Crystal structure of the wild-type human procathepsin B at 2.5 Å resolution reveals the native active site of a papain-like cysteine protease zymogen

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Synthesis of proteases as inactive zymogens is a very important mechanism for the regulation of their activity. For lysosomal proteases proteolytic cleavage of the propeptide is triggered by the acidic pH. By using fluorescence, circular dichroism, and NMR spectroscopy, we show that upon decreasing the pH from 6.5 to 3 the propeptide of cathepsin L loses most of the tertiary structure, but almost none of the secondary structure is lost. Another partially structured intermediate, prone to aggregation, was identified between pH 6.5 and 4. The conformation, populated below pH 4, where the activation of cathepsin L occurs, is not completely unfolded and has the properties of molten globule, including characteristic binding of the 1-anilinonaphthalene-8-sulfonic acid. This pH unfolding of the propeptide parallels a decrease of its affinity for cathepsin L and suggests the mechanism for the acidic zymogen activation. Addition of anionic polysaccharides that activate cathepsin L already at pH 5.5 unfolds the tertiary structure of the propeptide at this pH. Propeptide of human cathepsin L which is able to fold independently represents an evolutionary intermediate in the emergence of novel inhibitors originating from the enzyme proregions.All lysosomal and most other proteases are synthesized in the form of inactive precursors (1, 2). Propeptides are generally located N-terminal to the mature enzyme, and activation of the enzyme is accomplished by cis-or trans-cleavage of the propeptide. Propeptides vary from a few (e.g. trypsin) to more than 200 residues (e.g. cathepsin C). Longer propeptides are generally strong and specific inhibitors of their mature enzymes (3-7). In most cases propeptides are also indispensable for correct folding of the enzymes (8). In some enzymes folding of the mature form is extremely slow, and the propeptide assists in overcoming the kinetic barrier (9), which may also be overcome by physicochemical factors such as high ionic strength in subtilisin, for example (10). Proenzymes are quite often more stable than mature enzymes (11, 12) and can represent a pool of latent enzyme until the activation occurs in the proper conditions. Propeptides are also involved in targeting to specific organelles (13, 14); they can affect posttranslational modification such as glycosylation (15) and mediate interactions with other molecules (16,17). Propeptides can be cleaved either by other proteases or by intra-or intermolecular autocatalysis. pH change is one of the most common environmental parameters responsible for triggering the activation of proteases, occurring in cysteine, aspartic acid, and metalloproteases (1,18,19). Low pH is thought either to increase the susceptibility of the propeptide as a substrate due to the protonation of groups close to the cleavage site or to cause a conformational change in the propeptide or enzyme.Cathepsin L is one of the most active cysteine proteases and accounts for most of the lysosomal cysteine protease activity (20). It has been implicated in a range of processes incl...

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confidence: 99%

pH-induced Conformational Transitions of the Propeptide of Human Cathepsin L

Jerala

Žerovnik

Kidrič

et al. 1998

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“…Elucidation of the three-dimensional structure of human procathepsin L (14) and cathepsin B (29,30) revealed that this part of the propeptide is less structured and is readily accessible to proteases. Testament to this fact is the collective data showing that papain (32) cathepsin L (14), cathepsin B (29,30), cathepsin S (31), and cathepsin K (25) can all be activated to mature enzymes either by cis-or transcleavage at various bonds within this segment. Cathepsin B of the helminth parasite S. mansoni can also be activated by trans-cleavage at this segment (55,56).…”

Section: ϫ36mentioning

confidence: 99%

“…These observations suggest that processing and activation of procathepsin L1 occurs following secretion from these cells into the acidic gut lumen. Expression of the 37-kDa procathepsin L1 in Pichia pastoris showed that an intermolecular processing event within a conserved GXNXFXD motif in the propeptide generates an active 30 Fasciola hepatica is a helminth parasite that causes liver fluke disease in cattle and sheep worldwide and has recently emerged as an important pathogen of humans (1). Cathepsin L1, a major cysteine protease secreted by the parasite plays a pivotal role in various aspects of its pathogenicity.…”

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confidence: 99%

“…The precise mechanism of proteolytic conversion from proenzyme to mature enzyme is still actively debated. The threedimensional structure of procathepsin L (14) and procathepsin B (28,29,30) show that the N terminus of the mature enzyme is quite removed from the active site thus making it difficult to visualize an autocatalytic cleavage event. Moreover, circular dichroism studies reveal that activation does not involve significant conformational changes in the structure of procathepsin L (15) or procathepsin B (16).…”

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confidence: 99%

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Cathepsin L1, the Major Protease Involved in Liver Fluke (Fasciola hepatica) Virulence

Collins

Stack

O’Neill

et al. 2004

144

The secretion and activation of the major cathepsin L1 cysteine protease involved in the virulence of the helminth pathogen Fasciola hepatica was investigated. Only the fully processed and active mature enzyme can be detected in medium in which adult F. hepatica are cultured. However, immunocytochemical studies revealed that the inactive procathepsin L1 is packaged in secretory vesicles of epithelial cells that line the parasite gut. These observations suggest that processing and activation of procathepsin L1 occurs following secretion from these cells into the acidic gut lumen. Expression of the 37-kDa procathepsin L1 in Pichia pastoris showed that an intermolecular processing event within a con-

“…Studies on cathepsins B, L, and S demonstrated that the propeptides or parts of them are effective inhibitors of their cognate enzymes (2)(3)(4)(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).…”

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confidence: 99%

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

Müntener

Willimann

Zwicky

et al. 2005

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 ...