scholarly journals Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2′ and P3′ variations in extended substrates

2000 ◽  
Vol 347 (1) ◽  
pp. 123-129 ◽  
Author(s):  
Fernada C. Vieira PORTARO ◽  
Ana Beatriz F. SANTOS ◽  
Maria Helena S. CEZARI ◽  
Maria Aparecida JULIANO ◽  
Luiz JULIANO ◽  
...  
Keyword(s):  
Amino Acids ◽  
Significant Influence ◽  
Active Site ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Aminobenzoic Acid ◽  
Site Analysis ◽  
Hydrophobic Amino Acids ◽  
Human Cathepsin

We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S4 and S3 subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S2ʹ and S3ʹ. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P4, P3, P2ʹ and P3ʹ were made. The S4 to S2ʹ subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S3ʹ, indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S4, S3, S2ʹ and S3ʹ of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P2ʹ respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P3ʹ. Basic residues at P3 and P4 were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P2ʹ (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His111 (H111A) and His110 (H110A) of cathepsin B led to an increase in kcat values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.

scholarly journals Quantification of cathepsins B and L in cells

1998 ◽  
Vol 332 (2) ◽  
pp. 499-505 ◽  
Author(s):  
Ruye XING ◽  
Adele K. ADDINGTON ◽  
Robert W. MASON
Keyword(s):  
Active Site ◽  
Total Protein ◽  
Cathepsin B ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cathepsin L ◽  
Breast Tumour ◽  
Cathepsin S ◽  
Cysteine Proteinases ◽  
Breast Tumour Cells

A method for quantifying active cysteine proteinases in mammalian cells has been developed using an active-site-directed inhibitor. Fluoren-9-ylmethoxycarbonyl(di-iodotyrosylalanyl)-diazomethane (Fmoc-[I2]Tyr-Ala-CHN2) was prepared and shown to react irreversibly with cathepsins B and L, but not with cathepsin S. The non- and mono-iodo forms of the inhibitor reacted with all three enzymes. These results demonstrate that, unlike cathepsins B and L, cathepsin S has a restricted S2-binding site that cannot accommodate the bulky di-iodotyrosine. Fmoc-[I2]Tyr-Ala-CHN2 was able to penetrate cells and react with active enzymes within the cells. A radiolabelled form of the inhibitor was synthesized and the concentration of functional inhibitor was established by titration with papain. This inhibitor was used to quantify active cysteine proteinases in cultured cells. Active cathepsin B was found to be expressed by all of the cells studied, consistently with a housekeeping role for this enzyme. Active forms of cathepsin L were also expressed by all of the cells, but in different quantities. Two additional proteins were labelled in some of the cells, and these may represent other non-characterized proteinases. Higher levels of active cathepsins B and L, and an unidentified protein of Mr 39000, were found in breast tumour cells that are invasive, compared with those that are not invasive. From the data obtained, it can be calculated that the concentrations of both active cathepsins B and L in lysosomes can be as high as 1 mM, each constituting up to 20% of total protein in the organelle. This new technique provides a more direct procedure for determining the proteolytic potential of cellular lysosomes.

scholarly journals S3 to S3' subsite specificity of recombinant human cathepsin K and development of selective internally quenched fluorescent substrates

2003 ◽  
Vol 373 (3) ◽  
pp. 981-986 ◽  
Author(s):  
Marcio F. M. ALVES ◽  
Luciano PUZER ◽  
Simone S. COTRIN ◽  
Maria Aparecida JULIANO ◽  
Luiz JULIANO ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cathepsin K ◽  
Cysteine Proteases ◽  
Aminobenzoic Acid ◽  
Hydrophobic Amino Acids ◽  
Human Cathepsin ◽  
General Sequences ◽  
Natural Amino Acids ◽  
Positively Charged ◽  
K Activity

We have systematically examined the S3 to S3′ subsite substrate specificity requirements of cathepsin K using internally quenched fluorescent peptides derived from the lead sequence Abz-KLRFSKQ-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine]. We assayed six series of peptides, in which each position except Gln was substituted with various natural amino acids. The results indicated that the S3–S1 subsite requirements are more restricted than those of S1′–S3′. Cathepsin K preferentially accommodates hydrophobic amino acids with aliphatic side chains (Leu, Ile and Val) in the S2 site. Modifications at P1 residues also have a large influence on cathepsin K activity. Positively charged residues (Arg and Lys) represent the best accepted amino acids in this position, although a particular preference for Gly was found as well. Subsite S3 accepted preferentially basic amino acids such as Lys and Arg. A broad range of amino acids was accommodated in the remaining subsites. We further explored the acceptance of a Pro residue in the P2 position by cathepsin K in order to develop specific substrates for the enzyme. Two series of peptides with the general sequences Abz-KXPGSKQ-EDDnp and Abz-KPXGSKQ-EDDnp (where X denotes the position of the amino acid that is altered) were synthesized. The substrates Abz-KPRGSKQ-EDDnp and Abz-KKPGSKQ-EDDnp were cleaved by cathepsin K at the Arg–Gly and Gly–Ser bonds respectively, and have been shown to be specific for cathepsin K when compared with other lysosomal cysteine proteases such as cathepsins L and B and with the aspartyl protease cathepsin D.

