Crystal structures of human procathepsin B at 3.2 and 3.3 Å resolution reveal an interaction motif between a papain‐like cysteine protease and its propeptide

Turk, Dušan; Podobnik, M.; Kuhelj, Robert; Dolinar, Marko; Türk, Vito

doi:10.1016/0014-5793(96)00309-2

Cited by 137 publications

(135 citation statements)

References 28 publications

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“…This agrees with crystallographic data showing that interaction of the proregion with rat cathepsin B involves residues Leu-41p-Gly-47p which are deeply buried in the substrate binding site [13]. According to these data and those for human cathepsin B [13,14], Cys-42p, which is the residue closest to the oxyanion hole, significantly contributes to propeptide binding by interacting with several hydrophobic groups of cathepsin B. Alkylation of Cys-42p in peptide PB8 resulted in a loss of inhibition (peptide PB8Acm). Peptide PB8Acm was also cleaved at the G47p-G48p site upon incubation with cathepsin B, but much more rapidly than peptide PBS.…”

Section: Resultssupporting

confidence: 90%

“…This explains the resistance of peptide PB11 to cleavage by the mature enzyme. The results obtained here using overlapping peptides spanning the cysteine proteinase proregion agree with those reported from crystal structures of human and rat procathepsin B [13,14]. This promising approach to designing more selective inhibitors of cysteine proteinases will be extended to the proregions of other papain-like proteinases, especially those of protozoan parasites like trypanosomes, that are involved in both spreading and host infection [30,31], but have no specific substrate or inhibitors that can selectively control their activity.…”

Section: Resultssupporting

confidence: 78%

“…The recent elucidation of the 3D structures of human and rat procathepsin B is an essential step towards understanding the way the mature enzyme is inhibited by its propeptide. Three major areas in the protease take part in the interaction; two of them, a prosegment binding loop (PBL) and an occluding loop crevice, are remote from the active site, while the third is the substrate binding cleft [13,14]. However, whether all are required for inhibition or whether a minimal segment in the proregion can specifically interact with the corresponding enzyme is not known.…”

Section: Introductionmentioning

confidence: 99%

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Inhibition of cathepsin B by its propeptide: Use of overlapping peptides to identify a critical segment

et al. 1996

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Ten overlapping 15-mer peptides (peptidyl amides) spanning the proregion of rat cathepsin B (residues lp-60p) were constructed to identify minimal segments having inhibitory activity towards the mature enzyme, that could be used to develop a new generation of peptide-derived inhibitors specifically targeting the active site of the corresponding proteinase. Three synthetic peptides, containing the pentapeptide Leu-CysGly-Thr-Val (residues 41p-45p) in their sequence, inhibited cathepsin B with Ki values in the micromolar range. Alkylation of the thiol group of Cys-42p of peptide PB8 (36p-50p) resulted in its rapid proteolytic degradation, suggesting that this residue is essential for inhibition. The inhibition constant was slightly improved (Kt = 2 ltM) using a longer peptide (26p-50p) which was completely resistant to cleavage even after a prolonged incubation. Alkylation of its cysteinyl residue also resulted in rapid cleavage of the peptide chain. Peptides derived from the rat cathepsin B prosequence also inhibited human cathepsin B with similar Kl values. Unlike rat cathepsin B, which cleaves peptide PB8 at the G47p-G48p bond after prolonged incubation, the human enzyme cleaved both PB8 and PBll at the Lys-40p-Leu41p bond, in agreement with the different kinetic properties of these two proteinases. New probes with improved specificity for cysteine proteinases may therefore be designed based on the sequences of their propeptides.

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Section: Resultssupporting

confidence: 90%

Section: Resultssupporting

confidence: 78%

Section: Introductionmentioning

confidence: 99%

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Inhibition of cathepsin B by its propeptide: Use of overlapping peptides to identify a critical segment

et al. 1996

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“…In contrast to in vitro unfolding/refolding studies derived from the proregion of human procathepsin S and human procathepsin L (7,20), the insect cell system allows the assessment of protein folding under physiological conditions. Since folding was impaired in the case of the propeptide shortened by 23 amino acids and in the case of the propeptide mutated at position 23, we suggest that hydrophobic interactions between tryptophan 24p and tyrosine 183, as well as tyrosine 188 and phenylalanine 180, are necessary for folding and for anchoring the propeptide at the enzyme surface as suggested by Turk et al (21). In addition, tryptophan 24p was shown to form a hydrogen bond with the carbonyl group of glutamine 189 (21).…”

