Direct Link between RACK1 Function and Localization at the Ribosome In Vivo

The receptor for activated C-kinase (RACK1), a conserved protein implicated in numerous signaling pathways, is a stoichiometric component of eukaryotic ribosomes located on the head of the 40S ribosomal subunit. To test the hypothesis that ribosome association is central to the function of RACK1 in vivo, we determined the 2.1-Å crystal structure of RACK1 from Saccharomyces cerevisiae (Asc1p) and used it to design eight mutant versions of RACK1 to assess roles in ribosome binding and in vivo function. Conserved charged amino acids on one side of the β-propeller structure were found to confer most of the 40S subunit binding affinity, whereas an adjacent conserved and structured loop had little effect on RACK1-ribosome association. Yeast mutations that confer moderate to strong defects in ribosome binding mimic some phenotypes of a RACK1 deletion strain, including increased sensitivity to drugs affecting cell wall biosynthesis and translation elongation. Furthermore, disruption of RACK1''s position at the 40S ribosomal subunit results in the failure of the mRNA binding protein Scp160 to associate with actively translating ribosomes. These results provide the first direct evidence that RACK1 functions from the ribosome, implying a physical link between the eukaryotic ribosome and cell signaling pathways in vivo.Cells alter protein synthesis in response to stimuli whose effects are transmitted through established cell signaling pathways. Although the mechanisms of signal transduction to ribosomes remain unclear, the receptor for activated C-kinase (RACK1) has emerged as a possible molecular link that connects the signaling and translation machinery. RACK1, a highly conserved homologue of the β-subunit of heterotrimeric G proteins, was first identified over a decade ago as an anchoring protein for protein kinase C (33). Implicated as a scaffold in PDE4D5- and Src kinase-based signaling pathways (28), it functions in diverse developmental processes, such as sexual differentiation in Schizosaccharomyces pombe (29) and the control of cell proliferation in Drosophila melanogaster (26). The more recent discovery that RACK1 is a core component of the eukaryotic 40S ribosomal subunit (20, 24, 32) suggested that its signaling functions might directly influence the efficiency and specificity of translation.In support of this possibility, cryo-electron microscopy (cryo-EM) studies showed that RACK1 binds the 40S subunit near the mRNA exit tunnel in a location that is conserved from yeast to humans (35). The cryo-EM data verified RACK1''s architecture as a seven-bladed β-propeller and positioned the protein on the ribosome in such a way that much of its surface is exposed and available for interaction with other proteins and ligands. These structural data are consistent with the hypothesis that RACK1 might assemble signaling or other regulatory complexes directly on the ribosome (31). Indeed, various functions for RACK1 at the ribosome have been proposed, including roles in 40S and 60S subunit joining (8), the regulated translation of specific mRNAs (6, 36), and the localization of ribosomes for translation at specific sites within the cell (9, 10). Despite this abundance of hypothetical roles, the functional significance of RACK1 localization on the ribosome remains speculative.Here, we provide the first experimental evidence that RACK1''s position at the ribosome has biological importance in vivo. We determined the crystal structure of the full-length Saccharomyces cerevisiae RACK1 ortholog, Asc1p (henceforth RACK1), at 2.1-Å resolution. Using this structure and the cryo-EM model of the protein on the 40S ribosomal subunit, we analyzed the putative RACK1-40S subunit interface and generated eight RACK1 variants that have differing effects on ribosome binding in vivo. We show that yeast strains harboring even the most severely binding-defective RACK1 mutant fail to exhibit all of the phenotypes associated with RACK1 deletion. However, the efficiency of RACK1 binding to ribosomes correlates with a subset of growth behaviors observed for RACK1 deletion strains. These results indicate that although not required for all RACK1 activities, localization at ribosomes is integral to some aspects of RACK1 function.     

