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1.
Intracellular transport and processing of lysosomal cathepsin B   总被引:2,自引:0,他引:2  
Intracellular transport and processing of lysosomal cathepsin B was investigated in the subcellular fractions of rat liver by pulse-labeling experiments with [35S]methionine in vivo. A newly synthesized procathepsin B with a molecular weight of 39 kDa firstly appeared in the rough microsomal fraction at 10 min postinjection of label. This procathepsin B moved from the microsomal fractions to the Golgi subfractions at 30 min postinjection, and then a processed mature enzyme appeared in the lysosomal fraction at 60 min. These results suggest that the propeptide-processing of procathepsin B takes place in lysosomes in the course of intracellular transport from endoplasmic reticulum through Golgi complex to lysosomes.  相似文献   

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The cytotoxic lymphocyte protease granzyme B (GzmB) can promote apoptosis through direct processing and activation of members of the caspase family. GzmB can also cleave the BH3-only protein, BID, to promote caspase-independent mitochondrial permeabilization. Although human and mouse forms of GzmB exhibit extensive homology, these proteases diverge at residues predicted to influence substrate binding. We show that human and mouse GzmB exhibit radical differences in their ability to cleave BID, as well as several other key substrates, such as ICAD and caspase-8. Moreover, pharmacological inhibition of caspases clonogenically rescued human and mouse target cells from apoptosis initiated by mouse GzmB, but failed to do so in response to human GzmB. These data demonstrate that human and murine GzmB are distinct enzymes with different substrate preferences. Our observations also illustrate how subtle differences in enzyme structure can radically affect substrate selection.  相似文献   

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1. The specificity of cathepsin G, a neutral proteinase from human spleen, was examined by use of low-molecular-weight substrates. The enzyme was found to hydrolyse several synthetic substrates also hydrolysed by chymotrypsin, but with different kinetic constants. 2. Maximal activity against benzoyl-DL-phenylalanine 2-naphthol ester and azo-casein was in the range pH 7.5-8.0. 3. The sensitivity of cathepsin G to the action of potential inhibitors was determined, and compared with those of bovine chymotrypsin and subtilisin. Cathepsin G showed the characteristics of a serine proteinase, but was less affected by the chloromethyl ketone of tosylphenylalanine than was chymotrypsin. 4. A rabbit anti-(human cathepsin G) serum was raised, and precipitin lines formed in agarose gel were stained for activity of the enzyme. 5. Cathepsin G was shown to be immunologically identical with the chymotrypsin-like enzyme of the azurophil granules of the neutrophil granulocytes.  相似文献   

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Various biosynthetic forms of porcine spleen cathepsin D (Erickson, A. H. and Blobel, G. (1979) J. Biol. Chem. 254, 11771-11774), isolated by immunoprecipitation of in vivo- and in vitro-synthesized products, have been characterized by partial NH2-terminal sequence analysis. Two short lived and functionally distinct NH2-terminal sequence extensions, a "pre" sequence and a "pro" sequence, have been detected. Both sequence extensions are present in preprocathepsin D which is the primary translation product immunoprecipitated after translation of porcine spleen mRNA in a wheat germ cell-free system. Preprocathepsin D is not glycosylated and has an approximate Mr = 43,000. Its 20-residue pre sequence resembles the signal sequences of presecretory proteins in abundance of Leu residues (7 out of 20 residues). Addition of dog pancreatic microsomal vesicles to the translation system resulted in the cleavage of the pre sequence and yielded segregated and glycosylated procathepsin D (Mr = 46,000) that was indistinguishable from its in vivo-synthesized counterpart detected after pulse-labeling of cultured porcine kidney cells. Some of this in vivo-synthesized procathepsin D was secreted and persisted as such in the culture medium. The remainder was converted within a period of 15 min to 2 h to single chain cathepsin D (Mr = 44,000) by removal of a pro sequence which was estimated to be 44 residues. Its partial sequence showed considerable sequence homology to the 44-residue activation peptide of pepsinogen. It is possible, therefore, that the prosequence of procathepsin D serves as an activation peptide that keeps the enzyme inactive during intracellular transport to the lysosome. The enzymatically active single chain form of cathepsin D undergoes further cleavage into a light and a heavy chain (Mr = 15,000 and 30,000, respectively) over a period of 2-24 h after synthesis. The oligosaccharide moieties of procathepsin D and of the single chain and heavy chain forms of cathepsin D are cleaved by endoglycosidase H. Treatment of cells with tunicamycin arrests the biosynthetic pathway of cathepsin D at procathepsin D. The nonglycosylated procathepsin D is not proteolytically processed and its secretion is greatly inhibited.  相似文献   

