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I Y Gluzman

Publications and source records attributed to I Y Gluzman.

At least 19 recordsLinked to original sources
PubMed2001

Co-ordinated programme of gene expression during asexual intraerythrocytic development of the human malaria parasite Plasmodium falciparum revealed by microarray analysis.

Plasmodium falciparum is a protozoan parasite responsible for the most severe forms of human malaria. All the clinical symptoms and pathological changes seen during human infection are caused by the asexual blood stages of Plasmodium. Within host red blood cells, the parasite undergoes enormous developmental changes during its maturation. In order to analyse the expression of genes during intraerythrocytic development, DNA microarrays were constructed and probed with stage-specific cDNA. Developmental upregulation of specific mRNAs was found to cluster into functional groups and revealed a co-ordinated programme of gene expression. Those involved in protein synthesis (ribosomal proteins, translation factors) peaked early in development, followed by those involved in metabolism, most dramatically glycolysis genes. Adhesion/invasion genes were turned on later in the maturation process. At the end of intraerythrocytic development (late schizogony), there was a general shut-off of gene expression, although a small set of genes, including a number of protein kinases, were turned on at this stage. Nearly all genes showed some regulation over the course of development. A handful of genes remained constant and should be useful for normalizing mRNA levels between stages. These data will facilitate functional analysis of the P. falciparum genome and will help to identify genes with a critical role in parasite progression and multiplication in the human host.

Animals↗
PubMed1999

A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum.

Genomic information is rapidly accumulating for the human malaria pathogen, Plasmodium falciparum. Our ability to perform genetic manipulations to understand Plasmodium gene function is limited. Dihydrofolate reductase is the only selectable marker presently available for transfection of P. falciparum. Additional markers are needed for complementation and for expression of mutated forms of essential genes. We tested parasite sensitivity to different drugs for which selectable markers are available. Two of these drugs that were very effective as antiplasmodial inhibitors in culture, blasticidin and geneticin (G418), were selected for further study. The genes BSD, encoding blasticidin S deaminase of Aspergillus terreus, and NEO, encoding neomycin phosphotransferase II from transposon Tn 5, were expressed under the histidine-rich protein III (HRPIII) gene promoter and tested for their ability to confer resistance to blasticidin or G418, respectively. After transfection, blasticidin and G418-resistant parasites tested positive for plasmid replication and BSD or NEO expression. Cross-resistance assays indicate that these markers are independent. The plasmid copy number and the enzymatic activity depended directly on the concentration of the drug used for selection. These markers set the stage for new methods of functional analysis of the P. falciparum genome.

Aminohydrolases↗
PubMed1999

Potent, low-molecular-weight non-peptide inhibitors of malarial aspartyl protease plasmepsin II.

A number of single-digit nanomolar, low-molecular-weight plasmepsin II aspartyl protease inhibitors have been identified using combinatorial chemistry and structure-based design. By identifying multiple, small-molecule inhibitors using the parallel synthesis of several focused libraries, it was possible to select for compounds with desirable characteristics including enzyme specificity and minimal binding to serum proteins. The best inhibitors identified have Ki's of 2-10 nM, molecular weights between 594 and 650 Da, between 3- and 15-fold selectivity toward plasmepsin II over cathepsin D, the most closely related human protease, good calculated log P values (2.86-4.56), and no apparent binding to human serum albumin at 1 mg/mL in an in vitro assay. These compounds represent the most potent non-peptide plasmepsin II inhibitors reported to date.

Animals↗
PubMed1997

Probing the chloroquine resistance locus of Plasmodium falciparum with a novel class of multidentate metal(III) coordination complexes.

