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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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Inhibition of different proteases by the prodomain of falcipain-2.The inhibition of falcipain-2 (FP2), falcipain-2′ (FP2′), falcipain-3 (FP3), berghepain-2 (BP2), cruzain, human cathepsin B (Cath B), human cathepsin L (Cath L), bovine cathepsin C (Cath C), pepsin, α-chymotrypsin (α-Chymo), and collagenase was measured as described in Experimental Procedures. In each case activity was measured with and without the prodomain and the percentage inhibition calculated. Error bars represent standard deviations from two experiments, each performed in duplicate.
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pone-0005694-g003: Inhibition of different proteases by the prodomain of falcipain-2.The inhibition of falcipain-2 (FP2), falcipain-2′ (FP2′), falcipain-3 (FP3), berghepain-2 (BP2), cruzain, human cathepsin B (Cath B), human cathepsin L (Cath L), bovine cathepsin C (Cath C), pepsin, α-chymotrypsin (α-Chymo), and collagenase was measured as described in Experimental Procedures. In each case activity was measured with and without the prodomain and the percentage inhibition calculated. Error bars represent standard deviations from two experiments, each performed in duplicate.

Mentions: Cathepsin L sub-family protease prodomains generally inhibit only closely related proteases. For example, the prodomains of cathepsin L, cathepsin K, and cathepsin S are each potent inhibitors of all three proteases, but not of cathepsin B (10). In contrast, the falcipain-2 prodomain had a rather broad inhibitory specificity, with inhibition of the falcipain-2 homolog from Plasmodium berghei (berghepain-2), the Trypanosoma cruzi protease cruzain, cathepsin L, and cathepsin B (Fig. 3). The only tested papain-family cysteine protease that was not inhibited was the dipeptidyl peptidase cathepsin C. The aspartic protease pepsin, serine protease α-chymotrypsin, and metalloprotease collagenase were not inhibited by the falcipain-2 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)

Inhibition of different proteases by the prodomain of falcipain-2.The inhibition of falcipain-2 (FP2), falcipain-2′ (FP2′), falcipain-3 (FP3), berghepain-2 (BP2), cruzain, human cathepsin B (Cath B), human cathepsin L (Cath L), bovine cathepsin C (Cath C), pepsin, α-chymotrypsin (α-Chymo), and collagenase was measured as described in Experimental Procedures. In each case activity was measured with and without the prodomain and the percentage inhibition calculated. Error bars represent standard deviations from two experiments, each performed in duplicate.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005694-g003: Inhibition of different proteases by the prodomain of falcipain-2.The inhibition of falcipain-2 (FP2), falcipain-2′ (FP2′), falcipain-3 (FP3), berghepain-2 (BP2), cruzain, human cathepsin B (Cath B), human cathepsin L (Cath L), bovine cathepsin C (Cath C), pepsin, α-chymotrypsin (α-Chymo), and collagenase was measured as described in Experimental Procedures. In each case activity was measured with and without the prodomain and the percentage inhibition calculated. Error bars represent standard deviations from two experiments, each performed in duplicate.
Mentions: Cathepsin L sub-family protease prodomains generally inhibit only closely related proteases. For example, the prodomains of cathepsin L, cathepsin K, and cathepsin S are each potent inhibitors of all three proteases, but not of cathepsin B (10). In contrast, the falcipain-2 prodomain had a rather broad inhibitory specificity, with inhibition of the falcipain-2 homolog from Plasmodium berghei (berghepain-2), the Trypanosoma cruzi protease cruzain, cathepsin L, and cathepsin B (Fig. 3). The only tested papain-family cysteine protease that was not inhibited was the dipeptidyl peptidase cathepsin C. The aspartic protease pepsin, serine protease α-chymotrypsin, and metalloprotease collagenase were not inhibited by the falcipain-2 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