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R L Stein  A M Strimpler 《Biochemistry》1987,26(9):2611-2615
The microbial, peptide-derived aldehyde chymostatin is a potent, competitive inhibitor of chymotrypsin and cathepsin G: Ki = 4 X 10(-10) and 1.5 X 10(-7) M, respectively. Et is "slow-binding inhibitor" of both proteases and, as such, allows determination of rate constants for its association with and dissociation from these proteases. Inhibition kinetics indicate second-order rate constants for the association of chymostatin with chymotrypsin and cathepsin G of 360,000 and 2000 M-1 S-1, respectively and a first-order rate constant for the dissociation of both protease-chymostatin complexes of approximately 0.0002 s-1. Thus, the extreme difference in potency of chymostatin as an inhibitor of chymotrypsin and cathepsin G originates entirely in Kon. Solvent deuterium isotope effects (SIE) were determined to probe the reaction step that rate limits Kon. For the reaction of chymotrypsin with chymostatin, the SIE for Kon is 1.6 +/- 0.1, while for the reaction of chymotrypsin with the peptide substrates Ala-Ala-Phe-pNA and Suc-Ala-Ala-Pro-Phe-pNA, the SIE's for Kc/Km are 2.8 +/- 0.2 and 1.9 +/- 0.1, respectively. These results suggest that Kon for the association of chymotrypsin with chymostatin is at least partially rate limited by a reaction step involving proton transfer. Combined with results for the inhibition of chymotrypsin by Bz-Phe-H [Kennedy, W.P., & Schultz, R. M. (1979) Biochemistry 18, 349-356], these data suggest a mechanism for inhibition by chymostatin involving the general-base-catalyzed formation of an enzyme-bound hemiacetal, followed by a conformational change of this intermediate that produces the final, stable complex of enzyme and inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

11.
Human tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety. In eukaryotic cells, this type of linkage is found in stalled topoisomerase I-DNA covalent complexes, and Tdp1 has been implicated in the repair of such complexes in vivo. We confirm here that the Tdp1 catalytic cycle involves a covalent reaction intermediate in which a histidine residue is connected to a DNA 3'-phosphate through a phosphoamide linkage. Most surprisingly, this linkage can be hydrolyzed by Tdp1, and unlike a topoisomerase I-DNA complex, which requires modification to be an efficient substrate for Tdp1, the native form of Tdp1 can be removed from the DNA. The spinocerebellar ataxia with axonal neuropathy neurodegenerative disease is caused by the H493R mutant form of Tdp1, which shows reduced enzymatic activity and accumulates the Tdp1-DNA covalent intermediate. The ability of wild type Tdp1 to remove the stalled mutant protein from the DNA likely explains the recessive nature of spinocerebellar ataxia with axonal neuropathy. In addition to its activity on phosphotyrosine and phosphohistidine substrates, Tdp1 also possesses a limited DNA and RNA 3'-exonuclease activity in which a single nucleoside is removed from the 3'-hydroxyl end of the substrate. Furthermore, Tdp1 also removes a 3' abasic site and an artificial 3'-biotin adduct from the DNA. In combination with earlier data showing that Tdp1 can use 3'-phosphoglycolate as a substrate, these data suggest that Tdp1 may function to remove a variety of 3' adducts from DNA during DNA repair.  相似文献   

12.
The two-chain form of active cathepsin D, a glycosylated, lysosomal aspartic proteinase, has been isolated from human liver. Isoelectric focusing revealed two major species of enzyme that differed by approximately 0.2 pI unit. Crystals suitable for X-ray diffraction analysis were prepared from acidic solutions using precipitation with ammonium sulfate. The hexagonal crystals diffracted X-rays to beyond 3.1 A resolution and belonged to space group P6(1) (or P6(5)) with cell constants a = b = 125.9 A, c = 104.1 A, gamma = 120.0 degrees. The crystals likely contain two molecules in the asymmetric unit, giving a solvent content of 56% (v/w). Biochemical analysis of crystals indicated that both isoforms were present in approximately equimolar proportions. Full structure determination of the enzyme is underway.  相似文献   