The malaria organism Plasmodium falciparum detoxifies heme released during degradation of host erythrocyte hemoglobin by sequestering it within the parasite digestive vacuole as a polymer called hemozoin. Antimalarial agents such as chloroquine appear to work by interrupting the heme polymerization process, but their efficacy has been impaired by the emergence of drug-resistant organisms. We report here the identification of a new class of antimalarial compounds, hexadentate ethylenediamine-N, N'-bis[propyl(2-hydroxy-(R)-benzylimino)]metal(III) complexes [(R)-ENBPI-M(III)] and a corresponding ((R)-benzylamino)] analog [(R)-ENBPA-M(III)], a group of lipophilic monocationic leads amenable to metallopharmaceutical development. Racemic mixtures of Al(III), Fe(III), or Ga(III) but not In(III) (R)-ENBPI metallo-complexes killed intraerythrocytic malaria parasites in a stage-specific manner, the R = 4,6-dimethoxy-substituted ENBPI Fe(III) complex being most potent (IC50 approximately 1 microM). Inhibiting both chloroquine-sensitive and -resistant parasites, potency of these imino complexes correlated in a free metal-independent manner with their ability to inhibit heme polymerization in vitro. In contrast, the reduced (amino) 3-MeO-ENBPA Ga(III) complex (MR045) was found to be selectively toxic to chloroquine-resistant parasites in a verapamil-insensitive manner. In 21 independent recombinant progeny of a genetic cross, susceptibility to this agent mapped in perfect linkage with the chloroquine resistance phenotype suggesting that a locus for 3-MeO-ENBPA Ga(III) susceptibility was located on the same 36-kilobase segment of chromosome 7 as the chloroquine resistance determinant. These compounds may be useful as novel probes of chloroquine resistance mechanisms and for antimalarial drug development.

ATP-Binding Cassette Transporters↗
PubMed1997

Generation of hemoglobin peptides in the acidic digestive vacuole of Plasmodium falciparum implicates peptide transport in amino acid production.

Intraerythrocytic malaria parasites avidly consume hemoglobin as a source of amino acids for incorporation into parasite proteins. An acidic organelle, the digestive vacuole, is the site of hemoglobin proteolysis. Early events in hemoglobin catabolism have been well studied. Two aspartic proteases, plasmepsins I and II, and a cysteine protease, falcipain, cleave hemoglobin into peptides. While it has been presumed that hemoglobin peptide fragments are degraded to individual amino acids by exopeptidase activity in the digestive vacuole, this hypothesis lacks experimental support. Incubation of human hemoglobin with P. falciparum digestive vacuole lysate generated a series of discrete peptide fragments with cleavage sites an average of 8.4 amino acids apart. No free amino acids could be detected and there was no evidence of peptide heterogeneity due to exopeptidase trimming. These sites correspond to points of cleavage previously established for plasmepsin I, plasmepsin II, and falcipain as well as some novel sites that suggest the existence of an additional endoproteinase. By colorimetric assay, P. falciparum has abundant aminopeptidase activity but this activity is not found in the digestive vacuoles and the parasite lacks detectable carboxypeptidase activity altogether. These data support a model for hemoglobin catabolism wherein small peptides are formed from cleavage of hemoglobin by the enzymes of the digestive vacuole and then are transported through the membrane of the digestive vacuole to the cytoplasm. There, exopeptidase activity converts the peptides to individual amino acids for parasite growth and maturation.

Amino Acids↗
PubMed1996

Characterization of native falcipain, an enzyme involved in Plasmodium falciparum hemoglobin degradation.

In Plasmodium falciparum, a cysteine protease known as falcipain has been implicated in the essential metabolic process of hemoglobin degradation. Parallel lines of investigation, using native or recombinant enzyme, have led to differing conclusions about the specificity and role of this protease. We have now determined that (1) Native falcipain does not cleave hemoglobin unless this substrate has first been denatured by reducing agents, acid-acetone treatment or plasmepsin action. (2) Reducing agents such as glutathione cannot denature hemoglobin in the presence of catalase, which is accumulated in the digestive vacuole. (3) The purified native enzyme has kinetics similar to those obtained with trophozoite extract, but substantially different from those of recombinant enzyme. (4) Although there are numerous cysteine protease genes in the P. falciparum genome, the falcipain gene is the only one whose transcript can be detected in the early intraerythrocytic parasites. We conclude that falcipain likely works by degrading hemoglobin fragments after initial aspartic protease attack has denatured the substrate. We propose that falcipain inhibitors block the initial steps of degradation indirectly by promoting vacuolar accumulation of osmotically active hemoglobin peptides.

