The exopolyphosphatase TbrPPX1 of Trypanosoma brucei.

BACKGROUND: Exopolyphosphatases and pyrophosphatases play important but still incompletely understood roles in energy metabolism, and also in other aspects of cell biology such as osmoregulation or signal transduction. Earlier work has suggested that a human exopolyphosphatase, Prune, might exhibit cyclic nucleotide phosphodiesterase activity. RESULTS: The kinetoplastida, a large order of unicellular eukaryotes that contains many important pathogens such as Trypanosoma brucei (human sleeping sickness), Trypanosoma cruzi (Chagas disease) or Leishmania ssp (several clinically dinstinct leishmaniases) all contain several exo- and pyrophosphatases. The current study provides a systematic classification of these enzymes, which now allows to situate the information that is already available on some of these enzymes. It then analyses the exopolyphosphatase TbrPPX1 of T. brucei in detail, using RNA interference and genetic knockouts in an attempt to define its function, and immunofluorescence microscopy to study its subcellular localization.TbrPPX1 is an exopolyphosphatase that does hydrolyze pentasodium triphosphate, but not organic triphosphates such as ATP, pyrophosphate or long-chain polyphosphates. Finally, the study investigates the potential cyclic nucleotide phosphodiesterase activity of TbrPPX1. CONCLUSIONS: All kinetoplastid genomes that are currently available contain genes for an exopolyphosphatase and two classes of pyrophosphatases, one associated with the acidocalcisomes and one cytoplasmic. TbrPPX1 represents the T. brucei exopolyphosphatase. It is located throughout the cytoplasm, and its genetic ablation does not produce a dramatic phenotype. Importantly, TbrPPX1 does not exhibit any cyclic nucleotide specific phosphodiesterase activity, which definitively eliminates it as an additional player in cAMP signalling of the kinetoplastida.

Cyclic nucleotide-specific phosphodiesterases of Plasmodium falciparum: PfPDEalpha, a non-essential cGMP-specific PDE that is an integral membrane protein.

Cyclic nucleotide-specific phosphodiesterases (PDEs) have come into focus as interesting potential targets for PDE inhibitor-based anti-parasitic drugs. Genomes of the various agents of human malaria, most notably Plasmodium falciparum, all contain four genes for class 1 PDEs. The catalytic domains of these enzymes are closely related to those of the 11 human PDE families. This presents the possibility that the available vast expertise in developing drugs against human PDEs might now also be applied to developing compounds that are active against malarial PDEs. The current study identifies four Plasmodium genes that code for PfPDEalpha, PfPDEbeta, PfPDEgamma and PfPDEdelta, respectively. It further demonstrates that the PfPDEalpha polypeptide exists in two versions (PfPDEalphaA and PfPDEalphaB) that are generated by alternative splicing of the primary transcript. All malarial PDEs contain several transmembrane helices in their N-terminal regions, indicating that they are integral membrane proteins. In agreement with this prediction, essentially all PDE activity is associated with the cell membranes. PfPDEalpha was characterized as a cGMP-specific PDE that is not sensitive to a number of standard PDE inhibitors. Genetic ablation of the PfPDE1 gene produced no major phenotype in erythrocyte cultures.

A review of tobacco BY-2 cells as an excellent system to study the synthesis and function of sterols and other isoprenoids.

In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate (IPP), the universal precursor for isoprenoid biosynthesis. In this paper we review findings and observations made primarily with tobacco BY-2 cells (TBY-2), which have proven to be an excellent system in which to study the two biosynthetic pathways. A major advantage of these cells as an experimental system is their ability to readily take up specific inhibitors and stably- and/or radiolabeled precursors. This permits the functional elucidation of the role of isoprenoid end products and intermediates. Because TBY-2 cells undergo rapid cell division and can be synchronized within the cell cycle, they constitute a highly suitable test system for determination of those isoprenoids and intermediates that act as cell cycle inhibitors, thus giving an indication of which branches of the isoprenoid pathway are essential. Through chemical complementation; and use of precursors, intracellular compartmentation can be elucidated, as well as the extent to which the plastidial and cytosolic pathways contribute to the syntheses of specific groups of isoprenoids (e.g., sterols) via exchange of intermediates across membranes. These topics are discussed in the context of the pertinent literature.

Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase.

To get some insight into the regulatory mechanisms controlling the sterol branch of the mevalonate pathway, tobacco (Nicotiana tabacum cv Bright Yellow-2) cell suspensions were treated with squalestatin-1 and terbinafine, two specific inhibitors of squalene synthase (SQS) and squalene epoxidase, respectively. These two enzymes catalyze the first two steps involved in sterol biosynthesis. In highly dividing cells, SQS was actively expressed concomitantly with 3-hydroxy-3-methylglutaryl coenzyme A reductase and both sterol methyltransferases. At nanomolar concentrations, squalestatin was found to inhibit efficiently sterol biosynthesis as attested by the rapid decrease in SQS activity and [(14)C]radioactivity from acetate incorporated into sterols. A parallel dose-dependent accumulation of farnesol, the dephosphorylated form of the SQS substrate, was observed without affecting farnesyl diphosphate synthase steady-state mRNA levels. Treatment of tobacco cells with terbinafine is also shown to inhibit sterol synthesis. In addition, this inhibitor induced an impressive accumulation of squalene and a dose-dependent stimulation of the triacylglycerol content and synthesis, suggesting the occurrence of regulatory relationships between sterol and triacylglycerol biosynthetic pathways. We demonstrate that squalene was stored in cytosolic lipid particles, but could be redirected toward sterol synthesis if required. Inhibition of either SQS or squalene epoxidase was found to trigger a severalfold increase in enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, giving first evidence for a positive feedback regulation of this key enzyme in response to a selective depletion of endogenous sterols. At the same time, no compensatory responses mediated by SQS were observed, in sharp contrast to the situation in mammalian cells.