Folding Incompetence of Cathepsin L-Like Cysteine Proteases May Be Compensated by the Highly Conserved, Domain-Building N-Terminal Extension of the Proregion

Schilling, Klaus; Pietschmann, Sandra; Fehn, Marc; Wenz, Ingrid; Wiederanders, Bernd

doi:10.1515/bc.2001.105

Cited by 8 publications

(9 citation statements)

References 25 publications

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“…8). The kinetics of complex formation of the propeptide mutants Val44 → Ala and Asn48 → Ala with the maternal enzyme revealed the reason for this: dissociation of the complex is much faster (0.02 sec −1 and 0.03 sec −1 , respectively) than with other mutants and the wild‐type propeptide (0.001 sec −1 ; all data from Table 1 in Schilling et al 2001). Thus, these two residues, members of the ER(F/W)N(I/V)N motif, contribute to the tight binding of the P‐domain to the enzyme by stabilization of the hairpin‐anchor as well as by direct interaction with the harboring PBL.…”

Section: Discussionmentioning

confidence: 97%

“…This is deduced from two experimental series, where each of these tryptophan residues was alanine‐substituted by site‐directed mutagenesis. Figure 8 illustrates that tryptophan fluorescence emission spectra of only three propeptide mutants, Trp13p → Ala, Trp16p → Ala, and Trp37p → Ala, show a strong red shift (Schilling et al 2001). The three analogous zymogen mutants, when expressed in HEK cells, were degraded by the quality control machinery of the cell (Kreusch et al 2000).…”

Section: Discussionmentioning

confidence: 99%

“…The consequences of point mutations of highly conserved cathepsin S prosequence residues on refolding and inhibition have been studied systematically (Schilling et al 2001). These results will be discussed in the light of the 2.1 Å X‐ray structure of the Cys25 → Ala procathepsin S mutant presented here.…”

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confidence: 99%

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The crystal structure of a Cys25 → Ala mutant of human procathepsin S elucidates enzyme–prosequence interactions

et al. 2006

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The crystal structure of the active-site mutant Cys25 !Ala of glycosylated human procathepsin S is reported. It was determined by molecular replacement and refined to 2.1 Å resolution, with an R-factor of 0.198. The overall structure is very similar to other cathepsin L-like zymogens of the C1A clan. The peptidase unit comprises two globular domains, and a small third domain is formed by the N-terminal part of the prosequence. It is anchored to the prosegment binding loop of the enzyme. Prosegment residues beyond the prodomain dock to the substrate binding cleft in a nonproductive orientation. Structural comparison with published data for mature cathepsin S revealed that procathepsin S residues Phe146, Phe70, and Phe211 adopt different orientations. Being part of the S19 and S2 pockets, they may contribute to the selectivity of ligand binding. Regarding the prosequence, length, orientation and anchoring of helix a3p differ from related zymogens, thereby possibly contributing to the specificity of propeptide-enzyme interaction in the papain family. The discussion focuses on the functional importance of the most conserved residues in the prosequence for structural integrity, inhibition and folding assistance, considering scanning mutagenesis data published for procathepsin S and for its isolated propeptide.

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

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The crystal structure of a Cys25 → Ala mutant of human procathepsin S elucidates enzyme–prosequence interactions

et al. 2006

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“…Although α 1 does not interact with the mature portion (fig. 4) [21], it contributes to the inhibition of enzymatic activity [21] and to the maintenance of the global structure of the prodomain [27]. We assume that the initial step of maturation is a cleavage within the prosequence by activity of Pichia -derived protease activity in the culture supernatant, or by weak activity of Pro-Der p 1 and Pro-Der f 1.…”

Section: Discussionmentioning

confidence: 99%

Glycosylation of Recombinant Proforms of Major House Dust Mite Allergens Der p 1 and Der f 1 Decelerates the Speed of Maturation