scholarly journals The synthesis, kinetic characterization and application of a novel biotinylated affinity label for cathepsin B

1992 ◽  
Vol 283 (2) ◽  
pp. 449-453 ◽  
Author(s):  
B Walker ◽  
B M Cullen ◽  
G Kay ◽  
I M Halliday ◽  
A McGinty ◽  
...  
Keyword(s):  
Active Site ◽  
Cathepsin B ◽  
Solid Phase ◽  
Detection System ◽  
Kinetic Characterization ◽  
Cysteine Proteinases ◽  
Selective Labelling ◽  
Wide Range ◽  
Human Cathepsin ◽  
Biological Milieu

In this study we report on the synthesis, kinetic characterization and application of a novel biotinylated and active-site-directed inactivator of cathepsin B. Thus the peptidyldiazomethane biotinyl-Phe-Ala-diazomethane has been synthesized by a combination of solid-phase and solution methodologies and has been shown to be a very efficient inactivator of bovine and human cathepsin B. The respective apparent second-order rate constants (k0bs./[I]) for the inactivation of the human and bovine enzymes by this reagent, namely approximately 5.4 x 10(4) M-1.min-1 and approximately 7.8 x 10(4) M-1.min-1, compare very favourably with those values determined for the urethane-protected analogue benzyloxycarbonyl-Phe-Ala-chloromethane first described by Green & Shaw [(1981) J. Biol. Chem. 256, 1923-1928], thus demonstrating that the presence of the biotin moiety at the P3 position is compatible with inhibitor effectiveness. The utilization of this reagent for the detection of cathepsin B in electrophoretic gels, using Western blotting and in combination with a streptavidin/alkaline phosphatase detection system, is also demonstrated. Given that the peptidyldiazomethanes exhibit a pronounced reactivity towards cysteine proteinases, we feel that the present label may well constitute the archetypal example of a wide range of reagents for the selective labelling of this class of proteinase, even in a complex biological milieu containing additional classes of proteinases.

scholarly journals L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L

1982 ◽  
Vol 201 (1) ◽  
pp. 189-198 ◽  
Author(s):  
A J Barrett ◽  
A A Kembhavi ◽  
M A Brown ◽  
H Kirschke ◽  
C G Knight ◽  
...  
Keyword(s):  
Active Site ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Competitive Inhibitor ◽  
Serine Proteinases ◽  
Cysteine Proteinases ◽  
Leucocyte Elastase ◽  
Structural Analogues ◽  
Stoichiometric Reaction

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.

scholarly journals The use of benzyloxycarbonyl[125I]iodotyrosylalanyldiazomethane as a probe for active cysteine proteinases in human tissues

1989 ◽  
Vol 263 (3) ◽  
pp. 945-949 ◽  
Author(s):  
R W Mason ◽  
L T Bartholomew ◽  
B S Hardwick
Keyword(s):  
Tissue Distribution ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Molecular Size ◽  
Cathepsin S ◽  
Human Tissues ◽  
Cysteine Proteinases ◽  
Additional Protein ◽  
Human Cathepsin ◽  
Kidney Liver

The ability of benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 to label cysteine proteinases in a variety of human tissues was investigated. The inhibitor bound only to cathepsin B in tissues homogenized at pH 5.0. When liver was autolysed at pH 4.0 for up to 4 h, the inhibitor also bound to a protein of Mr 25,000. This was identified immunologically and chromatographically as cathepsin L. Both cathepsins B and L were found primarily in kidney, liver and spleen. In spleen, an additional protein of Mr 25,000 was also labelled. This protein could not be precipitated by antibodies to any of cathepsins B, H and L. This protein has tentatively been identified as human cathepsin S by its tissue distribution, chromatographic properties and molecular size. This work clearly shows that peptidyldiazomethanes are specific probes for cysteine proteinases, and that benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 binds to three such enzymes in human tissues.

