Active site specificity of plasmepsin II

Westling, Jennifer; Cipullo, Patty; Hung, Su-Hwi; Saft, Howard; Dame, John B.; Dunn, Ben M.

doi:10.1110/ps.8.10.2001

Cited by 59 publications

(60 citation statements)

References 27 publications

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“…There is abundant evidence that the proteases implicated in haemoglobin catabolism have a broad pH activity profile in vitro (Banerjee et al, 2002;Dorn et al, 1998;Francis et al, 1994;Gluzman et al, 1994;Goldberg et al, 1990;Moon et al, 1997;Murata and Goldberg, 2003;Shenai et al, 2000;Sijwali et al, 2001;Tyas et al, 1999;Westling et al, 1999) and there is also evidence for there being considerable redundancy among the various proteases (Malhotra et al, 2002;Sijwali et al, 2004;Liu et al, 2005;Omara-Opyene et al, 2004). Thus, our observation that the pH DV can apparently vary by at least 2 pH units without significant detriment to the parasite might be accounted for by the considerable functional overlap amongst the proteases present in this organelle permitting haemoglobin degradation to occur even when the pH DV was outside the optimum range for one or more of the vacuolar enzymes.…”

Section: Discussionmentioning

confidence: 99%

The pH of the digestive vacuole ofPlasmodium falciparumis not associated with chloroquine resistance

Hayward

Saliba

Kirk

2006

Journal of Cell Science

125

134

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noted that this hypothesis is Chloroquine resistance in the human malaria parasite, Plasmodium falciparum, arises from decreased accumulation of the drug in the 'digestive vacuole' of the parasite, an acidic compartment in which chloroquine exerts its primary toxic effect. It has been proposed that changes in the pH of the digestive vacuole might underlie the decreased accumulation of chloroquine by chloroquineresistant parasites. In this study we have investigated the digestive vacuole pH of a chloroquine-sensitive and a chloroquine-resistant strain of P. falciparum, using a range of dextran-linked pH-sensitive fluorescent dyes. The estimated digestive vacuole pH varied with the concentration and pK a of the dye, ranging from ~3.7-6.5. However, at low dye concentrations the estimated digestive vacuole pH of both the chloroquine-resistant and chloroquine-sensitive strains converged in the range 4.5-4.9. The results suggest that there is no significant difference in digestive vacuole pH of chloroquine-sensitive and chloroquine-resistant parasites, and that digestive vacuole pH does not play a primary role in chloroquine resistance.

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Section: Discussionmentioning

confidence: 99%

The pH of the digestive vacuole ofPlasmodium falciparumis not associated with chloroquine resistance

Hayward

Saliba

Kirk

2006

Journal of Cell Science

125

134

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“…Although plasmepsins from each of the four species have been cloned and expressed as recombinant enzymes (12,18,22,44,45), there is still very little known about many of the peptidases recently discovered from the P. falciparum genome-sequencing project (46). The most fully characterized plasmepsin is PfPM2, where numerous structures (18)(19)(20) and kinetic studies (21,31,45,47) have aided in identifying the determinants of substrate specificity.…”

Section: Discussionmentioning

confidence: 99%

Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries

et al. 2005

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Two targeted chromogenic octapeptide combinatorial libraries, comprised of 38 pools each containing 361 different peptides, were used to analyze the enzyme/substrate interactions of five plasmepsins. The first library (P1 library) was based on a good synthetic aspartic peptidase substrate [Westling, J., Cipullo, P., Hung, S. H., Saft, H., Dame, J. B., and Dunn, B. M. (1999) Protein Sci. 8, 2001-2009 Scarborough, P. E., and Dunn, B. M. (1994) Protein Eng. 7, 495-502] and had the sequence Lys-Pro-(Xaa)-Glu-P1*Nph-(Xaa)-Leu. The second library (P1′ library) incorporated results with the plasmepsins from the first library and had the sequence Lys-Pro-Ile-(Xaa)-Nph*P1′-Gln-(Xaa). In both cases, P1 and P1′ were fixed residues for a given peptide pool, where Nph was a para-nitrophenylalanine chromogenic reporter and Xaa was a mixture of 19 different amino acids. Kinetic assays monitoring the rates of cleavage of these libraries revealed the optimal P1 and P1′ residues for the five plasmepsins as hydrophobic substitutions. Extended specificity preferences were obtained utilizing liquid chromatographymass spectrometry (LC-MS) to analyze the cleavage products produced by enzyme-catalyzed digestion of the best pools of each peptide library. LC-MS analysis of the P1-Phe and P1′-Phe pools revealed the favored amino acids at the P3, P2, P2′, and P3′ positions. These analyses have provided new insights on the binding preferences of malarial digestive enzymes that were used to design specific methyleneamino peptidomimetic inhibitors of the plasmepsins. Some of these compounds were potent inhibitors of the five plasmepsins, and their possible binding modes were analyzed by computational methods.

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“…Inhibitors of PLM II that have low nanomolar inhibition constant values and are lethal against cultured malarial parasites have been identi®ed Carroll, Patel et al, 1998;Goldberg, 1992;Goldberg et al, 1991;Haque et al, 1999;Silva et al, 1996Silva et al, , 1998Westling et al, 1999). Furthermore, some aspartic and cysteine protease inhibitors demonstrate an apparent synergistic inhibition of hemoglobin degradation in both culture and a murine malaria model (Semenov et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Structures of Ser205 mutant plasmepsin II fromPlasmodium falciparumat 1.8 Å in complex with the inhibitors rs367 and rs370

Asojo

Afonina

Gulnik

et al. 2002

Acta Crystallogr D Biol Cryst

117

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Plasmepsin II is one of the four catalytically active plasmepsins found in the food vacuole of Plasmodium falciparum. These enzymes initiate hemoglobin degradation by cleavage at the -chain between Phe33 and Leu34. The crystal structures of Ser205 mutant plasmepsin II from P. falciparum in complex with two inhibitors have been re®ned at a resolution of 1.8 A Ê in the space group I222 and to R factors of 19.9 and 19.5%. Each crystal contains one monomer in the asymmetric unit. Both inhibitors have a Phe± Leu core and incorporate tetrahedral transition-state mimetic hydroxypropylamine. The inhibitor rs367 possesses a 2,6-dimethylphenyloxyacetyl group at the P2 position and 3-aminobenzamide at the P2 H position, while rs370 has the same P2 group but 4-aminobenzamide in the P2 H position. These complexes reveal key conserved hydrogen bonds between the inhibitor and the binding-cavity residues, notably with the¯ap residues Val78 and Ser79, the catalytic dyad Asp34 and Asp214 and the residues Ser218 and Gly36 that are in proximity to the catalytic dyad. The structures also show unexpected conformational variability of the binding cavity of plasmepsin II and may re¯ect the mode of binding of the hemoglobin -chain for cleavage.

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Active site specificity of plasmepsin II

Cited by 59 publications

References 27 publications

The pH of the digestive vacuole ofPlasmodium falciparumis not associated with chloroquine resistance

The pH of the digestive vacuole ofPlasmodium falciparumis not associated with chloroquine resistance

Active-Site Specificity of Digestive Aspartic Peptidases from the Four Species of Plasmodium that Infect Humans Using Chromogenic Combinatorial Peptide Libraries

Structures of Ser205 mutant plasmepsin II fromPlasmodium falciparumat 1.8 Å in complex with the inhibitors rs367 and rs370

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