13.
Incubation of either 125I-labelled or unlabelled Neisseria gonorrhoeae with enzymically active preparations of human polymorphonuclear leucocyte lysosomal cathepsin G revealed that surface-exposed outer-membrane proteins were susceptible to proteolytic modification. Electroimmunoblotting experiments confirmed that outer-membrane protein III (PIII) and the major iron-regulated protein (MIRP), two conserved gonococcal proteins, were cleaved by cathepsin G. A direct relationship was observed between susceptibility to the antibacterial properties of cathepsin G and cleavage of PIII among isogenic strains differing in their level of resistance to the bactericidal activity of cathepsin G. Although the antibacterial property of cathepsin G is known to be independent of serine-esterase activity, the data suggest that gonococcal outer-membrane proteins are involved in the binding of cathepsin G, and that variation in the level of resistance reflects the degree to which target outer-membrane proteins such as PIII are exposed.  相似文献   

14.
Cathepsin B (CTSB) and cathepsin L (CTSL) are two widely expressed cysteine proteases thought to predominantly reside within lysosomes. Functional analysis of CTSL in humans is complicated by the existence of two CTSL-like homologs (CTSL and CTSL2), in contrast to mice, which possess only one CTSL enzyme. Thus, transgenic expression of human CTSL in CTSL-deficient mice provides an opportunity to study the in vivo functions of this human protease without interference by its highly related homolog. While mice with single-gene deficiencies for murine CTSB or CTSL survive without apparent neuromuscular impairment, murine CTSB/CTSL double-deficient mice display degeneration of cerebellar Purkinje cells and neurons of the cerebral cortex, resulting in severe hypotrophy, motility defects, and lethality during their third to fourth week of life. Here we show that expression of human CTSL through a genomic transgene results in widespread expression of human CTSL in the mouse that is capable of rescuing the lethality found in CTSB/CTSL double-deficient animals. Human CTSL is expressed in the brain of these compound mutants, predominantly in neurons of the cerebral cortex and in Purkinje cells of the cerebellum, where it appears to prevent neuronal cell death.  相似文献   

15.
The antimicrobial and proinflammatory neutrophil granule protein cathepsin G (CaG) has been reported as a chemoattractant for human phagocytic leukocytes by using a putative G protein coupled receptor. In an effort to identify potential CaG receptor(s), we found that CaG-induced phagocyte migration was specifically attenuated by the bacterial chemotactic peptide fMLP, suggesting these two chemoattractants might share a receptor. In fact, CaG chemoattracts rat basophilic leukemia cells (RBL cells) expressing the high affinity human fMLP receptor FPR, but not parental RBL cells or cells transfected with other chemoattractant receptors. In addition, a specific FPR Ab and a defined FPR antagonist, cyclosporin H, abolished the chemotactic response of phagocytes and FPR-transfected cells to CaG. Furthermore, CaG down-regulated the cell surface expression of FPR in association with receptor internalization. Unlike fMLP, CaG did not induce potent Ca(2+) flux and was a relatively weaker activator of MAPKs through FPR. Yet CaG activated an atypical protein kinase C isozyme, protein kinase Czeta, which was essential for FPR to mediate the chemotactic activity of CaG. Thus, our studies identify CaG as a novel, host-derived chemotactic agonist for FPR and expand the functional scope of this receptor in inflammatory and immune responses.  相似文献   

16.
《Analytical biochemistry》1985,149(2):461-465
Peptide fluoromethyl ketones have been synthesized for the first time. The inhibitor 3-(N-benzyloxycarbonylphenylalanylamido)-dl-1-fluoro-2-butanone (Z-Phe-AlaCH2F) was found to be a 30-fold more potent inactivator of human cathepsin B than 3-(N-benzyloxycarbonylphenylalanylamido)-l-1-diazo-2-butanone (Z-Phe-AlaCHN2), but less reactive than 3-(N-benzyloxycarbonylphenylalanylamido)-l-1-chloro-2-butanone (Z-Phe-AlaCH2Cl). The fluoromethyl ketone's increased potency over the diazomethyl ketone is mainly due to its tighter binding to cathepsin B, with little difference between their respective k3 values. Both Z-Phe-AlaCH2 and Z-Phe-AlaCH2F were quite stable to high concentrations of dithiothreitol, while Z-Phe-AlaCH2Cl was rapidly destroyed by the thiol.  相似文献   

17.
A reproducible procedure for the isolation, from human placenta, of a cathepsin B1 in a homogeneous state, demonstrated by electrophoretic, ultracentrifugal and enzymic criteria, was carried out. The pH optimum was near pH5.5. The placental enzyme catalysed the release of acid-soluble u.v.-dense products from haemoglobin and myoglobin. It was inhibited by heavy metals and several compounds which react with the thiol groups. The optimum temperature was between 37° and 42°C. The molecular weight of the enzyme was calculated to be 24250.  相似文献   