Section: Discussionsupporting

confidence: 56%

Folding Competence of N-terminally Truncated Forms of Human Procathepsin B

Müntener

Willimann

Zwicky

et al. 2005

Journal of Biological Chemistry

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Besides acting as an inhibitor, the propeptide of human cathepsin B exerts an important auxiliary function as a chaperone in promoting correct protein folding. To explore the ability of N-terminally truncated forms of procathepsin B to fold into enzymatically active proteins, we produced procathepsin B variants progressively lacking N-terminal structural elements in baculovirus-infected insect cells. N-terminal truncation of the propeptide by up to 22 amino acids did not impair the production of activable procathepsin B. Secreted forms lacking the first 20, 21, or 22 amino acids spontaneously generated mature cathepsin B through autocatalytic processing, demonstrating that the first ␣-helix (Asp 11 -Arg 20 ) is necessary for efficient inhibition of the enzyme by its propeptide. In contrast, proenzymes lacking the N-terminal part including the first ␤-sheet (Trp 24 -Ala 26 ) of the propeptide or containing an amino acid mutation directly preceding this ␤-sheet were no longer properly folded. This shows that interactions between Trp 24 of the propeptide and Tyr 183 , Tyr 188 , and Phe 180 of the mature enzyme are important for stabilization and essential for procathepsin B folding. Thus, proenzyme forms missing more than the N-terminal 22 amino acids of the propeptide (notably truncated cathepsin B produced by the mRNA splice variant lacking exons 2 and 3, resulting in a propeptide shortened by 34 amino acids) are devoid of proteolytic activity because they cannot fold correctly. Thus, any pathophysiological involvement of truncated cathepsin B must be ascribed to properties other than proteolysis.The cysteine peptidase family C1 (the papain family, merops.sanger.ac.uk) contains two groups of enzymes within subfamily C1A characterized by the length of their propeptides: cathepsin B-like and cathepsin L-like enzymes. They show very little sequence homology in their proregions, and in contrast to cathepsin L-like proenzymes, which have a propeptide of about 100 amino acids in length, the propeptide region of the cathepsin B-like proenzymes is about 60 amino acids long (1). Studies on cathepsins B, L, and S demonstrated that the propeptides or parts of them are effective inhibitors of their cognate enzymes (2-5). The three-dimensional structure of human procathepsin B shows that the propeptide (Arg 1p-Lys 62p, where p stands for propeptide) is jacketed around the catalytic domain of the enzyme in a C-shaped fold, positioned in the direction opposite to bound substrates, thereby shielding the active site (6). Two regions of rat procathepsin B were found to be particularly important for inhibition, the NTTWQ sequence spanning residues 21p-25p, and the CGTVL sequence (amino acids 42p-46p) (2).Besides their function as intramolecular inhibitors, propeptides of several peptidases have been shown to assist in folding. Early in vitro refolding experiments showed that deletions in the propeptide of human cathepsin L resulted in the loss of enzymatic activity due to the lack of refolding capacity of the mature enzyme (7). In ...

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“…A similar but less systematic study of the interaction between cathepsin L and its propeptide also indicated that 20 and 15 amino acids could be removed from the N-and C-terminal parts of the propeptide without destroying the inhibitory activity [14]. Recently, the structure of human procathepsin B has been solved [18,19]. Assuming the propeptide interacts with cathepsin B in the same manner as the proregion in procathepsin B, the availability of the data presented in this work allows the functional evaluation of structural features evidenced by the proenzyme structure.…”

Section: N T T W Q Region C G T V L Regionmentioning

confidence: 99%

Delineating functionally important regions and residues in the cathepsin B propeptide for inhibitory activity

Chen¹,

Plouffe²,

Ménard³

et al. 1996

FEBS Letters

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Synthetic peptides derived from the proregion of rat cathepsin B were used to identify functionally important regions and residues for cathepsin B inhibition. Successive 5 amino acid deletions of a 56 amino acid propeptide from both the N-and Ctermini has allowed the identification of two regions important for inhibitory activity: the NTTWQ (residues 21p-25p) and CGTVL (42p--46p) regions. Alanine scanning of residues within these two regions indicates that Trp-24p and Cys-42p contribute strongly to inhibition, their replacement by Ala resulting in 160-and 140-fold increases in Ki, respectively.

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Crystal structures of human procathepsin B at 3.2 and 3.3 Å resolution reveal an interaction motif between a papain‐like cysteine protease and its propeptide

Cited by 137 publications

References 28 publications

Inhibition of cathepsin B by its propeptide: Use of overlapping peptides to identify a critical segment

Inhibition of cathepsin B by its propeptide: Use of overlapping peptides to identify a critical segment

Folding Competence of N-terminally Truncated Forms of Human Procathepsin B

Delineating functionally important regions and residues in the cathepsin B propeptide for inhibitory activity

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