Foldase Function of the Cathepsin S Proregion Is Strictly Based upon Its Domain Structure

Pietschmann, Sandra; Fehn, Marc; Kaulmann, Guido; Wenz, B. Wiederanders I.; Schilling, Klaus

doi:10.1515/bc.2002.165

Cited by 10 publications

(7 citation statements)

References 15 publications

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“…Because the pro‐tyrosinase form contains no predicted transmembrane helices and is indeed fully soluble in E. coli (see above), we suggest that the C‐terminal extension in this case has a purely inhibitory function and neither a significant role in stabilizing the enzyme, nor a chaperone‐like function during folding, as has been proposed for other N‐terminal/C‐terminal zymogen‐like systems [40]. It remains to be determined whether this is also the case for other pro‐tyrosinase forms.…”

Section: Resultsmentioning

confidence: 67%

Role of the C‐terminal extension in a bacterial tyrosinase

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Thöny‐Meyer

2010

The FEBS Journal

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The well studied bacterial tyrosinases from the Streptomyces sp. bacteria are distinguishable from their eukaryotic counterparts by the absence of a C‐terminal extension. In the present study, we report that the tyrosinase from the bacterium Verrucomicrobium spinosum also has such a C‐terminal extension, thus making it distinct from the Streptomyces enzymes. The entire tyrosinase gene from V. spinosum codes for a 57 kDa protein (full‐length unprocessed form), which has a twin arginine translocase type signal peptide, the two copper‐binding motifs typical of the tyrosinase protein family and the aforementioned C‐terminal extension. We expressed various mutants of the recombinant enzyme in Escherichia coli and found that removal of the C‐terminal extension by genetic engineering or limited trypsin digest of the pro‐form results in a more active enzyme (i.e. 30–100‐fold increase in monophenolase and diphenolase activities). Further studies also revealed the importance of a phenylalanine residue in this C‐terminal domain. These results demonstrate that the V. spinosum tyrosinase is a new example of this interesting family of enzymes. In addition, we show that this enzyme can be readily overproduced and purified and that it will prove useful in furthering the understanding of these enzymes, as well as their biotechnological application.

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Section: Resultsmentioning

confidence: 67%

Role of the C‐terminal extension in a bacterial tyrosinase

Fairhead

Thöny‐Meyer

2010

The FEBS Journal

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“…They seem to retain their secondary structure over a wide pH range from 6.5 to 3.0 (Jerala et al, 1998). Several experiments have shown that addition of recombinant propeptides can efficiently catalyze the refolding of denatured mature cysteine proteinases (Pietschmann et al, 2002;Yamamoto et al, 2002;Capetta et al, 2002). With respect to cathepsin S, this effect was strongly dependent on the three-dimensional structure of the propeptide.…”

Section: Folding Assistancementioning

confidence: 99%

“…With respect to cathepsin S, this effect was strongly dependent on the three-dimensional structure of the propeptide. Mutants affecting the aromatic Trp core of the propeptide showed a much weaker foldase effect than the wild type propeptide (Pietschmann et al, 2002). A mutation in the conserved GNFD motif (N70pI/F72pI) of the F. hepatica cathepsin L propeptide also diminished the foldase function of the propeptide (Capetta et al, 2002).…”

Section: Folding Assistancementioning

confidence: 99%

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Structure-function relationships in class CA1 cysteine peptidase propeptides.

Wiederanders¹

2003

Acta Biochim Pol

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Regulation of proteolytic enzyme activity is an essential requirement for cells and tissues because proteolysis at a wrong time and location may be lethal. Proteases are synthesized as inactive or less active precursor molecules in order to prevent such inappropriate proteolysis. They are activated by limited intra- or intermolecular proteolysis cleaving off an inhibitory peptide. These regulatory proenzyme regions have attracted much attention during the last decade, since it became obvious that they harbour much more information than just triggering activation. In this review we summarize the structural background of three functions of clan CA1 cysteine peptidase (papain family) proparts, namely the selectivity of their inhibitory potency, the participation in correct intracellular targeting and assistance in folding of the mature enzyme. Today, we know more than 500 cysteine peptidases of this family from the plant and animal kingdoms, e.g. papain and the lysosomal cathepsins L and B. As it will be shown, the propeptide functions are determined by certain structural motifs conserved over millions of years of evolution.

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“…We are at present investigating the propeptide of cathepsin S, a member of the cathepsin L-like papain subfamily (Karrer et al, 1993;Kirschke et al, 1986Kirschke et al, , 1989Wiederanders et al, 1992). The 99-residue cathepsin S propeptide (MW 12 kDa) can be regarded as a foldase which promotes the folding process of the mature enzyme (Kreusch et al, 2000;Schilling et al, 2001;Pietschmann et al, 2002). This function requires the tertiary structure of the isolated propeptide, which folds independently of the other parts of the molecule (Maubach et al, 1997;Schilling et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

An unfolding/refolding step helps in the crystallization of a poorly soluble protein

Kaulmann

Palm²,

Schilling³

et al. 2003

Acta Crystallogr D Biol Cryst

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Proteins that are unstable or poorly soluble often elude crystallization. Here, a novel strategy is presented that leads to the crystallization of the isolated N-terminal propeptide of human procathepsin S, a proteinase belonging to the cathepsin L-like endopeptidases of the clan CA1 cysteine peptidases. Being very hydrophobic, the propeptide is extremely poorly soluble in aqueous solvents at neutral pH. Solubility is much better at acidic pH, but the native structure is destroyed under these conditions. A novel approach to the crystallization of this poorly soluble protein is presented in which it is first unfolded in an acidic buffer (pH 4.5) and then mixed with a nearly neutral crystallization buffer (pH 6.75) in which the native conformation should form spontaneously. Crystals were grown at a high concentration of MES (1.14 M) with 10% 2-propanol as precipitant. They belong to a tetragonal space group, with unit-cell parameters a = b = 151.1, c = 75.8 A. Diffraction data to a resolution of 3.5 A were obtained.

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Foldase Function of the Cathepsin S Proregion Is Strictly Based upon Its Domain Structure

Cited by 10 publications

References 15 publications

Role of the C‐terminal extension in a bacterial tyrosinase

Role of the C‐terminal extension in a bacterial tyrosinase

Structure-function relationships in class CA1 cysteine peptidase propeptides.

An unfolding/refolding step helps in the crystallization of a poorly soluble protein

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