Labelling of four distinct trophozoite falcipains of Plasmodium falciparum by a cystatin-derived probe

Trophozoite cysteine protease (TCP) activity, isolated from Plasmodium falciparum soluble 100,000 g extracts, displayed native falcipain-1 kinetic parameters towards peptidyl substrates. The labelling of either isolated TCP or soluble 100,000 g extracts by a cystatin-derived probe (biotinyl-Leu-Val-Gly-CHN2) revealed a single band of ca. 30 kDa by SDS-PAGE, which was resolved into four spots displaying isoelectric points (pI) from 4.7 to 5.3 after two-dimensional separation. The molecular mass and pI correspond to those of falcipain-3, falcipain-2, falcipain-2' and falcipain-1, respectively. The two central spots were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry as falcipain-2 and falcipain-2'. This activity-based probe represents a potential tool for profiling active falcipains in parasites.     

Cysteine proteases of malaria parasites

A number of cysteine proteases of malaria parasites have been described, and many more putative cysteine proteases are suggested by analysis of the Plasmodium falciparum genome sequence. Studies with protease inhibitors have suggested roles for cysteine proteases in hemoglobin hydrolysis, erythrocyte rupture, and erythrocyte invasion by erythrocytic malaria parasites. The best characterised Plasmodium cysteine proteases are the falcipains, a family of papain-family (clan CA) enzymes. Falcipain-2 and falcipain-3 are hemoglobinases that appear to hydrolyse host erythrocyte hemoglobin in the parasite food vacuole. This function was recently confirmed for falcipain-2, with the demonstration that disruption of the falcipain-2 gene led to a transient block in hemoglobin hydrolysis. A role for falcipain-1 in erythrocyte invasion was recently suggested, but disruption of the falcipain-1 gene did not alter parasite development. Other papain-family proteases predicted by the genome sequence include dipeptidyl peptidases, a calpain homolog, and serine-repeat antigens. The serine-repeat antigens have cysteine protease motifs, but in some the active site Cys is replaced by a Ser. One of these proteins, SERA-5, was recently shown to have serine protease activity. As SERA-5 and some other serine-repeat antigens localise to the parasitophorous vacuole in mature parasites, they may play a role in erythrocyte rupture. The P. falciparum genome sequence also predicts more distantly related (clan CD and CE) cysteine proteases, but biochemical characterisation of these proteins has not been done. New drugs for malaria are greatly needed, and cysteine proteases may provide useful new drug targets. Cysteine protease inhibitors have demonstrated potent antimalarial effects, and the optimisation and testing of falcipain inhibitor antimalarials is underway.     

Circadian and senescence-enhanced expression of a tobacco cysteine protease gene

A cDNA clone encoding a cysteine protease was isolated from a tobacco cDNA library, utilizing as a probe a PCR fragment obtained from degenerated primers based on the conserved sequences of plant cysteine protease genes. A putative protein encoded by the clone NTCP-23 had an amino acid sequence with significant similarities to those of plant senescence-associated cysteine proteases and mammalian cathepsin H. Northern blot analysis showed that NTCP-23 mRNA is expressed in all organs and the mRNA and protein expression is enhanced during natural senescence. We propose that NTCP-23 is responsible for amino acid remobilization especially in senescencing leaves. Furthermore, it was found that the mRNA expression follows a circadian rhythm and is reduced by continuous darkness, wounding and hypersensitive reaction (HR). NTCP-23 is the first cysteine protease whose mRNA expression has been shown to be temporarily reduced by wounding.     

Falstatin, a cysteine protease inhibitor of Plasmodium falciparum, facilitates erythrocyte invasion

Erythrocytic malaria parasites utilize proteases for a number of cellular processes, including hydrolysis of hemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by malaria parasites to control protease activity have not been established. We report here the identification of an endogenous cysteine protease inhibitor of Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi cysteine protease inhibitor chagasin. Falstatin, expressed in Escherichia coli, was a potent reversible inhibitor of the P. falciparum cysteine proteases falcipain-2 and falcipain-3, as well as other parasite- and nonparasite-derived cysteine proteases, but it was a relatively weak inhibitor of the P. falciparum cysteine proteases falcipain-1 and dipeptidyl aminopeptidase 1. Falstatin is present in schizonts, merozoites, and rings, but not in trophozoites, the stage at which the cysteine protease activity of P. falciparum is maximal. Falstatin localizes to the periphery of rings and early schizonts, is diffusely expressed in late schizonts and merozoites, and is released upon the rupture of mature schizonts. Treatment of late schizionts with antibodies that blocked the inhibitory activity of falstatin against native and recombinant falcipain-2 and falcipain-3 dose-dependently decreased the subsequent invasion of erythrocytes by merozoites. These results suggest that P. falciparum requires expression of falstatin to limit proteolysis by certain host or parasite cysteine proteases during erythrocyte invasion. This mechanism of regulation of proteolysis suggests new strategies for the development of antimalarial agents that specifically disrupt erythrocyte invasion.     