Reduced ribosomes of the apicoplast and mitochondrion of Plasmodium spp. and predicted interactions with antibiotics

Apicomplexan protists such as Plasmodium and Toxoplasma contain a mitochondrion and a relic plastid (apicoplast) that are sites of protein translation. Although there is emerging interest in the partitioning and function of translation factors that participate in apicoplast and mitochondrial peptide synthesis, the composition of organellar ribosomes remains to be elucidated. We carried out an analysis of the complement of core ribosomal protein subunits that are encoded by either the parasite organellar or nuclear genomes, accompanied by a survey of ribosome assembly factors for the apicoplast and mitochondrion. A cross-species comparison with other apicomplexan, algal and diatom species revealed compositional differences in apicomplexan organelle ribosomes and identified considerable reduction and divergence with ribosomes of bacteria or characterized organelle ribosomes from other organisms. We assembled structural models of sections of Plasmodium falciparum organellar ribosomes and predicted interactions with translation inhibitory antibiotics. Differences in predicted drug–ribosome interactions with some of the modelled structures suggested specificity of inhibition between the apicoplast and mitochondrion. Our results indicate that Plasmodium and Toxoplasma organellar ribosomes have a unique composition, resulting from the loss of several large and small subunit proteins accompanied by significant sequence and size divergences in parasite orthologues of ribosomal proteins.     

Use of magnetically purified Plasmodium falciparum parasites improves the accuracy of erythrocyte invasion assays

Merozoite invasion of erythrocytes is a crucial step for the asexual cycle of Plasmodium falciparum. Multiple invasion pathways, which involve different ligand-receptor interactions, have been identified in P. falciparum by examining the entry of purified parasite into erythrocytes with different surface receptors, either mutant or under different enzyme treatments. The most critical step for a successful invasion assay is the isolation of erythrocytes infected with viable schizonts. Here, we applied a magnetic column to purify the schizonts for the erythrocyte invasion assay. Comparing to Percoll-sorbitol purification method, this modified approach showed great improvement on reproducibility and reliability of invasion assay, particularly for short-term, culture-adapted parasite isolates. The magnetic purification method is an excellent alternative for parasite isolates that do not tolerate or with unknown sensitivity to Percoll-sorbitol exposure.     

Acquisition of growth-inhibitory antibodies against blood-stage Plasmodium falciparum

Background

Antibodies that inhibit the growth of blood-stage Plasmodium falciparum may play an important role in acquired and vaccine-induced immunity in humans. However, the acquisition and activity of these antibodies is not well understood.

Methods

We tested dialysed serum and purified immunoglobulins from Kenyan children and adults for inhibition of P. falciparum blood-stage growth in vitro using different parasite lines. Serum antibodies were measured by ELISA to blood-stage parasite antigens, extracted from P. falciparum schizonts, and to recombinant merozoite surface protein 1 (42 kDa C-terminal fragment, MSP1-42).

Results

Antibodies to blood-stage antigens present in schizont protein extract and to recombinant MSP1-42 significantly increased with age and were highly correlated. In contrast, growth-inhibitory activity was not strongly associated with age and tended to decline marginally with increasing age and exposure, with young children demonstrating the highest inhibitory activity. Comparison of growth-inhibitory activity among samples collected from the same population at different time points suggested that malaria transmission intensity influenced the level of growth-inhibitory antibodies. Antibodies to recombinant MSP1-42 were not associated with growth inhibition and high immunoglobulin G levels were poorly predictive of inhibitory activity. The level of inhibitory activity against different isolates varied.

Conclusions

Children can acquire growth-inhibitory antibodies at a young age, but once they are acquired they do not appear to be boosted by on-going exposure. Inhibitory antibodies may play a role in protection from early childhood malaria.     

An interplay between 2 signaling pathways: Melatonin-cAMP and IP3–Ca signaling pathways control intraerythrocytic development of the malaria parasite Plasmodium falciparum

