC 1.1.1.27) and total soluble protein slightly. Leupeptin had apparently less effect on degradation of those enzymes, but significant effect on degradation of aldolase. These results indicate that proteinases, which are sensitive to inhibition by E-64 or leupeptin, especially cathepsin L and cathepsin B may be important in degradation of aldolase.

Gene structure of rat cathepsin H

The gene structure of rat cathepsin H was determined. It comprises at least 12 exons of various lengths (32–433 bp) spanning in total more than 17.5 kbp. The gene structure does not correspond well to the functional unit of the proteinase. The region around the active site Cys residue, the most conserved region among cysteine proteinases, is split by an intron. This is a common characteristic among the gene structures of cysteine proteinases.

A new enzyme that specifically inactivates apo-protein of NAD-dependent dehydrogenases

Abstract The existence of a new inactivating enzyme for NAD-dependent enzymes was recognized in small intestine of niacin-deficient rats. The increase of the enzyme activity was in parallel with the degree of niacin deficiency. The enzyme splits apo-NAD dependent enzymes to smaller molecular compounds. The inactivating reaction is prevented by the presence of NAD.