The translation initiation complex eIF3 in trypanosomatids and other pathogenic excavates – identification of conserved and divergent features based on orthologue analysis

Background

The initiation of translation in eukaryotes is supported by the action of several eukaryotic Initiation Factors (eIFs). The largest of these is eIF3, comprising of up to thirteen polypeptides (eIF3a through eIF3m), involved in multiple stages of the initiation process. eIF3 has been better characterized from model organisms, but is poorly known from more diverged groups, including unicellular lineages represented by known human pathogens. These include the trypanosomatids (Trypanosoma and Leishmania) and other protists belonging to the taxonomic supergroup Excavata (Trichomonas and Giardia sp.).

Results

An in depth bioinformatic search was carried out to recover the full content of eIF3 subunits from the available genomes of L. major, T. brucei, T. vaginalis and G. duodenalis. The protein sequences recovered were then submitted to homology analysis and alignments comparing them with orthologues from representative eukaryotes. Eleven putative eIF3 subunits were found from both trypanosomatids whilst only five and four subunits were identified from T. vaginalis and G. duodenalis, respectively. Only three subunits were found in all eukaryotes investigated, eIF3b, eIF3c and eIF3i. The single subunit found to have a related Archaean homologue was eIF3i, the most conserved of the eIF3 subunits. The sequence alignments revealed several strongly conserved residues/region within various eIF3 subunits of possible functional relevance. Subsequent biochemical characterization of the Leishmania eIF3 complex validated the bioinformatic search and yielded a twelfth eIF3 subunit in trypanosomatids, eIF3f (the single unidentified subunit in trypanosomatids was then eIF3m). The biochemical data indicates a lack of association of the eIF3j subunit to the complex whilst highlighting the strong interaction between eIF3 and eIF1.

Conclusions

The presence of most eIF3 subunits in trypanosomatids is consistent with an early evolution of a fully functional complex. Simplified versions in other excavates might indicate a primordial complex or secondary loss of selected subunits, as seen for some fungal lineages. The conservation in eIF3i sequence might indicate critical functions within eIF3 which have been overlooked. The identification of eIF3 subunits from distantly related eukaryotes provides then a basis for the study of conserved/divergent aspects of eIF3 function, leading to a better understanding of eukaryotic translation initiation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1175) contains supplementary material, which is available to authorized users.     

The effect of lysosomotrophic bases and inhibitors of transglutaminase on iron uptake by immature erythroid cells

The mechanism by which weak bases block iron uptake by immature erythroid cells was investigated using rabbit and rat reticulocytes and erythroblasts from the fetal rat liver. A large variety of bases was found to inhibit iron uptake but to have a much smaller or no effect on transferrin uptake by the cells. Quinacrine and chloroquine were active at the lowest concentrations. Dansylcadaverine, an inhibitor of transglutaminase, was also active at low concentration. However, the results do not indicate a role for transglutaminase in the iron uptake process. Instead they show that the major effect of the bases is to inhibit iron release from transferrin molecules on or within the cells. The possible mechanism of this effect was investigated by measurement of intracellular ATP levels, intracellular pH and by morphological studies utilizing fluorescent and electron microscopy. The bases caused little change in ATP levels, but elevated intracellular pH, probably due to accumulation within intracellular vesicles, which were shown to accumulate fluorescent weak bases, to swell under the action of the bases and to be the site of intracellular localization of transferrin. It is concluded that the bases tested in this work inhibit iron release from transferrin in intracellular vesicles by increasing their pH rather than by blocking transglutaminase and thereby restricting endocytosis. Reduction of transferrin uptake by the cells when it occurs is probably due to inhibition of recycling of transferrin receptors to the outer cell membrane.     

Relationship between lymph and tissue hyaluronan in skin and skeletal muscle

The size of hyaluronan was compared between tissue and lymph using a combination of agarose gel electrophoresis and radiometric assay. Prenodal lymph was collected from heel skin and the gastrocnemius muscle in anesthetized rabbits. The major fraction of hyaluronan in both tissues had a molecular weight >4 million. Lymph contained primarily low-molecular-weight hyaluronan (<0.79 x 10(6)), which was absent from tissue. Volume loading produced a preferential increase in the flux of low-molecular-weight hyaluronan, indicating that tissue contains a small quantity of mobile, low-molecular-weight hyaluronan. The maximum daily removal of hyaluronan by lymph was <1% of the tissue content. The amount of lysosomal hyaluronidase activity in tissue was more than enough to account for a rapid turnover of hyaluronan. The data support the conclusion that lymph drainage is not significant in the normal catabolism of hyaluronan and may represent a small amount that becomes detached from the pericellular and extracellular matrixes.     

Probing neural circuitry and function with electrical microstimulation

Since the discovery of the nervous system's electrical excitability more than 200 years ago, neuroscientists have used electrical stimulation to manipulate brain activity in order to study its function. Microstimulation has been a valuable technique for probing neural circuitry and identifying networks of neurons that underlie perception, movement and cognition. In this review, we focus on the use of stimulation in behaving primates, an experimental system that permits causal inferences to be made about the effect of stimulation-induced activity on the resulting behaviour or neural signals elsewhere in the brain.     

