Conversion of the cellular prion protein (PrP C ) into its altered conformation, PrPSc , is believed to be the major cause of prion diseases. Although PrP is the only identified agent for these diseases, there is increasing evidence that other molecules can modulate the conversion. We have found that interaction of PrP with double-stranded DNA leads to a protein with higher ␤-sheet content and characteristics similar to those of PrP Sc . RNA molecules can also interact with PrP and potentially modulate PrP C to PrP Sc conversion or even bind differentially to both PrP isoforms. Here, we investigated the interaction of recombinant murine PrP with synthetic RNA sequences and with total RNA extracted from cultured neuroblastoma cells (N2aRNA). We found that PrP interacts with N2aRNA with nanomolar affinity, aggregates upon this interaction, and forms species partially resistant to proteolysis. RNA does not bind to N-terminal deletion mutants of PrP, indicating that the N-terminal region is important for this process. Cell viability assays showed that only the N2aRNA extract induces PrP-RNA aggregates that can alter the homeostasis of cultured cells. Small RNAs bound to PrP give rise to nontoxic small oligomers. Nuclear magnetic resonance measurements of the PrP-RNA complex revealed structural changes in PrP, but most of its native fold is maintained. These results indicate that there is selectivity in the species generated by interaction with different molecules of RNA. The catalytic effect of RNA on the PrP C 3 PrP Sc conversion depends on the RNA sequence, and small RNA molecules may exert a protective effect.Prion diseases can be infectious, sporadic, or inherited (1). Regardless of their origin, they are related to modifications of a ubiquitous membrane-anchored protein, the prion protein (PrP).3 Through a poorly understood process, the cellular PrP isoform (PrP C ), an ␣-helix-rich protein, undergoes a profound conformational change, acquiring higher ␤-sheet content; the latter isoform is known as PrP Sc (Sc from scrapie) and is the only known component of the infectious prion particle (1-4).The protein-only hypothesis postulates that PrP Sc "multiplies" by catalyzing the conversion of PrP C into a likeness of itself, thus becoming responsible for its own propagation (5). This hypothesis is based strongly on the fact that PrP knock-out mice are resistant to prion infection, suggesting that endogenous PrP is necessary for prion propagation and infection (6). It was also suggested, however, that an additional unknown factor could influence the PrP C to PrP Sc conversion (7-10). This molecule would act by lowering the free energy barrier between PrP C and PrP Sc and triggering conversion (11,12). In this field, a great number of biological macromolecules have emerged as candidates for conversion catalysts. Cellular adhesion molecules, nucleic acids (NAs), basal membrane molecules, and sulfated glycans, among other biological macromolecules, have been reported to interact with PrP C and to induce its conversion in...

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Chagasin is a Trypanosoma cruzi protein that was recently characterized as a tight-binding inhibitor of papain-like cysteine proteases (CPs). Considering that parasite virulence and morphogenesis depend on the endogenous activity of lysosomal CPs of the cruzipain family, we sought to determine whether chagasin and cruzipain interact in the living cell. Ultrastructural studies showed that chagasin and cruzipain both localize to the Golgi complex and reservosomes (lysosome-like organelles), whereas free chagasin was found in small intracellular vesicles, suggesting that chagasin trafficking pathways might intersect with those of cruzipain. Taking advantage of the fact that sodium dodecyl sulphate and β-mercaptoethanol prevent binding between the isolated proteins but do not dismantle preformed cruzipain-chagasin complexes, we obtained direct evidence that chagasin-cruzipain complexes are indeed formed in epimastigotes. Chagasin transfectants (fourfold increase in CP inhibitory activity) displayed low rates of differentiation (metacyclogenesis) and exhibited increased resistance to a synthetic CP inhibitor. These phenotypic changes were accompanied by a drastic reduction of soluble cruzipain activity and by upregulated secretion of cruzipain-chagasin molecular complexes. Analysis of six T. cruzi strains revealed that expression levels of cruzipain and chagasin are variable, but the molar ratios are fairly stable (∼50:1) in most strains, with the exception of the G strain (5:1), which is poorly infective. On the same vein, we found that trypomastigotes overexpressing chagasin are less infective than wild-type parasites in vitro. The deficiency of chagasin overexpressers is caused by lower activity of membrane-associated CPs, because membranes recovered from wild-type trypomastigotes restored infectivity and this effect was nullified by the CP inhibitor E-64. In summary, our studies suggest that chagasin regulates the endogenous activity of CP, thus indirectly modulating proteolytic functions that are essential for parasite differentiation and invasion of mammalian cells.

Trypanosoma cruzi, the agent of Chagas' disease, alternates between different morphogenetic stages that face distinct physiological conditions in their invertebrate and vertebrate hosts, likely in the availability of glucose. While the glucose transport is well characterized in epimastigotes of T. cruzi, nothing is known about how the mammalian stages acquire this molecule. Herein glucose transport activity and expression were analyzed in the three developmental stages present in the vertebrate cycle of T. cruzi. The infective trypomastigotes showed the highest transport activity (V(max)=5.34+/-0.54 nmol/min per mg of protein; K(m)=0.38+/-0.01 mM) when compared to intracellular epimastigotes (V(max)=2.18+/-0.20 nmol/min per mg of protein; K(m)=0.39+/-0.01 mM). Under the conditions employed no transport activity could be detected in amastigotes. The gene of the glucose transporter is expressed at the mRNA level in trypomastigotes and in intracellular epimastigotes but not in amastigotes, as revealed by real-time PCR. In both trypomastigotes and intracellular epimastigotes protein expression could be detected by Western blot with an antibody raised against the glucose transporter correlating well with the transport activity measured experimentally. Interestingly, anti-glucose transporter antibodies showed a strong reactivity with glycosome and reservosome organelles. A comparison between proline and glucose transport among the intracellular differentiation forms is presented. The data suggest that the regulation of glucose transporter reflects different energy and carbon requirements along the intracellular life cycle of T. cruzi.