Animals↗
PubMed1996

On the molecular mechanism of chloroquine's antimalarial action.

Chloroquine is thought to exert its antimalarial effect by preventing the polymerization of toxic heme released during proteolysis of hemoglobin in the Plasmodium digestive vacuole. The mechanism of this blockade has not been established. We incubated cultured parasites with subinhibitory doses of [3H]chloroquine and [3H] quinidine. These [3H]quinoline compounds became associated with hemozoin as assessed by electron microscope autoradiography and subcellular fractionation. In vitro, binding of [3H]quinoline inhibitors to the hemozoin chain depended on the addition of heme substrate. These data counter previous conclusions regarding the lack of quinoline association with hemozoin, explain the exaggerated accumulation of quinolines in the plasmodium digestive vacuole, and suggest that a quinoline heme complex incorporates into the growing polymer to terminate chain extension, blocking further sequestration of toxic heme.

Animals↗
PubMed1996

Structure and inhibition of plasmepsin II, a hemoglobin-degrading enzyme from Plasmodium falciparum.

Plasmodium falciparum is the major causative agent of malaria, a disease of worldwide importance. Resistance to current drugs such as chloroquine and mefloquine is spreading at an alarming rate, and our antimalarial armamentarium is almost depleted. The malarial parasite encodes two homologous aspartic proteases, plasmepsins I and II, which are essential components of its hemoglobin-degradation pathway and are novel targets for antimalarial drug development. We have determined the crystal structure of recombinant plasmepsin II complexed with pepstatin A. This represents the first reported crystal structure of a protein from P. falciparum. The crystals contain molecules in two different conformations, revealing a remarkable degree of interdomain flexibility of the enzyme. The structure was used to design a series of selective low molecular weight compounds that inhibit both plasmepsin II and the growth of P. falciparum in culture.

Amino Acid Sequence↗
PubMed1996

Plasmodium hemozoin formation mediated by histidine-rich proteins.

The digestive vacuole of Plasmodium falciparum is the site of hemoglobin degradation, heme polymerization into crystalline hemozoin, and antimalarial drug accumulation. Antibodies identified histidine-rich protein II (HRP II) in purified digestive vacuoles. Recombinant or native HRP II promoted the formation of hemozoin, and chloroquine inhibited the reaction. The related HRP III also polymerized heme, and an additional HRP was identified in vacuoles. It is proposed that after secretion by the parasite into the host erythrocyte cytosol, HRPs are brought into the acidic digestive vacuole along with hemoglobin. After hemoglobin proteolysis, HRPs bind the liberated heme and mediate hemozoin formation.

Amino Acid Sequence↗
PubMed1996

Kinetic analysis of plasmepsins I and II aspartic proteases of the Plasmodium falciparum digestive vacuole.

Plasmepsins I and II are Plasmodium falciparum aspartic proteases implicated in hemoglobin degradation. Using a synthetic fluorogenic peptide substrate based on the initial hemoglobin cleavage site, we have analyzed kinetic parameters of the two enzymes in native and recombinant forms. Both native plasmepsins cleave the model substrate well. Recombinant plasmepsin II behaves similarly to native enzyme, substantiating its usefulness for inhibition and structural studies. In contrast, recombinant plasmepsin I does not resemble its native homolog kinetically. A hybrid molecule, in which the polyproline loop of plasmepsin I has been replaced by the homologous sequence from plasmepsin II, still maintains the specificity/kinetics of plasmepsin II. This suggests that the polyproline loop, important for substrate recognition in the mammalian aspartic protease renin, does not play a similar role in the plasmepsins.

Amino Acid Sequence↗
PubMed1994

Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase.

