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Regulatory elements within the prodomain of Falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum.

Pandey KC, Barkan DT, Sali A, Rosenthal PJ - PLoS ONE (2009)

Bottom Line: We identified a C-terminal segment (Leu(155)-Asp(243)) of the prodomain, including two motifs (ERFNIN and GNFD) that are conserved in cathepsin L sub-family papain family proteases, as the mediator of prodomain inhibitory activity.The falcipain-2 prodomain also efficiently inhibited other papain family proteases, including cathepsin K, cathepsin L, cathepsin B, and cruzain, but it did not inhibit cathepsin C or tested proteases of other classes.A structural model of pro-falcipain-2 was constructed by homology modeling based on crystallographic structures of mature falcipain-2, procathepsin K, procathepsin L, and procaricain, offering insights into the nature of the interaction between the prodomain and mature domain of falcipain-2 as well as into the broad specificity of inhibitory activity of the falcipain-2 prodomain.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Falcipain-2, a papain family cysteine protease of the malaria parasite Plasmodium falciparum, plays a key role in parasite hydrolysis of hemoglobin and is a potential chemotherapeutic target. As with many proteases, falcipain-2 is synthesized as a zymogen, and the prodomain inhibits activity of the mature enzyme. To investigate the mechanism of regulation of falcipain-2 by its prodomain, we expressed constructs encoding different portions of the prodomain and tested their ability to inhibit recombinant mature falcipain-2. We identified a C-terminal segment (Leu(155)-Asp(243)) of the prodomain, including two motifs (ERFNIN and GNFD) that are conserved in cathepsin L sub-family papain family proteases, as the mediator of prodomain inhibitory activity. Circular dichroism analysis showed that the prodomain including the C-terminal segment, but not constructs lacking this segment, was rich in secondary structure, suggesting that the segment plays a crucial role in protein folding. The falcipain-2 prodomain also efficiently inhibited other papain family proteases, including cathepsin K, cathepsin L, cathepsin B, and cruzain, but it did not inhibit cathepsin C or tested proteases of other classes. A structural model of pro-falcipain-2 was constructed by homology modeling based on crystallographic structures of mature falcipain-2, procathepsin K, procathepsin L, and procaricain, offering insights into the nature of the interaction between the prodomain and mature domain of falcipain-2 as well as into the broad specificity of inhibitory activity of the falcipain-2 prodomain.

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Circular dichroism analysis of prodomain constructs.Different falcipain-2 prodomain constructs (200 μg/ml) were incubated in 20 mM sodium phosphate, pH 5.8, and absorbance between 195 and 240 nm was measured.
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pone-0005694-g004: Circular dichroism analysis of prodomain constructs.Different falcipain-2 prodomain constructs (200 μg/ml) were incubated in 20 mM sodium phosphate, pH 5.8, and absorbance between 195 and 240 nm was measured.

Mentions: Structure-function studies identified a discrete portion of the falcipain-2 prodomain required for inhibition of the cognate mature protease. Prior work with other cathepsin L sub-family proteases suggests key roles for conserved hydrophobic amino acids as well as the ERFNIN and GNFD motifs in maintaining prodomain structure (10). We explored the roles of different domains in maintaining prodomain structure by circular dichroism analysis (Fig. 4). Secondary structure was seen in a fragment with potent inhibitory activity (Leu155-Asp243), but not in two larger constructs that lacked any sequence downstream of the ERFNIN and GNFD motifs (Tyr54-Leu206; Tyr54-Asn180) or in a peptide spanning the ERFNIN and GNFD motifs (Tyr176-Asp216). These results indicate that the ERFNIN and GNFD motifs and an upstream region including conserved Phe residues are required for proper folding or maintenance of secondary structure of the prodomain.


Regulatory elements within the prodomain of Falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum.

Pandey KC, Barkan DT, Sali A, Rosenthal PJ - PLoS ONE (2009)

Circular dichroism analysis of prodomain constructs.Different falcipain-2 prodomain constructs (200 μg/ml) were incubated in 20 mM sodium phosphate, pH 5.8, and absorbance between 195 and 240 nm was measured.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2682653&req=5

pone-0005694-g004: Circular dichroism analysis of prodomain constructs.Different falcipain-2 prodomain constructs (200 μg/ml) were incubated in 20 mM sodium phosphate, pH 5.8, and absorbance between 195 and 240 nm was measured.
Mentions: Structure-function studies identified a discrete portion of the falcipain-2 prodomain required for inhibition of the cognate mature protease. Prior work with other cathepsin L sub-family proteases suggests key roles for conserved hydrophobic amino acids as well as the ERFNIN and GNFD motifs in maintaining prodomain structure (10). We explored the roles of different domains in maintaining prodomain structure by circular dichroism analysis (Fig. 4). Secondary structure was seen in a fragment with potent inhibitory activity (Leu155-Asp243), but not in two larger constructs that lacked any sequence downstream of the ERFNIN and GNFD motifs (Tyr54-Leu206; Tyr54-Asn180) or in a peptide spanning the ERFNIN and GNFD motifs (Tyr176-Asp216). These results indicate that the ERFNIN and GNFD motifs and an upstream region including conserved Phe residues are required for proper folding or maintenance of secondary structure of the prodomain.

Bottom Line: We identified a C-terminal segment (Leu(155)-Asp(243)) of the prodomain, including two motifs (ERFNIN and GNFD) that are conserved in cathepsin L sub-family papain family proteases, as the mediator of prodomain inhibitory activity.The falcipain-2 prodomain also efficiently inhibited other papain family proteases, including cathepsin K, cathepsin L, cathepsin B, and cruzain, but it did not inhibit cathepsin C or tested proteases of other classes.A structural model of pro-falcipain-2 was constructed by homology modeling based on crystallographic structures of mature falcipain-2, procathepsin K, procathepsin L, and procaricain, offering insights into the nature of the interaction between the prodomain and mature domain of falcipain-2 as well as into the broad specificity of inhibitory activity of the falcipain-2 prodomain.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Falcipain-2, a papain family cysteine protease of the malaria parasite Plasmodium falciparum, plays a key role in parasite hydrolysis of hemoglobin and is a potential chemotherapeutic target. As with many proteases, falcipain-2 is synthesized as a zymogen, and the prodomain inhibits activity of the mature enzyme. To investigate the mechanism of regulation of falcipain-2 by its prodomain, we expressed constructs encoding different portions of the prodomain and tested their ability to inhibit recombinant mature falcipain-2. We identified a C-terminal segment (Leu(155)-Asp(243)) of the prodomain, including two motifs (ERFNIN and GNFD) that are conserved in cathepsin L sub-family papain family proteases, as the mediator of prodomain inhibitory activity. Circular dichroism analysis showed that the prodomain including the C-terminal segment, but not constructs lacking this segment, was rich in secondary structure, suggesting that the segment plays a crucial role in protein folding. The falcipain-2 prodomain also efficiently inhibited other papain family proteases, including cathepsin K, cathepsin L, cathepsin B, and cruzain, but it did not inhibit cathepsin C or tested proteases of other classes. A structural model of pro-falcipain-2 was constructed by homology modeling based on crystallographic structures of mature falcipain-2, procathepsin K, procathepsin L, and procaricain, offering insights into the nature of the interaction between the prodomain and mature domain of falcipain-2 as well as into the broad specificity of inhibitory activity of the falcipain-2 prodomain.

Show MeSH
Related in: MedlinePlus