Limits...
Cross-talk between malarial cysteine proteases and falstatin: the BC loop as a hot-spot target.

Sundararaj S, Saxena AK, Sharma R, Vashisht K, Sharma S, Anvikar A, Dixit R, Rosenthal PJ, Pandey KC - PLoS ONE (2014)

Bottom Line: Using site-directed mutagenesis, hemoglobin hydrolysis assays and peptide inhibition studies, we demonstrate that the BC loop, but not the DE or FG loops, inhibits cysteine proteases of P. falciparum and P. vivax via hydrogen bonds.These results suggest that the BC loop of falstatin acts as a hot-spot target for inhibiting malarial cysteine proteases.This finding suggests new strategies for the development of anti-malarial agents based on protease-inhibitor interactions.

View Article: PubMed Central - PubMed

Affiliation: Host-Parasite Interaction Biology Group, National Institute of Malaria Research, Indian Council of Medical Research, Dwarka, New Delhi, India.

ABSTRACT
Cysteine proteases play a crucial role in the development of the human malaria parasites Plasmodium falciparum and Plasmodium vivax. Our earlier studies demonstrated that these enzymes are equipped with specific domains for defined functions and further suggested the mechanism of activation of cysteine proteases. The activities of these proteases are regulated by a new class of endogenous inhibitors of cysteine proteases (ICPs). Structural studies of the ICPs of Trypanosoma cruzi (chagasin) and Plasmodium berghei (PbICP) indicated that three loops (termed BC, DE, and FG) are crucial for binding to target proteases. Falstatin, an ICP of P. falciparum, appears to play a crucial role in invasion of erythrocytes and hepatocytes. However, the mechanism of inhibition of cysteine proteases by falstatin has not been established. Our study suggests that falstatin is the first known ICP to function as a multimeric protein. Using site-directed mutagenesis, hemoglobin hydrolysis assays and peptide inhibition studies, we demonstrate that the BC loop, but not the DE or FG loops, inhibits cysteine proteases of P. falciparum and P. vivax via hydrogen bonds. These results suggest that the BC loop of falstatin acts as a hot-spot target for inhibiting malarial cysteine proteases. This finding suggests new strategies for the development of anti-malarial agents based on protease-inhibitor interactions.

Show MeSH

Related in: MedlinePlus

Only the BC loop appears to plays a central role in protease inhibition.Peptides based on the DE, FG and L0 loops of falstatin also incubated with VP2 (Fig. 5A) or FP2 (Fig. 5B) or FP3 (Fig. 5C, Fig. 5D add E), and hemoglobin hydrolysis patterns were observed in 15% SDS-PAGE. As a control the wild type peptide was pre-incubated with trypsin, pepsin, and plasmepsin- 4 of P.vivax (PvPM4) under optimal conditions and hydrolysis pattern was observed in 15% SDS-PAGE (5F). The hydrolysis of hemoglobin and fluorogenic substrate was done with two independent batches of each enzyme.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3974720&req=5

pone-0093008-g005: Only the BC loop appears to plays a central role in protease inhibition.Peptides based on the DE, FG and L0 loops of falstatin also incubated with VP2 (Fig. 5A) or FP2 (Fig. 5B) or FP3 (Fig. 5C, Fig. 5D add E), and hemoglobin hydrolysis patterns were observed in 15% SDS-PAGE. As a control the wild type peptide was pre-incubated with trypsin, pepsin, and plasmepsin- 4 of P.vivax (PvPM4) under optimal conditions and hydrolysis pattern was observed in 15% SDS-PAGE (5F). The hydrolysis of hemoglobin and fluorogenic substrate was done with two independent batches of each enzyme.

Mentions: In contrast, peptides based on the DE, FG and L0 loops of falstatin did not inhibit hemoglobin hydrolysis by VP2 (Fig. 5A) or FP2 (Fig. 5B) or FP3 (Fig. 5C and Fig. 5D and 5E). Although, there was a difference in inhibitory potency between the wild type peptide and falstatin, but both were efficient inhibitors of malarial cysteine proteases. The difference of inhibition between the wild type and the mutant peptide in two different assays (Fig. 4B and 4E) was due to different level of sensitivity. The hydrolysis of hemoglobin, by proteases of other classes, the serine protease trypsin and the aspartic proteases pepsin and P.vivax plasmepsin-4 (PvPM4), [27] was unaffected by the peptide (Fig. 5F). Thus, the BC loop peptide mimicked wild falstatin to block hemoglobin hydrolysis by plasmodial cysteine proteases.