Inactivation of the Potassium Conductance and Related Phenomena Caused by Quaternary Ammonium Ion Injection in Squid Axons

Several analogues of the tetraethylammonium (TEA⁺) ion were injected into the giant axon of the squid, and the resultant changes in time course and magnitude of the potassium current (I_K) were studied. For all the analogues used, three of the ethyl side chains of TEA⁺ were left unchanged, while the fourth chain was either lengthened or shortened. Increasing the length of this chain increased binding to the blocking site in the channel by a factor of roughly two for each added CH₂ group. The effect on the rate of entry into the blocking site was relatively slight. Thus the concentration for half-suppression of g_K decreased by about the same factor of two for each added CH₂. All the analogues caused anomalous or ingoing rectification. The longest chain analogue used, pentyltriethylammonium ion, caused rapid inactivation of g_K, and this inactivation had properties quite similar to g_Na inactivation. The anomalous rectification and the g_K inactivation caused by these compounds have the same basic mechanism.     

Thrombospondin 2 inhibits microvascular endothelial cell proliferation by a caspase-independent mechanism

The matricellular protein thrombospondin 2 (TSP2) regulates a variety of cell-matrix interactions. A prominent feature of TSP2-null mice is increased microvascular density, particularly in connective tissues synthesized after injury. We investigated the cellular basis for the regulation of angiogenesis by TSP2 in cultures of murine and human fibroblasts and endothelial cells. Fibroblasts isolated from murine and human dermis synthesize TSP2 mRNA and secrete significant amounts of immunoreactive TSP2, whereas endothelial cells from mouse lung and human dermis did not synthesize TSP2 mRNA or protein. Recombinant mouse TSP2 inhibited growth of human microvascular endothelial cells (HMVECs) mediated by basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, and vascular endothelial growth factor (VEGF). HMVECs exposed to TSP2 in the presence of these growth factors had a decreased proportion of cells in S and G2/M phases. HMVECs cultured with a combination of basic fibroblast growth factor, insulin-like growth factor-1, and epidermal growth factor displayed an increased proportion of nonviable cells in the presence of TSP2, but the addition of VEGF blocked this TSP2-mediated impairment of cell viability. TSP2-mediated inhibition of DNA synthesis by HMVECs in the presence of VEGF was not affected by the broad-spectrum caspase inhibitor zVAD-fmk. Similar findings were obtained with TSP1. Taken together, these observations indicate that either TSP2 or TSP1 can inhibit HMVEC proliferation by inhibition of cell cycle progression and induction of cell death, but the mechanisms responsible for TSP2-mediated inhibition of cell cycle progression are independent from those leading to cell death.     

Insight into the catalytic mechanism of Pseudomonas aeruginosa exotoxin A. Studies of toxin interaction with eukaryotic elongation factor-2

The molecular nature of the protein-protein interactions between the catalytic domain from Pseudomonas aeruginosa exotoxin A (PE24H) and its protein substrate, eukaryotic elongation factor-2 (eEF-2) were probed using a fluorescence resonance energy transfer method. Single cysteine mutant proteins of PE24H were prepared and site-specifically labeled with the donor fluorophore IAEDANS (5-(2-iodoacetylaminoethylamino)-1-napthalenesulfonic acid), whereas eEF-2 was labeled with the acceptor fluorophore fluorescein. The association was found to be independent of ionic strength and of the co-substrate, NAD(+) but dependent upon pH. The lack of requirement for NAD(+) to produce the toxin-eEF-2 complex demonstrates that the catalytic process is a random order mechanism, thereby disputing the current model. The previously observed pH dependence for catalytic function can be assigned to the toxin-eEF-2 binding event, as the pH dependence of binding observed in this study showed a strong correlation with enzymatic activity. The ability of the toxin to bind eEF-2 with bound GTP/GDP was assessed using nonhydrolyzable analogues. The results from the substrate binding and catalytic activity experiments indicate that PE24H is able to interact and bind with eEF-2 in all of its guanyl nucleotide-induced conformational states. Thus, the toxin ribosylates eEF-2 regardless of the nucleotide-charged state of eEF-2. These results represent the first detailed characterization of the molecular details and physiological conditions governing this protein-protein interaction.     

Interactions between androgen and growth factors in granulosa cell subtypes of porcine antral follicles

Androgens acting via the androgen receptor (AR) have been implicated in regulation of folliculogenesis in many animal species. These effects are possibly mediated via enhancement of FSH and/or insulin-like growth factor (IGF)-I activity in granulosa cells, which contain high levels of AR protein. We examined the in vitro effect of dihydrotestosterone (DHT) on DNA synthesis and progesterone secretion by follicular cells in response to FSH and IGF-I, alone or in combination. Cells from separate pools of 1- to 3-mm and 3- to 5-mm antral follicles were aspirated from gilt ovaries and fractioned into mural granulosa cells (MGCs) and cumulus-oocyte complexes (COCs) for subsequent cell culture. Androgen alone or with any combination of mitogen had minimal effect on proliferative and no effect on steroidogenic responses of MGCs from 3- to 5-mm antral follicles. Conversely, in MGCs from 1- to 3-mm follicles, DHT significantly enhanced IFG-I-stimulated proliferation and had variable influence on progesterone secretion. The effects of DHT on proliferative responses of COCs were also dependent on follicle size: DHT significantly augmented either IGF-I-stimulated proliferation (1- to 3-mm follicles) or FSH-stimulated proliferation (3- to 5-mm follicles). However, the steroidogenic responses of all COCs were identical, whereby DHT significantly suppressed progesterone secretion, predominantly in the presence of FSH. Addition of an AR antagonist, hydroxyflutamide, generally reversed the proliferative responses invoked by DHT but not the steroidogenic responses. We conclude that androgen-receptor-mediated activity in granulosa cells of antral follicles is dependent on follicle size, is influenced by proximity of cells to the oocyte, and possibly involves both classic and nonclassic steroid mechanisms.