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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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Inhibitory activity of profalcipain-2 constructs.The domains of falcipain-2 and the studied constructs are represented diagrammatically. Abbreviations: Cyto, cytosolic domain; TM, transmembrane domain; Hb, hemoglobin. The residues contained in each construct are shown, and the inhibitory capacity of mature falcipain-2 for each construct is indicated. The data provided are the Ki values for each polypeptide construct. Results are from two experiments, each performed in duplicate.
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pone-0005694-g002: Inhibitory activity of profalcipain-2 constructs.The domains of falcipain-2 and the studied constructs are represented diagrammatically. Abbreviations: Cyto, cytosolic domain; TM, transmembrane domain; Hb, hemoglobin. The residues contained in each construct are shown, and the inhibitory capacity of mature falcipain-2 for each construct is indicated. The data provided are the Ki values for each polypeptide construct. Results are from two experiments, each performed in duplicate.

Mentions: We previously showed that the prodomain of falcipain-2 is a potent reversible inhibitor of the protease (6). To characterize the requirements for inhibition, we expressed a series of prodomain fragments in E. coli (Figure S1) and evaluated inhibition of falcipain-2 by each of the fragments (Fig. 2). All peptides were soluble in the buffers used for our experiments and stable under our experimental conditions. As we hypothesized, the large upstream portion of the prodomain, which includes a transmembrane domain flanked by cytosolic and lumenal segments, and which mediates trafficking of falcipain-2 to the food vacuole (9), is not required for enzyme inhibition. Inhibitory potency was the same for a prodomain construct lacking only the upstream cytosolic and transmembrane domains (Tyr54-Asp243) and for constructs lacking the upstream 104 (Ser105-Asp243), 126 (Leu127-Asp243), or 154 (Leu155-Asp243) amino acids of the prodomain (Fig. 2). All of these constructs were very potent inhibitors of falcipain-2, with Ki < 1 nM. Removal of the 27 C-terminal amino acids of the prodomain (Tyr54-Asp216) did not affect inhibitory potency, but removal of the 37 C-terminal amino acids (Tyr54-Leu206) led to an ∼2000-fold loss of inhibitory potency, and removal of the 63 C-terminal amino acids (Tyr54-Asn180) led to a complete loss of inhibitory activity. A peptide spanning the ERFNIN and GNFD motifs (Tyr176-Asp216) demonstrated no inhibitory activity. These results allow identification of a minimum inhibitory domain for falcipain-2 (Leu155-Asp216), which includes two hydrophobic residues (Phe165 and Phe168 in falcipain-2; Phe182 and Phe185 in falcipain-3) and the ERFNIN and GNFD motifs, all of which are highly conserved among other cathepsin L sub-family proteases. We could not directly test the inhibitory activity of this minimum inhibitory peptide, as production of the recombinant peptide was unsuccessful.


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)

Inhibitory activity of profalcipain-2 constructs.The domains of falcipain-2 and the studied constructs are represented diagrammatically. Abbreviations: Cyto, cytosolic domain; TM, transmembrane domain; Hb, hemoglobin. The residues contained in each construct are shown, and the inhibitory capacity of mature falcipain-2 for each construct is indicated. The data provided are the Ki values for each polypeptide construct. Results are from two experiments, each performed in duplicate.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005694-g002: Inhibitory activity of profalcipain-2 constructs.The domains of falcipain-2 and the studied constructs are represented diagrammatically. Abbreviations: Cyto, cytosolic domain; TM, transmembrane domain; Hb, hemoglobin. The residues contained in each construct are shown, and the inhibitory capacity of mature falcipain-2 for each construct is indicated. The data provided are the Ki values for each polypeptide construct. Results are from two experiments, each performed in duplicate.
Mentions: We previously showed that the prodomain of falcipain-2 is a potent reversible inhibitor of the protease (6). To characterize the requirements for inhibition, we expressed a series of prodomain fragments in E. coli (Figure S1) and evaluated inhibition of falcipain-2 by each of the fragments (Fig. 2). All peptides were soluble in the buffers used for our experiments and stable under our experimental conditions. As we hypothesized, the large upstream portion of the prodomain, which includes a transmembrane domain flanked by cytosolic and lumenal segments, and which mediates trafficking of falcipain-2 to the food vacuole (9), is not required for enzyme inhibition. Inhibitory potency was the same for a prodomain construct lacking only the upstream cytosolic and transmembrane domains (Tyr54-Asp243) and for constructs lacking the upstream 104 (Ser105-Asp243), 126 (Leu127-Asp243), or 154 (Leu155-Asp243) amino acids of the prodomain (Fig. 2). All of these constructs were very potent inhibitors of falcipain-2, with Ki < 1 nM. Removal of the 27 C-terminal amino acids of the prodomain (Tyr54-Asp216) did not affect inhibitory potency, but removal of the 37 C-terminal amino acids (Tyr54-Leu206) led to an ∼2000-fold loss of inhibitory potency, and removal of the 63 C-terminal amino acids (Tyr54-Asn180) led to a complete loss of inhibitory activity. A peptide spanning the ERFNIN and GNFD motifs (Tyr176-Asp216) demonstrated no inhibitory activity. These results allow identification of a minimum inhibitory domain for falcipain-2 (Leu155-Asp216), which includes two hydrophobic residues (Phe165 and Phe168 in falcipain-2; Phe182 and Phe185 in falcipain-3) and the ERFNIN and GNFD motifs, all of which are highly conserved among other cathepsin L sub-family proteases. We could not directly test the inhibitory activity of this minimum inhibitory peptide, as production of the recombinant peptide was unsuccessful.

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