Plasmodium falciparum spends most of its asexual life cycle within human erythrocytes, where proliferation and maturation occur. Development into the mature forms of P. falciparum causes severe symptoms due to its distinctive sequestration capability. However, the physiological roles and the molecular mechanisms of signaling pathways that govern development are poorly understood. Our previous study showed that P. falciparum exhibits stage-specific spontaneous Calcium (Ca²⁺) oscillations in ring and early trophozoites, and the latter was essential for parasite development. In this study, we show that luzindole (LZ), a selective melatonin receptor antagonist, inhibits parasite growth. Analyses of development and morphology of LZ-treated P. falciparum revealed that LZ severely disrupted intraerythrocytic maturation, resulting in parasite death. When LZ was added at ring stage, the parasite could not undergo further development, whereas LZ added at the trophozoite stage inhibited development from early into late schizonts. Live-cell Ca²⁺ imaging showed that LZ treatment completely abolished Ca²⁺ oscillation in the ring forms while having little effect on early trophozoites. Further, the melatonin-induced cAMP increase observed at ring and late trophozoite stage was attenuated by LZ treatment. These suggest that a complex interplay between IP₃–Ca²⁺ and cAMP signaling pathways is involved in intraerythrocytic development of P. falciparum.     

Plasmodium falciparum: loss of knobs on the infected erythrocyte surface after long-term cultivation.

After long-term in vitro cultivation in human erythrocytes, variants of three strains of the malaria parasite Plasmodium falciparum no longer produce the “knob” alterations on the host erythrocyte surface. The time in continuous culture before knobs failed to appear ranged from 18 months for the Gambian strain FCR-4 to 33 months for the Vietnamese strain FCR-1. The loss of knobs is correlated with the inability to concentrate trophozoites, schizonts, and segmenters from these variant lines by the use of gelatin-containing media. This is the first report of a change in Plasmodium falciparum or its host cell as a consequence of long-term culture.     

Effects of novel triple-stage antimalarial ionic liquids on lipid membrane models

Primaquine-based ionic liquids, obtained by acid-base reaction between parent primaquine and cinnamic acids, were recently found as triple-stage antimalarial hits. These ionic compounds displayed significant activity against both liver- and blood-stage Plasmodium parasites, as well as against stage V P. falciparum parasites. Remarkably, blood-stage activity of the ionic liquids against both chloroquine-sensitive (3D7) and resistant (Dd2) P. falciparum strains was clearly superior to those of the respective covalent (amide) analogues and of parent primaquine. Having hypothesized that such behaviour might be ascribed to an enhanced ability of the ionic compounds to permeate into Plasmodium-infected erythrocytes, we have carried out a differential scanning calorimetry-based study of the interactions between the ionic liquids and membrane models. Results provide evidence, at the molecular level, that the primaquine-derived ionic liquids may contribute to an increased permeation of the parent drug into malaria-infected erythrocytes, which has relevant implications towards novel antimalarial approaches based on ionic liquids.     

Plasmodium falciparum Dynein Light Chain 1 Interacts with Actin/Myosin during Blood Stage Development

Dynein light chain 1 (LC1), a member of the leucine-rich repeat protein family, has been shown to be engaged in controlling flagellar motility in Chlamydomonas reinhardtii and Trypanosoma brucei via its interaction with the dynein γ heavy chain. In Plasmodium falciparum, we have identified the LC1 ortholog, designated Pfdlc1. Negative attempts to disrupt the dlc1 gene by reverse genetic approaches in both P. falciparum and P. berghei suggest either its essentiality for parasite survival or the inaccessibility of its locus. Expression studies revealed high levels of DLC1 protein in late trophozoites and schizonts, pointing to an unexpected role of this protein in blood-stage parasites as they do not have flagella. Interactions studies and co-immunoprecipitation experiments revealed that PfDLC1 was able to bind to P. falciparum myosin A and actin 1. The PfDLC1 interacting domains present in P. falciparum myosin A and actin 1 were mapped to sequences containing SDIE and/or EEMKT motifs present in the upper 50-kDa segment of the myosin A head domain and in the subdomain IV of actin 1, respectively. Detection of PfDLC1 by fluorescence tagging and immunofluorescence staining using specific antibodies showed a cytoplasmic location similar to actin and immunofluorescence studies showed a co-localization of PfDLC1 and myosin A. Taken together, these findings suggest that PfDLC1 might play an important role in P. falciparum erythrocytic stages by its interaction with myosin A and actin 1, known to be essential for parasite development.     