Takai

Mizuuchi²,

Kikuchi³

et al. 2006

Int Arch Allergy Immunol

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Background: The efficient manufacture of recombinant Der p 1 and Der f 1 has been an important bottleneck in the study of house dust mite allergies and the development of applications for allergen engineering. While Der f 1 has only one N-glycosylation motif in the mature sequence, Der p 1 has two motifs, one in the prosequence and the other in the mature sequence. To test whether inefficient maturation of a recombinant Pro-Der p 1 versus Pro-Der f 1 is due to N-glycosylation, the maturation speed of N-glycosylation motif mutants was compared. Methods: Expression vectors for the mutants, in which the motif in the Der p 1 prodomain was disrupted or a motif was created within the Der f 1 prodomain, were constructed by site-directed mutagenesis of preproforms with or without the motif within the mature portion. Culture supernatants of yeast Pichia pastoris transfectant cells containing proforms were buffer exchanged by gel filtration and incubated for maturation. Samples from the reactions were collected every 20 min and subjected to electrophoresis. The maturation speed was compared based on the band densities of the pro- and mature forms. Results: Disruption of the motif in the mature portion decreased the productivity and accelerated the maturation. Maturation was also accelerated by disruption of the other motif in the Der p 1 prodomain and slowed down by introduction of the motif into the Der f 1 prodomain. Conclusions: Maturation systems using Pro-Der p 1 without the prodomain glycosylation are useful for the efficient preparation of a recombinant mature allergen. In addition, these results demonstrated that the maturation of cysteine protease could be controlled through glycosylation of the prodomain.

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“…In addition to its role in autoinhibition, the N-terminal proregion of cathepsin L appears to be necessary for the correct folding of the protein (35), a finding that is also true for papain (36). Mutations destabilizing the interface between the helices prevent correct folding of the protein (37,38). Folding of the proregion may precede folding of the full protein, thereby providing a scaffold that then directs the folding of the remaining domains.…”

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confidence: 99%

Specialized roles for cysteine cathepsins in health and disease

Reiser

Adair²,

Reinheckel³

2010

J. Clin. Invest.

502

403

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A primer on cathepsin biology Cathepsin L transcription and translation. Substantial work has beendone to analyze the promoter regions of the human cathepsin L gene (CTSL) promoter as well as to understand the regulation of different splice variants within the 5′ untranslated region of the transcript (21,22). Of note, one of the splice variants contains a functional internal ribosomal entry site that enables ongoing translation of human cathepsin L under stress conditions, and hypoxia can shut down cap-dependent translation initiation (23). More recent work has focused on the regulation of cathepsin L alternative translation. According to the presence of different forms of cathepsin L in distinct subcellular and extracellular compartments, cathepsin L proteins can be initiated from downstream AUG sites (10), omitting the signal peptide that is normally present at the N terminus of lysosomal cathepsin L that routes the protein to the ER during its synthesis (Figure 2) (10, 24-26 Cathepsins were originally identified as proteases that act in the lysosome. Recent work has uncovered nontraditional roles for cathepsins in the extracellular space as well as in the cytosol and nucleus. There is strong evidence that subspecialized and compartmentalized cathepsins participate in many physiologic and pathophysiologic cellular processes, in which they can act as both digestive and regulatory proteases. In this review, we discuss the transcriptional and translational control of cathepsin expression, the regulation of intracellular sorting of cathepsins, and the structural basis of cathepsin activation and inhibition. In particular, we highlight the emerging roles of various cathepsin forms in disease, particularly those of the cardiac and renal systems.

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Folding Incompetence of Cathepsin L-Like Cysteine Proteases May Be Compensated by the Highly Conserved, Domain-Building N-Terminal Extension of the Proregion

Cited by 8 publications

References 25 publications

The crystal structure of a Cys25 → Ala mutant of human procathepsin S elucidates enzyme–prosequence interactions

The crystal structure of a Cys25 → Ala mutant of human procathepsin S elucidates enzyme–prosequence interactions

Glycosylation of Recombinant Proforms of Major House Dust Mite Allergens Der p 1 and Der f 1 Decelerates the Speed of Maturation

Specialized roles for cysteine cathepsins in health and disease

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