18.
Abstract

Unregulated uptake of oxidized LDL by the scavenger receptor (s) of macrophages is thought to be an early event in atherosclerotic lesion development. Accumulation of oxidized LDL within macrophages may result from resistance of the modified LDL to enzymatic hydrolysis or from direct inactivation of lysosomal enzymes by reactive LDL-associated moieties. Since HOCl-modified LDL has been detected in vivo, the effects of HOCl-modified LDL on the activities of the cysteine protease cathepsin B and the aspartyl protease cathepsin D were investigated. LDL (0.5 mg protein/ml), which had been exposed to HOCl (25–200 µM), caused rapid dose-dependent inactivation of cathepsin B, but not of cathepsin D. Exposure of LDL to HOCl results primarily in the formation of LDL-associated chloramines, and the model chloramine N-acetyl-lysine chloramine also caused dose-dependent inactivation of cathepsin B. Incubation of HOCl-modified LDL with ascorbic and lipoic acids (25–200 µM) resulted in dose-dependent reduction of LDL-associated chloramines and concomitant protection against cathepsin B inactivation. Thus, the data indicate that HOCl-modified LDL inactivates cathepsin B by a chloramine-dependent mechanism, most likely via oxidation of the enzyme's critical cysteine residue. Furthermore, small molecule antioxidants, such as ascorbic and lipoic acids, may be able to inhibit this potentially proatherogenic process by scavenging LDL-associated chloramines.  相似文献   

19.
Emerging studies indicate that intracellular eukaryotic ceramide species directly activate cathepsin B (CatB), a lysosomal‐cysteine‐protease, in the cytoplasm of osteoclast precursors (OCPs) leading to elevated RANKL‐mediated osteoclastogenesis and inflammatory osteolysis. However, the possible impact of CatB on osteoclastogenesis elevated by non‐eukaryotic ceramides is largely unknown. It was reported that a novel class of phosphoglycerol dihydroceramide (PGDHC), produced by the key periodontal pathogen Porphyromonas gingivalis upregulated RANKL‐mediated osteoclastogenesis in vitro and in vivo. Therefore, the aim of this study was to evaluate a crosstalk between host CatB and non‐eukaryotic PGDHC on the promotion of osteoclastogenesis. According to a pulldown assay, high affinity between PGDHC and CatB was observed in RANKL‐stimulated RAW264.7 cells in vitro. It was also demonstrated that PGDHC promotes enzymatic activity of recombinant CatB protein ex vivo and in RANKL‐stimulated osteoclast precursors in vitro. Furthermore, no or little effect of PGDHC on the RANKL‐primed osteoclastogenesis was observed in male and female CatB‐knock out mice compared with their wild type counterparts. Altogether, these findings demonstrate that bacterial dihydroceramides produced by Pgingivalis elevate RANKL‐primed osteoclastogenesis via direct activation of intracellular CatB in OCPs.  相似文献   

20.
Unregulated uptake of oxidized LDL by the scavenger receptor(s) of macrophages is thought to be an early event in atherosclerotic lesion development. Accumulation of oxidized LDL within macrophages may result from resistance of the modified LDL to enzymatic hydrolysis or from direct inactivation of lysosomal enzymes by reactive LDL-associated moieties. Since HOCl-modified LDL has been detected in vivo, the effects of HOCI-modified LDL on the activities of the cysteine protease cathepsin B and the aspartyl protease cathepsin D were investigated. LDL (0.5 mg protein/ml), which had been exposed to HOCl (25-200 microM), caused rapid dose-dependent inactivation of cathepsin B, but not of cathepsin D. Exposure of LDL to HOCl results primarily in the formation of LDL-associated chloramines, and the model chloramine N(alpha)-acetyl-lysine chloramine also caused dose-dependent inactivation of cathepsin B. Incubation of HOCl-modified LDL with ascorbic and lipoic acids (25-200 microM) resulted in dose-dependent reduction of LDL-associated chloramines and concomitant protection against cathepsin B inactivation. Thus, the data indicate that HOCl-modified LDL inactivates cathepsin B by a chloramine-dependent mechanism, most likely via oxidation of the enzyme's critical cysteine residue. Furthermore, small molecule antioxidants, such as ascorbic and lipoic acids, may be able to inhibit this potentially pro-atherogenic process by scavenging LDL-associated chloramines.  相似文献   

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