Biomedical subjects

Eva S Istvan

Publications and source records attributed to Eva S Istvan.

4 recordsLinked to original sources

PubMed2006

Hemoglobin-degrading plasmepsin II is active as a monomer.

A family of aspartic proteases called plasmepsins is important for hemoglobin degradation in intraerythrocytic Plasmodium parasites. Plasmepsin II (PM II) is the best studied member of this family. PM II and its close orthologs and paralogs form homodimers with extensive interfaces in all known crystal structures. This raised the question whether the homodimer is the functional subunit of plasmepsins in solution. We have used gel filtration chromatography, site-directed mutagenesis, and analytical ultracentrifugation to study the oligomeric status of PM II in solution. Our results reveal that PM II exists mainly as a monomer in solution and that the monomer is fully functional for catalysis. A hydrophobic loop at the PM II monomer surface, which would be buried in a PM II dimer, is shown to be essential for the hemoglobin degradation capability of PM II.

Aspartic Acid Endopeptidases↗

PubMed2006

Plasmodium falciparum ensures its amino acid supply with multiple acquisition pathways and redundant proteolytic enzyme systems.

Degradation of host hemoglobin by the human malaria parasite Plasmodium falciparum is a massive metabolic process. What role this degradation plays and whether it is essential for parasite survival have not been established, nor have the roles of the various degradative enzymes been clearly defined. We report that P. falciparum can grow in medium containing a single amino acid (isoleucine, the only amino acid missing from human hemoglobin). In this medium, growth of hemoglobin-degrading enzyme gene knockout lines (missing falcipain-2 and plasmepsins alone or in combination) is impaired. Blockade of plasmepsins with the potent inhibitor pepstatin A has a minimal effect on WT parasite growth but kills falcipain-2 knockout parasites at low concentrations and is even more potent on falcipain-2, plasmepsin I and IV triple knockout parasites. We conclude that: (i) hemoglobin degradation is necessary for parasite survival; (ii) hemoglobin degradation is sufficient to supply most of the parasite's amino acid requirements; (iii) external amino acid acquisition and hemoglobin digestion are partially redundant nutrient pathways; (iv) hemoglobin degradation uses dual protease families with overlapping function; and (v) hemoglobin-degrading plasmepsins are not promising drug targets.

Amino Acids↗

PubMed2004

Distal substrate interactions enhance plasmepsin activity.

Plasmepsin II (PM II) is an aspartic protease active in hemoglobin (Hb) degradation in the protozoan parasite Plasmodium falciparum. A fluorescence-quenched octapeptide substrate based on the initial hemoglobin cleavage site is recognized well by PM II. C-terminal extension of this peptide has little effect, but N-terminal extension results in higher maximal velocity and dramatic concentration-dependent substrate inhibition. This inhibition, but not the rate stimulation, depends on the presence of a DABCYL group on the peptide N terminus. Using site-directed mutagenesis, we have identified PM II residues that interact with N-terminal amino acids of peptide substrates. The same residues influence degradation of Hb by PM II. Cathepsin E (CatE), a related mammalian aspartic protease, is also stimulated by N-terminally extended substrates. This suggests that distal substrate interactions as far out as P6 may be a general property of aspartic proteases and may be important in substrate and inhibitor recognition. Although PM II and CatE are similar in their ability to cleave Hb-based peptides and Hb alpha-chains, CatE is not able to degrade native Hb, which is a substrate for PM II. Based on these results, we propose that PM II may have the special feature of being a Hb denaturase.

Amino Acid Sequence↗

PubMed2002

Structural mechanism for statin inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) catalyzes the committed step in cholesterol biosynthesis. HMGR is the target of compounds (HMGR inhibitors, commonly referred to as statins) that are very effective in lowering serum cholesterol levels. These inhibitors have K(i) values in the nanomolar range and are widely prescribed in the treatment of hypercholesterolemia. We have determined structures of this enzyme in complexes with 6 different statins (compactin, simvastatin, fluvastatin, cerivastatin, atorvastatin, and rosuvastatin). The statins occupy a portion of the binding site of HMG-CoA, thus blocking access of this substrate to the active site. The nicotinamide binding pocket of NADP(H) (the second substrate of the enzyme) is unoccupied by the inhibitor molecules. Near the C-terminus of HMGR, several catalytically relevant residues are disordered in the enzyme-statin complexes. The flexibility of these residues is critical for binding with statins; if ordered, they would sterically hinder such binding.

Animals↗