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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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Alignment of C-terminal amino acid residues of the prodomains of falcipain-2 and related cysteine proteases.The sequences of falcipain-2 (FP2), falcipain-3 (FP3), berghepain-2 (BP2), human cathepsin K (Cath K), human cathepsin L (Cath L), human cathepsin B (Cath B), and papain were aligned using Expassy (European Bioinformatics Institute). Amino acids comprising the ERFNIN and GNFD motifs are labeled with stars, and conserved hydrophobic residues are indicated by arrows. Amino acids that are identical or similar to those of falcipain-2 are highlighted.
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pone-0005694-g001: Alignment of C-terminal amino acid residues of the prodomains of falcipain-2 and related cysteine proteases.The sequences of falcipain-2 (FP2), falcipain-3 (FP3), berghepain-2 (BP2), human cathepsin K (Cath K), human cathepsin L (Cath L), human cathepsin B (Cath B), and papain were aligned using Expassy (European Bioinformatics Institute). Amino acids comprising the ERFNIN and GNFD motifs are labeled with stars, and conserved hydrophobic residues are indicated by arrows. Amino acids that are identical or similar to those of falcipain-2 are highlighted.

Mentions: Falcipain-2 and homologs from related plasmodia have much larger prodomains than those of most papain-family proteases. The upstream portion of the falcipain-2 prodomain bears no obvious resemblance to sequences of non-plasmodial proteases, and mediates enzyme trafficking to the parasite food vacuole (9). In contrast, the downstream portion of the falcipain-2 prodomain is similar to that of papain, and in particular to the cathepsin L sub-family of papain-family proteases (Fig. 1). Sequence identity for this region between falcipain-2 and human cathepsin L is ∼21%, and residues that have been identified as playing key roles in the functions of papain family prodomains are generally conserved in falcipain-2 and plasmodial homologs. The well characterized ERFNIN and GNFD domains (10), which contribute to proenzyme stability, are both fully conserved in falcipain-3, but falcipain-2 differs from the consensus sequence at one ERFNIN (I→V) and one GNFD (G→E) residue. Two highly conserved Trp residues (at positions 19 and 22, of procathepsin L), which also contribute to the stability of cathepsin L sub-family proteases (12), are each replaced by Phe in both falcipain-2 and falcipain-3 (Fig. 1; falcipain-2 positions 165 and 168).


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)

Alignment of C-terminal amino acid residues of the prodomains of falcipain-2 and related cysteine proteases.The sequences of falcipain-2 (FP2), falcipain-3 (FP3), berghepain-2 (BP2), human cathepsin K (Cath K), human cathepsin L (Cath L), human cathepsin B (Cath B), and papain were aligned using Expassy (European Bioinformatics Institute). Amino acids comprising the ERFNIN and GNFD motifs are labeled with stars, and conserved hydrophobic residues are indicated by arrows. Amino acids that are identical or similar to those of falcipain-2 are highlighted.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005694-g001: Alignment of C-terminal amino acid residues of the prodomains of falcipain-2 and related cysteine proteases.The sequences of falcipain-2 (FP2), falcipain-3 (FP3), berghepain-2 (BP2), human cathepsin K (Cath K), human cathepsin L (Cath L), human cathepsin B (Cath B), and papain were aligned using Expassy (European Bioinformatics Institute). Amino acids comprising the ERFNIN and GNFD motifs are labeled with stars, and conserved hydrophobic residues are indicated by arrows. Amino acids that are identical or similar to those of falcipain-2 are highlighted.
Mentions: Falcipain-2 and homologs from related plasmodia have much larger prodomains than those of most papain-family proteases. The upstream portion of the falcipain-2 prodomain bears no obvious resemblance to sequences of non-plasmodial proteases, and mediates enzyme trafficking to the parasite food vacuole (9). In contrast, the downstream portion of the falcipain-2 prodomain is similar to that of papain, and in particular to the cathepsin L sub-family of papain-family proteases (Fig. 1). Sequence identity for this region between falcipain-2 and human cathepsin L is ∼21%, and residues that have been identified as playing key roles in the functions of papain family prodomains are generally conserved in falcipain-2 and plasmodial homologs. The well characterized ERFNIN and GNFD domains (10), which contribute to proenzyme stability, are both fully conserved in falcipain-3, but falcipain-2 differs from the consensus sequence at one ERFNIN (I→V) and one GNFD (G→E) residue. Two highly conserved Trp residues (at positions 19 and 22, of procathepsin L), which also contribute to the stability of cathepsin L sub-family proteases (12), are each replaced by Phe in both falcipain-2 and falcipain-3 (Fig. 1; falcipain-2 positions 165 and 168).

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