Identification of the versatile scaffold protein RACK1 on the eukaryotic ribosome by cryo-EM

RACK1 serves as a scaffold protein for a wide range of kinases and membrane-bound receptors. It is a WD-repeat family protein and is predicted to have a beta-propeller architecture with seven blades like a Gbeta protein. Mass spectrometry studies have identified its association with the small subunit of eukaryotic ribosomes and, most recently, it has been shown to regulate initiation by recruiting protein kinase C to the 40S subunit. Here we present the results of a cryo-EM study of the 80S ribosome that positively locate RACK1 on the head region of the 40S subunit, in the immediate vicinity of the mRNA exit channel. One face of RACK1 exposes the WD-repeats as a platform for interactions with kinases and receptors. Using this platform, RACK1 can recruit other proteins to the ribosome.     

An assay of malaria parasite invasion into human erythrocytes: The effects of chemical and enzymatic modification of erythrocyte membrane components

Invasion of erythrocytes by malaria parasites is known to be blocked by proteolytic digestion of merozoite receptors allegedly present in red cell membranes. This information was used in the present work to develop a simple and convenient assay for parasite invasion into red blood cells and for evaluating the role played by red cell membrane components in this process. Synchronized in vitro cultures of Plasmodium falciparum containing only ring stages were subjected to either trypsin or pronase digestion, a treatment that neither affected ring development into schizonts nor mature merozoite release. Cells from this culture were not invaded by the released merozoites. However, upon addition of untreated human red blood cells, marked invasion was observed, either microscopically or as [³H]isoleucine incorporation. The new assay circumvents the need for separating schizonts from uninfected cells and provides a convenient means for assessing how chemical and biochemical manipulation of red blood cells affects their invasiveness by parasites. Using this assay, we verified that sheep and rabbit erythrocytes were resistant to invasion, as were human erythrocytes which had been treated with trypsin, pronase or neuraminidase. Chymotrypsin digestion of human erythrocytes was without effect on invasion. Human erythrocytes which were chemically modified with the impermeant amino reactive reagent H₂DIDS, or with the crosslinker of spectrin, TCEA, were found to resist invasion. The results underscore the involvement of surface membrane components as well as of elements of the cytoskeleton in the process of parasite invasion into erythrocytes.     

Separation and Concentration of Schizonts of Plasmodium falciparum by Percoll Gradients1

A technique for the separation of schizonts of Plasmodium falciparum is described. The different stages of the asexual cell cycle of the parasite were positioned according to their density in a continuous gradient of Percoll. Young trophozoites coincided with erythrocytes in a broad band corresponding to densities from 1.075 to 1.100 g/ml, whereas schizonts were concentrated at a density approximating 1.062 g/ml. The viability of the parasites was unimpaired by this procedure. Young trophozoites and schizonts continued their normal life cycle when cultured after the separation procedure. The percentage of recovery was high, reaching 80% of the initial quantity. Possible applications of the technique are discussed.     

Absence of Erythrocyte Sequestration and Lack of Multicopy Gene Family Expression in Plasmodium falciparum from a Splenectomized Malaria Patient

Background

To avoid spleen-dependent killing mechanisms parasite-infected erythrocytes (IE) of Plasmodium falciparum malaria patients have the capacity to bind to endothelial receptors. This binding also known as sequestration, is mediated by parasite proteins, which are targeted to the erythrocyte surface. Candidate proteins are those encoded by P. falciparum multicopy gene families, such as var, rif, stevor or PfMC-2TM. However, a direct in vivo proof of IE sequestration and expression of multicopy gene families is still lacking. Here, we report on the analysis of IE from a black African immigrant, who received the diagnosis of a malignant lymphoproliferative disorder and subsequently underwent splenectomy. Three weeks after surgery, the patient experienced clinical falciparum malaria with high parasitemia and circulating developmental parasite stages usually sequestered to the vascular endothelium such as late trophozoites, schizonts or immature gametocytes.

Methodology/Principal Findings

Initially, when isolated from the patient, the infected erythrocytes were incapable to bind to various endothelial receptors in vitro. Moreover, the parasites failed to express the multicopy gene families var, A-type rif and stevor but expression of B-type rif and PfMC-2TM genes were detected. In the course of in vitro cultivation, the parasites started to express all investigated multicopy gene families and concomitantly developed the ability to adhere to endothelial receptors such as CD36 and ICAM-1, respectively.