Intraerythrocytic malaria parasites rapidly degrade virtually all of the host cell hemoglobin. We have cloned the gene for an aspartic hemoglobinase that initiates the hemoglobin degradation pathway in Plasmodium falciparum. It encodes a protein with 35% homology to human renin and cathepsin D, but has an unusually long pro-piece that includes a putative membrane spanning anchor. Immunolocalization studies place the enzyme in the digestive vacuole and throughout the hemoglobin ingestion pathway, suggesting an unusual protein targeting route. A peptidomimetic inhibitor selectively blocks the aspartic hemoglobinase, prevents hemoglobin degradation and kills the organism. We conclude that Plasmodium hemoglobin catabolism is a prime target for antimalarial chemotherapy and have identified a lead compound towards this goal.

Amino Acid Sequence↗
PubMed1994

Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway.

The human malaria parasite, Plasmodium falciparum, degrades nearly all its host cell hemoglobin during a short segment of its intraerythrocytic development. This massive catabolic process occurs in an acidic organelle, the digestive vacuole. Aspartic and cysteine proteases have been implicated in this pathway. We have isolated three vacuolar proteases that account for most of the globin-degrading activity of the digestive vacuole. One is the previously described aspartic hemoglobinase that initiates hemoglobin degradation. A second aspartic protease is capable of cleaving hemoglobin with an overlapping specificity, but seems to prefer acid-denatured globin. The third is a cysteine protease that does not recognize native hemoglobin but readily cleaves denatured globin. It is synergistic with the aspartic hemoglobinase, both by in vitro assay of hemoglobin degradation, and by isobologram analysis of protease inhibitor-treated parasites in culture. The cysteine protease is highly sensitive to chloroquine-heme complex, suggesting a possible mechanism of 4-aminoquinoline antimalarial action. The data suggest an ordered pathway of hemoglobin catabolism that presents an excellent target for chemotherapy.

Amino Acid Sequence↗
PubMed1992

Energy dependence of chloroquine accumulation and chloroquine efflux in Plasmodium falciparum.

Chloroquine inhibits the growth of susceptible malaria parasites at low (nanomolar) concentrations because of an energy-requiring drug-concentrating mechanism in the parasite secondary lysosome (food vacuole) which is dependent on the acidification of that vesicle. Chloroquine resistance results from another energy-requiring process: efflux of chloroquine from the resistant parasite with a half-time of 2 min. Chloroquine efflux is inhibited reversibly by the removal of metabolizable substrate (glucose); it is also reduced by the ATPase inhibitor vanadate. These results suggest that chloroquine efflux is an energy-requiring process dependent on the generation and hydrolysis of ATP. Chloroquine efflux cannot be explained by differences in drug accumulation between chloroquine-susceptible and -resistant parasites because the 40-50-fold difference in initial efflux rates between -susceptible and -resistant parasites is unchanged when both parasites contain the same amount of chloroquine. Although chloroquine efflux is phenotypically similar to the efflux of anticancer drugs from multidrug-resistant (mdr) mammalian cells, it is not linked to either of the mdr-like genes of the parasite.

Adenosine Triphosphatases↗
PubMed1991

Calcium and the malaria parasite: parasite maturation and the loss of red cell deformability.

In the studies reported here, we examined the role of calcium in the maturation of the human malaria parasite Plasmodium falciparum, and in the loss of red cell deformability associated with parasite maturation. P. falciparum alters the permeability of its host red cell, which normally maintains submicromolar cytoplasmic concentrations of calcium. Infection of the red cell and parasite maturation produce a 30-fold increase in calcium uptake. Both parasite maturation and the loss of red cell deformability are blocked by EGTA (by extracellular-free calcium concentrations less than or equal to 35 microM) and by other calcium antagonists. The loss of red cell deformability that occurs with parasite maturation is accompanied by alterations in the cytoskeletal proteins of parasitized red cells similar to those produced by the calcium ionophore A23187 (reductions in bands 2.1 [ankyrin], 4.1, and 5 [actin]). These results establish that parasite development and the loss of red cell deformability are calcium-dependent. They suggest that parasite-induced changes in the calcium permeability of the red cell activate endogenous transglutaminase activity by raising the free calcium concentration of the red cell cytoplasm.

Animals↗