HER-2͞neu (erbB-2) encodes an 185-kDa orphan receptor tyrosine kinase that is constitutively active as a dimer and displays potent oncogenic activity when overexpressed. Here we describe a secreted protein of Ϸ68 kDa, designated herstatin, as the product of an alternative HER-2 transcript that retains intron 8. This alternative transcript specifies 340 residues identical to subdomains I and II from the extracellular domain of p185HER-2 followed by a unique C-terminal sequence of 79 aa encoded by intron 8. The recombinant product of the alternative transcript specifically binds to HER-2-transfected cells with a K D of Ϸ14 nM and was chemically crosslinked to p185HER-2, whereas the intron encoded sequence alone also binds with high affinity to transfected cells and associates with p185 solubilized from cell extracts. The herstatin mRNA is expressed in normal human fetal kidney and liver, but is at reduced levels relative to p185HER-2 mRNA in carcinoma cells that contain an amplified HER-2 gene. Herstatin appears to be an inhibitor of p185HER-2, because it disrupts dimers, reduces tyrosine phosphorylation of p185, and inhibits the anchorageindependent growth of transformed cells that overexpress HER-2.

Trypanosomatids, the etiologic agents of sleeping sickness, leishmaniasis, and Chagas' disease, compartmentalize glycolysis within glycosomes, metabolic organelles related to peroxisomes. Here, we identify a trypanosome homologue of PEX14, one of the components of the peroxisomal protein import docking complex. We have used double-stranded RNA interference to target the PEX14 transcript for degradation. Glycosomal matrix protein import was compromised, and both glycolytic bloodstream stage parasites and mitochondrially respiring procyclic stage parasites were killed. Thus, unlike peroxisomes, glycosomes are essential organelles. Surprisingly, procyclic forms, which can grow in the absence of glucose, were killed by PEX14 RNA interference only when simple sugars were present. Thus, interference with glycosome protein import makes glucose toxic to trypanosomes.

Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25–26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 proteins, the majority of which lack a signal peptide and are thus thought to be secreted via nonconventional mechanisms. Interestingly, this protein release is accompanied by alterations in parasite morphology including an augmentation in the budding of exovesicles from its surface. Here we show that the exoproteome of L. mexicana upon TS induces cleavage and activation of the host protein tyrosine phosphatases, specifically SHP-1 and PTP1-B, in a murine bone-marrow-derived macrophage cell line. Furthermore, translocation of prominent inflammatory transcription factors, namely NF-κB and AP-1 is altered. The exoproteome also caused inhibition of nitric oxide production, a crucial leishmanicidal function of the macrophage. Overall, our results provide strong evidence that within early moments of interaction with the mammalian host, L. mexicana rapidly releases proteins and exovesicles that modulate signalling and function of the macrophage. These modulations can result in attenuation of the inflammatory response and deactivation of the macrophage aiding the parasite in the establishment of infection.

A knockout strain of Leishmania donovani lacking both ornithine decarboxylase (ODC) alleles has been created by targeted gene replacement. Growth of ⌬odc cells in polyamine-deficient medium resulted in a rapid and profound depletion of cellular putrescine pools, although levels of spermidine were relatively unaffected. Concentrations of trypanothione, a spermidine conjugate, were also reduced, whereas glutathione concentrations were augmented. The ⌬odc L. donovani exhibited an auxotrophy for polyamines that could be circumvented by the addition of the naturally occurring polyamines, putrescine or spermidine, to the culture medium. Whereas putrescine supplementation restored intracellular pools of both putrescine and spermidine, exogenous spermidine was not converted back to putrescine, indicating that spermidine alone is sufficient to meet the polyamine requirement, and that L. donovani does not express the enzymatic machinery for polyamine degradation. The lack of a polyamine catabolic pathway in intact parasites was confirmed radiometrically. In addition, the ⌬odc strain could grow in medium supplemented with either 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), but polyamine auxotrophy could not be overcome by other aliphatic diamines or spermine. These data establish genetically that ODC is an essential gene in L. donovani, define the polyamine requirements of the parasite, and reveal the absence of a polyamine-degradative pathway.Polyamines are cationic compounds that play essential roles in cell proliferation, differentiation, and macromolecular synthesis (1-3). Ornithine decarboxylase (ODC) 1 catalyzes the conversion of ornithine to putrescine (1,4-diaminobutane) and is the initial and rate-limiting enzyme in polyamine biosynthesis in most organisms (4). The ODC enzyme of protozoan parasites is a novel therapeutic target, because D,L-␣-difluoromethylornithine (DFMO; eflornithine), an irreversible inhibitor of ODC (5), exhibits notable efficacy against the central nervous system phase of African sleeping sickness caused by Trypanosoma brucei gambiense (3, 6). DFMO is also active against T. b. rhodesiense and T. congolense in murine models and has proven effective against other genera of protozoan parasites in vivo and in vitro, including Plasmodia (7), Giardia (8), and Leishmania (9). DFMO has been shown to induce a lethal polyamine depletion in both T. brucei (10) and L. donovani (9), the etiologic agent of visceral leishmaniasis, and toxicity to both species is ameliorated by polyamine addition (3, 9).The ability of trypanosomatids to undergo a very high frequency of homologous recombination allows the disruption of chromosomal loci with transfected drug resistance cassettes (11,12) and permits a direct test of gene function. This enables the creation of conditionally lethal parasite strains whose survival and ability to propagate are dependent upon the provision of compounds that can ameliorate the consequences of the genetic lesion. This genetic approach is predicated on the availability of c...

