The Trypanosoma brucei sphingolipid synthase, an essential enzyme and drug target

Abstract

Publisher's copyright statement:Additional information: Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. AbstractSphingolipids are important components of eukaryotic membranes, particularly the plasma membrane, and are involved in a diverse array of signal transduction processes.In the Eukaryota the biosynthetic pathway for the formation of these lipid species is largely conserved. However, in contrast to mammals which produce sphingomyelin (SM), several pathogenic fungi and protozoa synthesize inositol phosphorylceramide 3 Key wordsTrypanosoma, trypanosomiasis, sphingolipid synthase, drug target FootnoteSince the initial submission of this work to Molecular and Biochemical Parasitology a paper has been published in Molecular Microbiology identifying and characterising the same family of enzymes in Trypanosoma brucei:

A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase

“…With a Z' value of 0.58 (Zhang, et al 1999), the assay system described lends itself to high-throughput screening for inhibitors of a protozoal enzyme activity that has been demonstrated to be essential for kinetoplastid pathogens (Mina, et al 2009;Sutterwala, et al 2008). The assay of ceramide analogues demonstrated this potential and provides the backbone for consideration of rationally designed enzyme inhibitors.…”

“…Adopting this approach and employing microsomes from LmjIPCS complemented HIS-GAL-AUR1 yeast, washed with CHAPS as described, the parasite enzyme assay proved to be dependent not only on the addition of the fluorescent acceptor substrate, NBD-C 6 -ceramide, but now also more than 90% dependent on the addition of the donor substrate (bovine liver PI; figure 2A). It has previously been demonstrated that the S. cerevisiae orthologous enzyme, AUR1p, is unable to utilise bovine liver PI (Mina, et al 2009). In the present assay system, the CHAPS-washed microsomes also demonstrated an approximately 2-fold higher level of enzyme turnover than equivalent quantities of crude material ( figure 2B).…”

“…In addition, this compound has been demonstrated to inhibit the growth and infectivity of L. amazonensis (Tanaka, et al 2007). Furthermore, recent work in our laboratory (Mina, et al 2009) and elsewhere (Sutterwala, et al 2008) has both genetically and pharmacologically validated the kinetoplastid enzyme as a target in T.…”

Functional analyses of differentially expressed isoforms of the Arabidopsis inositol phosphorylceramide synthase

“…In the experiment ( figure 2A cerevisiae AUR1p (Mina, et al 2009). Despite this, these data confirm AtIPCS1-3 as functional orthologues of AUR1p, forming at least part of an IPC synthase.…”

“…For in vitro assay microsomal membranes were prepared from YPH499-HIS-GAL-AUR1 pRS246 AtIPCS1-3 and YPH499-HIS-GAL-AUR1 pRS246 AUR1 and assayed as described (Mina, et al 2009). In brief, assays were performed in 50µl of 100mM Tris HCl pH 7.4, 10mM EDTA and 6mg/ml defatted BSA, with or without 1mM PI (soybean, Avanti Polar Lipids; predominant species C16:0-C18:2) and 5µM aureobsidin A, and with 2 µl of microsomes (10 mg/ml protein) and 2µl of 5mM NBD C 6 -Ceramide ( (Synergy HT, Bio-tek) samples were analysed by high performance thin-layer chromatography (Denny, et al 2001;Ralton and McConville 1998), imaged using a FLA3000 scanner (Fuji) and quantified using the Aida V3.11 software package.…”