FATTY ACID METABOLISM IN APICOMPLEXAN PARASITES
Embargo until
2015-05-01
Date
2014-02-28
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Publisher
Johns Hopkins University
Abstract
Plasmodium falciparum and Toxoplasma gondii are human parasites that belong to the phylum Apicomplexa and are the causative agents of malaria and toxoplasmosis, respectively. These parasites infect billions of people annually worldwide causing significant morbidity. We made use of biochemical and biophysical methods to better understand essential fatty acid metabolism of these pathogens, such as Fatty Acid Synthesis type II (FAS-II) and lipoate metabolism. The apicoplast, a plastid organelle, harbors the machinery for FAS-II which is essential for the survival of T. gondii and for liver stage malaria parasites. A small biocide, triclosan, is known to target the last enzyme in FAS-II with high specificity. Several modifications were made to this scaffold molecule to increase its druggability properties, while retaining specificity. We designed a method to measure the inhibition and IC50 values of triclosan analogues in 96-well plate format. Also, we modified a thermal shift assay to measure their binding affinity to the enzyme and determine their inhibition mode of action. The FAS-II produces octanoyl-ACP, which is the substrate of downstream enzymes to synthesize lipoate on pyruvate dehydrogenase (PDH). Contrary to the apicoplast, the mitochondrion relies on lipoate scavenged from the host which is essential for the survival of these parasites. In both parasites, two lipoate ligases are localized to the mitochondrion, LipL1 and LipL2. These ligases are believed to catalyze the attachment of lipoate to three lipoate-requiring substrates in the mitochondrion: the H-protein, the branch chain amino acid dehydrogenase (BCDH) and the α-ketoglutarate dehydrogenase (KDH). We show that LipL1 is the sole lipoate ligase in the mitochondrion with specificity for the H-protein. The BCDH and KDH are lipoylated through a concerted mechanism: LipL1 produces the conjugate lipoyl-AMP which is transferred to LipL2 to lipoylate the BCDH and KDH. LipL2 is unable to produce the lipoyl-AMP conjugate, but instead acts as a lipoyl-AMP:N-lysine lipoyltransferase. The two step LipL1+LipL2 lipoylation mechanism appears to be conserved in most apicomplexan parasites, since the genes encoding both enzymes are conserved. Furthermore, lipoate scavenging shows redox sensitivity for the lipoate redox state -a phenomenon that has not been previously described-. Last, we can target the mitochondrial proteins with lipoate analogues and inhibit their activities, causing cell growth inhibition. The lipoylation mechanism found in apicomplexan parasites differs from that found in metazoans, making it an attractive drug target.
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Keywords
malaria, lipoate