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Autophagy is a ubiquitous eukaryotic process that also occurs in trypanosomatid parasites, protist organisms belonging to the supergroup Excavata, distinct from the supergroup Opistokontha that includes mammals and fungi. Half of the known yeast and mammalian AuTophaGy (ATG) proteins were detected in trypanosomatids, although with low sequence conservation. Trypanosomatids such as Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for serious tropical diseases in humans. The parasites are transmitted by insects and, consequently, have a complicated life cycle during which they undergo dramatic morphological and metabolic transformations to adapt to the different environments. Autophagy plays a major role during these transformations. Since inhibition of autophagy affects the transformation, survival and/or virulence of the parasites, the ATGs offer promise for development of drugs against tropical diseases. Furthermore, various trypanocidal drugs have been shown to trigger autophagy-like processes in the parasites. It is inferred that autophagy is used by the parasites in an—not always successful—attempt to cope with the stress caused by the toxic compounds.

Glycosomes are peroxisome-like organelles essential for trypanosomatid parasites. Glycosome biogenesis is mediated by proteins called "peroxins," which are considered to be promising drug targets in pathogenic Trypanosomatidae. The first step during protein translocation across the glycosomal membrane of peroxisomal targeting signal 1 (PTS1)-harboring proteins is signal recognition by the cytosolic receptor peroxin 5 (PEX5). The C-terminal PTS1 motifs interact with the PTS1 binding domain (P1BD) of PEX5, which is made up of seven tetratricopeptide repeats. Obtaining diffraction-quality crystals of the P1BD of Trypanosoma brucei PEX5 (TbPEX5) required surface entropy reduction mutagenesis. Each of the seven tetratricopeptide repeats appears to have a residue in the alpha(L) conformation in the loop connecting helices A and B. Five crystal structures of the P1BD of TbPEX5 were determined, each in complex with a hepta- or decapeptide corresponding to a natural or nonnatural PTS1 sequence. The PTS1 peptides are bound between the two subdomains of the P1BD. These structures indicate precise recognition of the C-terminal Leu of the PTS1 motif and important interactions between the PTS1 peptide main chain and up to five invariant Asn side chains of PEX5. The TbPEX5 structures reported here reveal a unique hydrophobic pocket in the subdomain interface that might be explored to obtain compounds that prevent relative motions of the subdomains and interfere selectively with PTS1 motif binding or release in trypanosomatids, and would therefore disrupt glycosome biogenesis and prevent parasite growth.

In this paper, we report the subcellular distribution of phosphoglycerate kinase (PGK) in epimastigotes of Trypanosoma cruzi. Approximately 80% of the PGK activity was found in the cytosol, 20% in the glycosomes. Western blot analysis suggested that two isoenzymes of 56 and 48 kDa, respectively, are responsible for the glycosomal PGK activity, whereas the cytosolic activity should be attributed to a single PGK of 48 kDa. In analogy to the situation previously reported for PGK in Trypanosoma brucei, these isoenzymes were called PGKA, C and B, respectively. However, in T. cruzi, PGKA seems not to be a minor enzyme like its counterpart in T. brucei. Whereas PGKC behaved as a soluble glycosomal matrix protein, PGKA appeared to be present at the inner surface of the organelle's membrane. After alkaline carbonate treatment, the enzyme remained associated with the particulate fraction of the organelles. Upon solubilization of glycosomes with Triton X-114, PGKA was recovered from the detergent phase, indicating its (partial) hydrophobic character and therefore, a possible hydrophobic interaction with the membrane. The PGKA gene was cloned and sequenced, but the predicted amino-acid sequence did not reveal an obvious clue as to the mechanism by which the enzyme is attached to the glycosomal membrane, (C) 2001 Elsevier Science B.V. All rights reserved.

Trypanosoma brucei contains an ATP-dependent phosphofructokinase (PFK), located in its glycosomes, which are peroxisome-like organelles sequestering the majority of its glycolytic enzymes. In this paper, we report the cloning and sequencing of the single-copy gene encoding this enzyme. Its amino-acid sequence is more similar to pyrophosphate (PPi)-dependent PFKs than to other ATP-dependent PFKs. A phylogenetic analysis suggests that the enzyme must have been derived from a PPi-dependent ancestral PFK, which changed its phospho-donor specificity during evolution. The enzyme is no longer capable of using PPi as phospho substrate, nor can it catalyze the reverse reaction as PPi-PFKs generally can. Moreover, the presence of a high pyrophosphatase activity in the cell renders it unlikely that PPi can function as free-energy source in present-day trypanosomes. It remains to be determined which mutations were responsible for the change in phospho-substrate specificity of the trypanosomatid PFK. As a result of its particular evolutionary history, the T. brucei PFK shows many structural differences, even at the active site, when compared with other ATP-dependent PFKs. These differences offer great potential for the structure-based design of trypanocidal drugs.

Biosynthetic studies using both [14C]- and [32P]-labelled substrates and a cell-free system to synthesise 1-O-alkyl moieties in glycerolipids, have shown that the three initial steps in ether-lipid biosynthesis in Leishmania mexicana promastigotes resemble those described for mammals and are associated with glycosomes. Purified glycosomes were able to sequentially synthesise the first intermediates of the ether-lipid biosynthetic pathway [acyl-dihydroxyacetonephosphate (DHAP), alkyl-DHAP and acyl/alkyl-glycerol-3-phosphate (G3P)] when incubated in the presence of radiolabelled DHAP, palmitoyl-CoA, hexadecanol and NADPH. However, when glycosomes were incubated under the same conditions in the presence of radiolabelled G3P, a rapid synthesis of acyl-G3P and phosphatidic acid was observed without any formation of alkyl-G3P, suggesting that the enzyme alkyl-synthase recognises only acyl-DHAP as substrate. Both the DHAP acyltransferase (DHAP-AT) and alkyl-DHAP synthase activities were located inside glycosomes whereas the alkyl/acyl-DHAP oxidoreductase activity was associated with the cytoplasmic face of the glycosomal membrane. The G3P acyltransferase (G3P-AT) and lyso-phosphatidic acid acyltransferase activities were not found inside glycosomes. The results suggest that the DHAP-AT and G3P-AT activities are catalysed by two distinct enzymes associated with different sub-cellular compartments.