Conclusion/Significance

This case strongly supports the hypothesis that parasite surface proteins such as PfEMP1, A-type RIFIN or STEVOR are involved in interactions of infected erythrocytes with endothelial receptors mediating sequestration of mature asexual and immature sexual stages of P. falciparum. In contrast, multicopy gene families coding for B-type RIFIN and PfMC-2TM proteins may not be involved in sequestration, as these genes were transcribed in infected but not sequestered erythrocytes.     

Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing

Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3′-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation.     

Antimalarial drug prescribing practice in private and public health facilities in South-east Nigeria: a descriptive study

The development of acquired protective immunity to Plasmodium falciparum infection in young African children is considered in the context of three current strategies for malaria prevention: insecticide-impregnated bed nets or curtains, anti-sporozoite vaccines and intermittent preventive therapy. Evidence is presented that each of these measures may permit attenuated P. falciparum blood-stage infections, which do not cause clinical malaria but can act as an effective blood-stage "vaccine". It is proposed that the extended serum half-life, and rarely considered liver-stage prophylaxis provided by the anti-folate combination sulphadoxine-pyrimethamine frequently lead to such attenuated infections in high transmission areas, and thus contribute to the sustained protection from malaria observed among children receiving the combination as intermittent preventative therapy or for parasite clearance in vaccine trials.     

Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements

Our understanding of the mechanism of protein synthesis has undergone rapid progress in recent years as a result of low-resolution X-ray and cryo-EM structures of ribosome functional complexes and high-resolution structures of ribosomal subunits and vacant ribosomes. Here, we present the crystal structure of the Thermus thermophilus 70S ribosome containing a model mRNA and two tRNAs at 3.7 A resolution. Many structural details of the interactions between the ribosome, tRNA, and mRNA in the P and E sites and the ways in which tRNA structure is distorted by its interactions with the ribosome are seen. Differences between the conformations of vacant and tRNA-bound 70S ribosomes suggest an induced fit of the ribosome structure in response to tRNA binding, including significant changes in the peptidyl-transferase catalytic site.     

The plasmodium receptor for activated C kinase protein inhibits Ca signaling in mammalian cells

Plasmodium falciparum, the most lethal malarial parasite, expresses an ortholog for the protein kinase C (PKC) activator RACK1. However, PKC has not been identified in this parasite, and the mammalian RACK1 can interact with the inositol 1,4,5-trisphosphate receptor (InsP3R). Therefore we investigated whether the Plasmodium ortholog PfRACK also can affect InsP3R-mediated Ca²⁺ signaling in mammalian cells. GFP-tagged PfRACK and endogenous RACK1 were expressed in a similar distribution within cells. PfRACK inhibited agonist-induced Ca²⁺ signals in cells expressing each isoform of the InsP3R, and this effect persisted when expression of endogenous RACK1 was reduced by siRNA. PfRACK also inhibited Ca²⁺ signals induced by photorelease of caged InsP3. These findings provide evidence that PfRACK directly inhibits InsP3-mediated Ca²⁺ signaling in mammalian cells. Interference with host cell signaling pathways to subvert the host intracellular milieu may be an important mechanism for parasite survival.     

Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

The prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodium parasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novo via the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum, the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparum by assessing the functional consequences of deleting fabI and fabB/F. Targeted deletion or disruption of these genes in P. falciparum did not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodium parasites P. berghei and P. yoelii, where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anopheles mosquito midgut.     

Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2

BackgroundIn falciparum malaria sequestration of erythrocytes containing mature forms of Plasmodium falciparum in the microvasculature of vital organs is central to pathology, but quantitation of this hidden sequestered parasite load in vivo has not previously been possible. The peripheral blood parasite count measures only the circulating, relatively non-pathogenic parasite numbers. P. falciparum releases a specific histidine-rich protein (PfHRP2) into plasma. Quantitative measurement of plasma PfHRP2 concentrations may reflect the total parasite biomass in falciparum malaria.ConclusionPlasma PfHRP2 concentrations may be used to estimate the total body parasite biomass in acute falciparum malaria. Severe malaria results from extensive sequestration of parasitised erythrocytes.