Cross-talk between malarial cysteine proteases and falstatin: the BC loop as a hot-spot target.

Sundararaj S, Saxena AK, Sharma R, Vashisht K, Sharma S, Anvikar A, Dixit R, Rosenthal PJ, Pandey KC - PLoS ONE (2014)

Only the BC loop appears to plays a central role in protease inhibition.Peptides based on the DE, FG and L0 loops of falstatin also incubated with VP2 (Fig. 5A) or FP2 (Fig. 5B) or FP3 (Fig. 5C, Fig. 5D add E), and hemoglobin hydrolysis patterns were observed in 15% SDS-PAGE. As a control the wild type peptide was pre-incubated with trypsin, pepsin, and plasmepsin- 4 of P.vivax (PvPM4) under optimal conditions and hydrolysis pattern was observed in 15% SDS-PAGE (5F). The hydrolysis of hemoglobin and fluorogenic substrate was done with two independent batches of each enzyme.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974720&req=5

pone-0093008-g005: Only the BC loop appears to plays a central role in protease inhibition.Peptides based on the DE, FG and L0 loops of falstatin also incubated with VP2 (Fig. 5A) or FP2 (Fig. 5B) or FP3 (Fig. 5C, Fig. 5D add E), and hemoglobin hydrolysis patterns were observed in 15% SDS-PAGE. As a control the wild type peptide was pre-incubated with trypsin, pepsin, and plasmepsin- 4 of P.vivax (PvPM4) under optimal conditions and hydrolysis pattern was observed in 15% SDS-PAGE (5F). The hydrolysis of hemoglobin and fluorogenic substrate was done with two independent batches of each enzyme.
Mentions: In contrast, peptides based on the DE, FG and L0 loops of falstatin did not inhibit hemoglobin hydrolysis by VP2 (Fig. 5A) or FP2 (Fig. 5B) or FP3 (Fig. 5C and Fig. 5D and 5E). Although, there was a difference in inhibitory potency between the wild type peptide and falstatin, but both were efficient inhibitors of malarial cysteine proteases. The difference of inhibition between the wild type and the mutant peptide in two different assays (Fig. 4B and 4E) was due to different level of sensitivity. The hydrolysis of hemoglobin, by proteases of other classes, the serine protease trypsin and the aspartic proteases pepsin and P.vivax plasmepsin-4 (PvPM4), [27] was unaffected by the peptide (Fig. 5F). Thus, the BC loop peptide mimicked wild falstatin to block hemoglobin hydrolysis by plasmodial cysteine proteases.

Bottom Line: Using site-directed mutagenesis, hemoglobin hydrolysis assays and peptide inhibition studies, we demonstrate that the BC loop, but not the DE or FG loops, inhibits cysteine proteases of P. falciparum and P. vivax via hydrogen bonds.These results suggest that the BC loop of falstatin acts as a hot-spot target for inhibiting malarial cysteine proteases.This finding suggests new strategies for the development of anti-malarial agents based on protease-inhibitor interactions.

View Article: PubMed Central - PubMed

Affiliation: Host-Parasite Interaction Biology Group, National Institute of Malaria Research, Indian Council of Medical Research, Dwarka, New Delhi, India.

ABSTRACT
Cysteine proteases play a crucial role in the development of the human malaria parasites Plasmodium falciparum and Plasmodium vivax. Our earlier studies demonstrated that these enzymes are equipped with specific domains for defined functions and further suggested the mechanism of activation of cysteine proteases. The activities of these proteases are regulated by a new class of endogenous inhibitors of cysteine proteases (ICPs). Structural studies of the ICPs of Trypanosoma cruzi (chagasin) and Plasmodium berghei (PbICP) indicated that three loops (termed BC, DE, and FG) are crucial for binding to target proteases. Falstatin, an ICP of P. falciparum, appears to play a crucial role in invasion of erythrocytes and hepatocytes. However, the mechanism of inhibition of cysteine proteases by falstatin has not been established. Our study suggests that falstatin is the first known ICP to function as a multimeric protein. Using site-directed mutagenesis, hemoglobin hydrolysis assays and peptide inhibition studies, we demonstrate that the BC loop, but not the DE or FG loops, inhibits cysteine proteases of P. falciparum and P. vivax via hydrogen bonds. These results suggest that the BC loop of falstatin acts as a hot-spot target for inhibiting malarial cysteine proteases. This finding suggests new strategies for the development of anti-malarial agents based on protease-inhibitor interactions.

Show MeSH
Related in: MedlinePlus