Clickable, Selective, and Cell-Permeable Activity-Based Probe of Human Cathepsin B – Minimalistic Approach for Enhanced Selectivity

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.

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

Biological Chemistry ◽

10.1515/hsz-2018-0186 ◽

2018 ◽

Vol 399 (10) ◽

pp. 1223-1235 ◽

Cited By ~ 5

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.

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Murine and human cathepsin B exhibit similar properties: possible implications for drug discovery

Biological Chemistry ◽

10.1515/bc.2009.021 ◽

2009 ◽

Vol 390 (2) ◽

pp. 175-179 ◽

Cited By ~ 5

Author(s):

Dejan Caglič ◽

Gregor Kosec ◽

Lea Bojič ◽

Thomas Reinheckel ◽

Vito Turk ◽

...

Keyword(s):

Mouse Models ◽

Cathepsin B ◽

Drug Targets ◽

Biochemical Properties ◽

Disease Models ◽

Preclinical Studies ◽

Animal Disease ◽

Transgenic Mouse Models ◽

Animal Disease Models ◽

Human Cathepsin

Abstract Validation of drug targets and subsequent preclinical studies are usually carried out on animal disease models, with mouse being the most commonly used. However, results from mouse models cannot always be directly related to human disease. Major discrepancies between the properties of murine and human variants were observed during the evaluation of compounds targeting cathepsins S and K. It is important, therefore, to know whether similar differences exist between murine and human cathepsin B. Thus, both enzymes were expressed and biochemically characterized. The enzymes exhibited similar biochemical properties, indicating that cathepsin B transgenic mouse models could be useful for studying its role in human pathologies.

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A catalytically active high-Mr form of human cathepsin B from sputum

Biochemical Journal ◽

10.1042/bj2540693 ◽

1988 ◽

Vol 254 (3) ◽

pp. 693-699 ◽

Cited By ~ 23

Author(s):

D J Buttle ◽

B C Bonner ◽

D Burnett ◽

A J Barrett

Keyword(s):

Lysosomal Enzyme ◽

Active Site ◽

Cathepsin B ◽

Human Liver ◽

Cysteine Proteinase ◽

Truncated Form ◽

Physiological Importance ◽

Catalytically Active ◽

Human Cathepsin ◽

Chicken Cystatin

A cysteine proteinase from purulent sputum was partially purified by a method involving affinity chromatography on Sepharose-aminohexanoylphenylalanylglycinaldehyde semicarbazone. It was immunologically related to lysosomal cathepsin B from human liver and was similar in many, but not all, other aspects. It was catalytically active, as demonstrated by active-site-directed radioiodination, and hydrolysed three cathepsin B substrates, two with Km values similar to those of lysosomal cathepsin B. In addition, the rates of inactivation of the sputum and lysosomal forms of the enzyme by L-3-carboxy-2,3-transepoxypropionyl-leucylamido(4-guanidino) butane (Compound E-64) were very similar. However, the sputum enzyme differed from lysosomal cathepsin B in the following respects. Inhibition by chicken cystatin was much weaker for sputum cathepsin B than for the lysosomal enzyme. Sputum cathepsin B had greater stability at pH 7.5 and a higher apparent Mr, even after deglycosylation, than lysosomal cathepsin B. We conclude that the form of cathepsin B found in sputum is probably a truncated form of human procathepsin B, with some differences in properties that could be of physiological importance.

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Human tumour cathepsin B. Comparison with normal liver cathepsin B

Biochemical Journal ◽

10.1042/bj2850427 ◽

1992 ◽

Vol 285 (2) ◽

pp. 427-434 ◽

Cited By ~ 84

Author(s):

K Moin ◽

N A Day ◽

M Sameni ◽

S Hasnain ◽

T Hirama ◽

...

Keyword(s):

Cathepsin B ◽

Human Liver ◽

Normal Liver ◽

Polyacrylamide Gels ◽

Single Chain ◽

Human Tumour ◽

Double Chain ◽

Normal Human Liver ◽

Human Cathepsin ◽

Chain Form

Cathepsin B was purified from normal human liver and several human tumour tissues and partially characterized. Three forms of cathepsin B, with molecular masses of 25 kDa, 26 kDa (the two appearing as a doublet) and 30 kDa, were detected in SDS/polyacrylamide gels. The 25-26 kDa doublet was associated with the fractions from tumours and normal liver containing the highest cathepsin B activity. Cathepsin B from both sources showed similar pH optima. Both normal liver and tumour cathepsin B exhibited similar kinetics against selected synthetic substrates. At neutral pH and 24 degrees C, cathepsin B from both normal liver and tumour exhibited a lower Km and a higher kcat./Km than at pH 6.0. Their inhibitory profiles against synthetic inhibitors were also similar. Immunological studies with a monospecific antibody against the mature double-chain form of human liver cathepsin B and an antibody against a cathepsin B-derived synthetic peptide established the immunological similarity of liver and tumour enzymes. The N-terminal sequences of the 25 kDa and 26 kDa forms were identical with that of the heavy chain of the mature double-chain form of human cathepsin B, whereas the N-terminal sequence of the 30 kDa species was identical with that of the single-chain form of human cathepsin B. Treatment of the double-chain form of cathepsin B from normal liver and tumours with the endoglycosidase peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase converted the 26 kDa form into 25 kDa in SDS/polyacrylamide gels, suggesting that cathepsin B may exist as both glycosylated and unglycosylated forms. Our results, in contrast with those reported earlier for mouse cathepsin B, indicate that human liver and tumour cathepsin B are similar.

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