A role of falcipain-2, principal cysteine proteases of Plasmodium falciparum in merozoite egression

The process of merozoite release in Plasmodium falciparum involves rupture of the parasitophorous vacuole membrane and erythrocyte plasma membrane. Through the use of protease inhibitors that halt the merozoite release, a number of parasite proteases, especially serine, aspartic, and cysteine proteases, have been implicated in the schizont rupture. To understand the precise role of cysteine proteases in the merozoite release, in the present study, we treated P. falciparum cultures with siRNAs corresponding to falcipain-1, falcipain-2, and falcipain-3, the three papain-family proteases of the parasite. Treatment of malaria parasites with either of the falcipain siRNAs considerably reduced parasite growth. Morphological examination of the siRNA treated parasite cultures revealed that most of the parasites in falcipain-2 siRNA treated cultures were arrested at schizont stage. Analysis of a transgenic P. falciparum line expressing chimeric-GFP upon treatment with falcipain-2 siRNA revealed block in the rupture of erythrocyte membrane at the time of merozoite egression. These results suggest that falcipain-2 is an important parasitic protease that participates in hemoglobin degradation and in the merozoite release.     

Plasmodium falciparum: biochemical characterization of the cysteine protease falcipain-2'

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 are hemoglobinases and potential antimalarial drug targets. The falcipain-2' gene was identified recently and is nearly identical in sequence to falcipain-2. The product of this gene has not been studied previously. The mature protease domain of falcipain-2' was expressed in Escherichia coli, purified, and refolded to active enzyme. Functional analysis revealed similar biochemical properties to those of falcipain-2, including pH optima (pH 5.5-7.0), reducing requirements, and substrate preference. Studies with cysteine protease inhibitors showed similar inhibition of falcipain-2 and falcipain-2', although specificities were not identical. Considering activity against the presumed biological substrate, both enzymes readily hydrolyzed hemoglobin. Our results confirm that falcipain-2' is an active hemoglobinase and suggest that falcipain-2 and falcipain-2' play similar roles in erythrocytic parasites but that, for promising cysteine protease inhibitors, it will be important to confirm activity against this additional target.     

Characterization of native and recombinant falcipain-2, a principal trophozoite cysteine protease and essential hemoglobinase of Plasmodium falciparum

Trophozoites of the malaria parasite Plasmodium falciparum hydrolyze erythrocyte hemoglobin in an acidic food vacuole to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block hemoglobin degradation, indicating that a cysteine protease plays a key role in this process. A principal trophozoite cysteine protease was purified by affinity chromatography. Sequence analysis indicated that the protease is encoded by a previously unidentified gene, falcipain-2. Falcipain-2 was predominantly expressed in trophozoites, was concentrated in food vacuoles, and was responsible for at least 93% of trophozoite soluble cysteine protease activity. A construct encoding mature falcipain-2 and a small portion of the prodomain was expressed in Escherichia coli and refolded to active enzyme. Specificity for the hydrolysis of peptide substrates by native and recombinant falcipain-2 was very similar, and optimal at acid pH in a reducing environment. Under physiological conditions (pH 5.5, 1 mm glutathione), falcipain-2 hydrolyzed both native hemoglobin and denatured globin. Our results suggest that falcipain-2 can initiate cleavage of native hemoglobin in the P. falciparum food vacuole, that, following initial cleavages, the protease plays a key role in rapidly hydrolyzing globin fragments, and that a drug discovery effort targeted at this protease is appropriate.     

Structure-based approach to falcipain-2 inhibitors: synthesis and biological evaluation of 1,6,7-trisubstituted dihydroisoquinolines and isoquinolines

1,4,7-Trisubstituted isoquinolines were designed, synthesized and evaluated for their inhibition against Plasmodium falciparum cysteine protease falcipain-2. The 1-benzyloxyphenyl-dihydroisoquinoline and -isoquinoline derivatives were found to exhibit better activity against falcipain-2 than their corresponding 1-hydroxyphenyl or 1-methoxyphenyl analogues. The docking scores correlate with the IC(50) values of compounds and give a high coefficient correlation of 0.94.     

黄瓜叶片细菌性角斑病侵染初期cDNA文库分析

文章以接种细菌性角斑病原菌48 h的抗病品种‘D0462’黄瓜叶片为材料, 构建了黄瓜叶片cDNA文库, 文库插入片段大小为0.45~2.1 kb之间, 平均长度为1 kb。随机选取2 966个克隆进行测序, 共拼接出2 352个假定独立转录本(TUTs), 其中包括282个重叠群(Contigs), 2 070个单拷贝EST(Singlets)。经BlastX分析共获得已知功能和未知功能基因1 848个, 无序列相似性新基因504个。文库中含有大量防御/抗病基因, 如金属硫蛋白、谷胱甘肽-S-转移酶、泛素、b-1, 3-葡聚糖酶、锌指蛋白和半胱氨酸蛋白酶等, 这些基因很可能参与了植物与病原菌互作过程。     

cDNA cloning of a novel cdc2+/CDC28-related protein kinase from rice

A cDNA clone, named R2, has been isolated by screening a rice cell cDNA library with a redundant oligonucleotide probe derived from the conserved ATP binding site of cdc2+/CDC28 protein kinases. The cDNA contained the entire coding sequence for a 424 amino acid polypeptide with a molecular mass of 47.6 kDa. The R2 mRNA, 2.1 kb in size, was expressed in both cultured rice cells and rice seedlings at similar levels. The predicted R2 protein has canonical motifs for ATP binding and catalysis, and is significantly homologous (up to 47%) to members of the cdc2+/CDC28 subfamily of serine/threonine protein kinase. The R2 protein is a novel member of the subfamily.     