scholarly journals Elastinolytic activity of human cathepsin L

1986 ◽  
Vol 233 (3) ◽  
pp. 925-927 ◽  
Author(s):  
R W Mason ◽  
D A Johnson ◽  
A J Barrett ◽  
H A Chapman
Keyword(s):  
Cathepsin B ◽  
Specific Activity ◽  
Cathepsin L ◽  
Cysteine Proteinases ◽  
Pancreatic Elastase ◽  
Optimum Ph ◽  
Human Cathepsin ◽  
Hydrolysis Of

The hydrolysis of a tritiated elastin substrate by the human cysteine proteinases cathepsins B and L has been studied. Cathepsin L was found to be at least 100-fold more active on this substrate than cathepsin B. The specific activity of cathepsin L at pH 5.5 for hydrolysis of elastin was about the same as that of pig pancreatic elastase at its optimum pH of 8.8.

scholarly journals The active-site residue Cys-29 is responsible for the neutral-pH inactivation and the refolding barrier of human cathepsin B

FEBS Letters ◽  
2000 ◽  
Vol 475 (3) ◽  
pp. 157-162 ◽  
Author(s):  
Jianxing Song ◽  
Ping Xu ◽  
Hui Xiang ◽  
Zhengding Su ◽  
Andrew C. Storer ◽  
...  
Keyword(s):  
Active Site ◽  
Cathepsin B ◽  
Active Site Residue ◽  
Neutral Ph ◽  
Ph Inactivation ◽  
Human Cathepsin

The cysteine proteinases of Schistosoma mansoni cercariae

Parasitology ◽  
1997 ◽  
Vol 114 (2) ◽  
pp. 105-112 ◽  
Author(s):  
J. P. DALTON ◽  
K. A. CLOUGH ◽  
M. K. JONES ◽  
P. J. BRINDLEY
Keyword(s):  
Schistosoma Mansoni ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Serine Proteinase ◽  
Skin Penetration ◽  
Scanning Electron Micrographs ◽  
Cysteine Proteinases ◽  
Similar Function ◽  
Substrate Preferences ◽  
Serine Proteinase Activity

Based on substrate preferences, cercariae of Schistosoma mansoni were seen to express both cathepsin L and cathepsin B cysteine proteinases, although the former activity was many -fold greater. Two cathepsin L activities identified in cercarial extracts by zymography co-migrated with activities in extracts of 3 h and 24 h schisotosomula and in extracts of adult worms. Since these enzymes have been implicated in haemoglob in digestion by adult worms, they may perform a similar function in schistosomula. Immunolocalization using scanning electron micrographs showed that cathepsin L and cathepsin B proteinases were present in the cercarial post-acetabular glands. In addition, cercarial serine proteinase activities considered to facilitate skin penetration efficiently cleaved the substrates Z-Gly-Pro-Arg-NHMec and Z-Gly-Pro-Lys-NHMec. Cercariae release most of this serine proteinase activity when induced to secrete the contents of their acetabular glands. In contrast, newly transformed 3 h and 24 h schistosomula did not express this activity.

scholarly journals Purification of cathepsin B by a new form of affinity chromatography

1986 ◽  
Vol 235 (3) ◽  
pp. 731-734 ◽  
Author(s):  
D H Rich ◽  
M A Brown ◽  
A J Barrett
Keyword(s):  
Affinity Chromatography ◽  
Cathepsin B ◽  
Single Step ◽  
Starting Material ◽  
High Yield ◽  
Cysteine Proteinases ◽  
New Form ◽  
Human Cathepsin ◽  
Sepharose 4B

Human cathepsin B was purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal linked to Sepharose 4B, with elution by 2,2′-dipyridyl disulphide at pH 4.0. The product obtained in high yield by the single step from crude starting material was 80-100% active cathepsin B. The possibility that this new form of affinity chromatography may be of general usefulness in the purification of cysteine proteinases is discussed.

scholarly journals The two cathepsin B-like proteases of Arabidopsis thaliana are closely related enzymes with discrete endopeptidase and carboxydipeptidase activities

2018 ◽  
Vol 399 (10) ◽  
pp. 1223-1235 ◽  
Author(s):  
Andreas Porodko ◽  
Ana Cirnski ◽  
Drazen Petrov ◽  
Teresa Raab ◽  
Melanie Paireder ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Active Site ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Substrate Specificities ◽  
Active Site Cleft ◽  
Molecular Modeling Studies ◽  
Human Cathepsin

Abstract The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336→Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.

Sign in / Sign up

Export Citation Format

Share Document