Exosomes are small vesicles of endocytic origin, which are released into the extracellular environment and mediate a variety of physiological and pathological conditions. Here we show that Schistosoma mansoni releases exosome-like vesicles in vitro. Vesicles were purified from culture medium by sucrose gradient fractionation and fractions containing vesicles verified by western blot analyses and electron microscopy. Proteomic analyses of exosomal contents unveiled 130 schistosome proteins. Among these proteins are common exosomal markers such as heat shock proteins, energy-generating enzymes, cytoskeletal proteins, and others. In addition, the schistosome extracellular vesicles contain proteins of potential importance for host-parasite interaction, notably peptidases, signaling proteins, cell adhesion proteins (e.g., integrins) and previously described vaccine candidates, including glutathione-S-transferase (GST), tetraspanin (TSP-2) and calpain. S. mansoni exosomes also contain 143 microRNAs (miRNA), of which 25 are present at high levels, including miRNAs detected in sera of infected hosts. Quantitative PCR analysis confirmed the presence of schistosome-derived miRNAs in exosomes purified from infected mouse sera. The results provide evidence of vesicle-mediated secretion in these parasites and suggest that schistosome-derived exosomes could play important roles in host-parasite interactions and could be a useful tool in the development of vaccines and therapeutics.

Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade.

Purine nucleotides function in a variety of vital cellular and metabolic processes including energy production, cell signaling, synthesis of vitamin-derived cofactors and nucleic acids, and as determinants of cell fate. Unlike their mammalian and insect hosts, Leishmania cannot synthesize the purine ring de novo and are absolutely dependent upon them to meet their purine requirements. The obligatory nature of purine salvage in these parasites, therefore, offers an attractive paradigm for drug targeting, and consequently, the delineation of the pathway has been under scientific investigation for over 30 years. Here we review recent developments that reveal how purines flux in Leishmania and offer a potential ‘Achilles’ heel’ for future validation.

A novel membrane molecule, previously observed to be co-isolated with lipophosphoglycan and called lipophosphoglycan-associated protein, has been detected in Leishmania donovani promastigotes and amastigotes. This kinetoplastid membrane protein (KMP-11) has been purified by preparative SDS/PAGE after organic solvent extraction of promastigote membranes. Isoelectric-focusing experiments indicated that this was an acidic protein with an isoelectric point of 4.8. Immunoblot analysis of subcellular fractions, together with 125I-labelling experiments, showed this molecule to be associated with the promastigote cell surface membrane. KMP-11 was expressed at a copy number similar to that of lipophosphoglycan (1 x 10(6)-2 x 10(6) molecules per cell), making this glycoprotein one of the major features on the parasite cell surface. The primary structure, less a blocked N-terminal region, was determined by automated Edman degradation of peptides derived from CNBr or enzymic fragmentation. Several post-translational modifications were also found during these studies, including an O-linked oligosaccharide and an NG-monomethylarginine functionality which was verified by m.s. Finally, a set of sequential synthetic peptides was made based on the established partial sequence allowing structural determination of two distinct antibody-binding sites for the monoclonal antibodies L98 and L157.