Synthesis and biological evaluation of phenolic Mannich bases of benzaldehyde and (thio)semicarbazone derivatives against the cysteine protease falcipain-2 and a chloroquine resistant strain of Plasmodium falciparum

A targeted series of phenolic Mannich bases of benzaldehyde and (thio)semicarbazone derivatives were synthesized and evaluated in vitro against the malarial cysteine protease falcipain-2 and a chloroquine resistant strain (W2) of Plasmodium falciparum. A novel series of 4-aminoquinoline semicarbazones were the most effective inhibitors of falcipain-2 (most potent inhibitor had IC(50)=0.63microM) while a bisquinoline semicarbazone compound 8f was the most potent antimalarial compound with an IC(50) of 0.07microM against W2. Compound 8f also weakly inhibited falcipain-2, with an IC(50) of 3.16microM, although its principal antiparasitic activity did not appear to be due to inhibition of this enzyme.     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

An mRNA from human brain encodes an isoform of the B subunit of the vacuolar H(+)-ATPase

The B subunit (approximately 60 kDa) of the vacuolar H(+)-ATPase is one of the two major subunits comprising the hydrophilic catalytic complex of the enzyme. Using left and catalytic complex of the enzyme. Using left and right primers which bind two highly conserved sequences of the B subunit, an 836-base pair fragment was amplified from human brain cDNA by the polymerase chain reaction. The amplified fragment was used to probe a Northern blot and to screen a brain cDNA library. A single RNA band, 3.2 kilobases (kb) in length, was detected on Northern blots. A positive cDNA clone containing a 2.5-kb insert was isolated and sequenced. It included a long 3'-untranslated region (greater than 1.2 kb) and was missing a minor portion of the 5'-end of the coding region. The coding region of the brain cDNA sequence was 77% identical at the nucleotide level and 90% identical at the amino acid level to the previously reported sequence for the B subunit of the vacuolar H(+)-ATPase from human kidney (Sudhof, T. C., Fried, V. A., Stone, D. K., Johnston, P. A., and Xie, X.-S. (1989) Proc. Natl. Acad. Sci, U. S. A. 86, 6067-6071). Within the coding region of the brain cDNA, which is 6 amino acid residues shorter at the 3'-end than the kidney sequence, an 11% difference in the GC content was calculated. The 3'-noncoding sequence of the brain cDNA was completely unrelated to that of kidney and was three times longer. We conclude that the B subunit cDNAs from human kidney and brain represent different isoforms. This is the first demonstration of an isoform of a vacuolar H(+)-ATPase subunit.     

Isolation and characterization of a cDNA for rat liver cysteine dioxygenase

Cysteine dioxygenase is a key enzyme of cysteine metabolism in mammals. The cDNA clones for rat liver cysteine dioxygenase were isolated by immunological screening and plaque hybridization from a rat liver cDNA library. The longest clone contained an insert of 1458 bp and encoded a polypeptide of 200 amino acids. The clone included the corresponding nucleotide sequence to amino acid sequences obtained from four lysyl endopeptidase-digested fragments of purified rat liver cysteine dioxygenase. The calculated molecular weight of rat liver cysteine dioxygenase was 23,025. Northern blot analysis revealed a single cysteine dioxygenase mRNA species of about 1.7 kb. A computer homology search indicated that this protein showed no homology with any known protein.     

Molecular cloning and expression analysis of novel wheat cysteine protease

A cDNA clone encoding a novel papain-like cysteine protease was isolated from wheat germ (Triticum aestivum). This cDNA encoded a 371-residue protein, designated WCP2, composed of signal peptide followed by a propeptide and a mature protease containing active site residues that are highly conserved among the papain family. The mature WCP2 protein (26 kDa) was detected in the quiescent embryo and its level of expression in the germinating embryo was greatly increased.     

Cloning and sequencing of protochlorophyllide reductase.

Putative protochlorophyllide reductase cDNA clones (252 and 113) were isolated from an etiolated-oat (Avena sativa) cDNA library. These were used to indirectly characterize a further clone, p127, isolated from a lambda-phage gt11 cDNA library. The latter (1.15 kb in length) was sequenced, and the derived amino acid sequence was shown to be remarkably similar to that derived from chemical analysis of a CNBr-cleavage fragment of the purified reductase, p127 codes for more than 95% of the reductase protein.