Characterization of the S3 Subsite Specificity of Cathepsin B

Taralp, Alpay; Kaplan, Harvey; Sytwu, Iou-Iou; Vlattas, Isidoros; Bohacek, Regine S.; Knap, Anna K.; Hirama, Takashi; Huber, C. P.; Hasnain, Sadiq

doi:10.1074/jbc.270.30.18036

Cited by 43 publications

(22 citation statements)

References 26 publications

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“…The efficient cleavage of P 2 ϭ Lys substrates can be attributed to the presence of Glu 205 in the P 2 pocket (papain numbering), which can mediate the charge of the basic residues via salt bridging (63)(64)(65). Cathepsin B demonstrated specificity for Lys, Leu, and Pro in the P 3 position, which is consistent with literature reports (59,66).…”

Section: Resultssupporting

confidence: 80%

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Gosalia

Salisbury

Ellman

et al. 2005

Molecular & Cellular Proteomics

154

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Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format AcAla-X-X-(Arg/Lys)-coumarin was synthesized (X ‫؍‬ all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXa␤, factor XIa and factor ␣ XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates ؋ 24 different proteases ؋ 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P 2 preference for Leu, Val, Phe, and Tyr in both the P 1 ‫؍‬ Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage. Molecular & Cellular Proteomics 4:626 -636, 2005.Because of their critical roles in biological pathways like hormone activation, proteasomal degradation, and apoptosis, proteases are essential for cellular function and viability. Proteases regulate hormonal activation, cellular homeostasis, apoptosis, and coagulation and play an important role in the pathogenicity and progression of many diseases (1). Proteases comprise one of the largest protein families in organisms from Escherichia coli to humans (2-4). Improved understanding of proteases will provide insight into biological systems and will likely provide a number of important new therapeutic targets (1).To properly function, proteases must preferentially cleave their target substrates in the presence of other proteins. While many factors impact protease substrate selection, one of the key aspects is the complementary nature of the enzymeactive site with the residues surrounding the cleaved bond in the substrate. As such, determination of the residues that comprise the preferred cleavage site of a protease provides critical information regarding substrate selection. Furthermore, determination of substrate specificity also provides a framework for the design of potent and selective inhibitors.Here we exploit solution-phase substrate nanodroplet microarrays (5), in which fluorogenic substrates suspended in glycerol droplets are treated with aerosolized aqueous enzyme solutions, to provide protease substrate specificity profiles (6). These arrays allow high throughput characterization of the preferred residues on the P side (7) of the substrate in a highl...

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Section: Resultssupporting

confidence: 80%

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Gosalia

Salisbury

Ellman

et al. 2005

Molecular & Cellular Proteomics

154

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“…In addition, both cathepsins L and B have been activated in the presence of Brij 35 (4 -6), while cathepsin S has been activated in the presence of Triton X-100 (4). Sometimes cathepsins are even assayed in the absence of detergents (7)(8)(9). Although the properties and utilization of detergents in biochemistry have been reviewed extensively (10 -12), their influence on enzyme catalysis has not yet been fully examined.…”

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confidence: 98%

Optimization of Detergents for the Assay of Cathepsins B, L, S, and K

Krupa

Mort

2000

Analytical Biochemistry

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“…Essentially no effect is seen for the generic cysteine protease substrate Z-FR-Amc or for the amidated form of the quenched fluorescence substrate Abz-FRF(4NO # )A. These compounds are neither typical exonor endo-peptidase substrates since, although they are short (thus mimicking exopeptidase substrates), no terminal charge is present, and in the case of Z-FR-Amc, the leaving group does not reach into the S # subsite [39]. Correct orientation of the loop is critical for exopeptidase action, as demonstrated by the almost complete lack of activity of the D22A\H110A mutant with the Abz-FRF(4NO # )A exopeptidase substrate, which is an excellent wild-type cathepsin B substrate.…”

Section: Discussionmentioning

confidence: 99%

“…In the present study, cathepsin B was shown to have relatively low (20-fold) specificity in the P # subsite. Cathepsin B has been shown to have a similar level of specificity in the P $ subsite [39]. As a general principle, the major specificity determinant of the papain family is an extremely high selectivity for large hydrophobic residues in P # , for example 2700-and 12 000-fold higher activity for Leu compared with Arg in the case of cathepsins S and cathepsin K respectively [40,41], and similar patterns for cathepsin L and cathepsin L2 (cathepsin V) [42].…”

Section: Discussionmentioning

confidence: 99%

S′2 substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B

Krupa

Hasnain

Nägler

et al. 2002

Biochemical Journal

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The ability of the lysosomal cysteine protease cathepsin B to function as a peptidyldipeptidase (removing C-terminal dipeptides) has been attributed to the presence of two histidine residues (His110 and His111) present in the occluding loop, an extra peptide segment located in the primed side of the active-site cleft. Whereas His111 is unpaired, His110 is present as an ion pair with Asp22 on the main body of the protease. This ion pair appears to act as a latch to hold the loop in a closed position. The exopeptidase activity of cathepsin B, examined using quenched fluorescence substrates, was shown to have a 20-fold preference for aromatic side chains in the P′3 position relative to glutamic acid as the least favourable residue. Site-directed mutagenesis demonstrated that His111 makes a positive 10-fold contribution to the exopeptidase activity, whereas His110 is critical for this action with the Asp22—His110 ion pair stabilizing the electrostatic interaction by a maximum of 13.9kJ/mol (3.3kcal/mol). These studies showed that cathepsin B is optimized to act as an exopeptidase, cleaving dipeptides from protein substrates in a successive manner, because of its relaxed specificity in P′3 and its other subsites.

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Characterization of the S3 Subsite Specificity of Cathepsin B

Cited by 43 publications

References 26 publications

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Optimization of Detergents for the Assay of Cathepsins B, L, S, and K

S′2 substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B

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