Regulating the expression of eEF1A to decipher its non canonical functions in eukaryotic cells

The canonical function of eEF1A is delivery of the aminoacylated tRNA to the A site of the ribosome during protein translation, however, it is also known to be an actin binding protein. As well as this actin binding function, eEF1A has been shown to be involved in other cellular processes such as cell proliferation and apoptosis. It has long been thought that the actin cytoskeleton and protein synthesis are linked and eEF1A has been suggested to be a candidate protein to form this link, though very little is understood about the relationship between its two functions. Overexpression of eEF1A has also been shown to be implicated in many different types of cancers, especially cancers that are metastatic, therefore it is important to further understand how eEF1A can affect both translation and the organisation of the actin cytoskeleton. To this end, we aimed to determine the effects of reduced expression of eEF1A on both translation and its non canonical functions in CHO cells. We have shown that reduced expression of eEF1A in this cell system results in no change in protein synthesis, however results in an increased number of actin stress fibres and other proteins associated with these fibres such as myosin IIA, paxillin and vinculin. Cell motility and attachment are also affected by this reduction in eEF1A protein expression. The organisational and motility phenotypes were found to be specific to eEF1A by transforming the cells with plasmids containing either human eEF1A1 or eEF1A2. Though the mechanisms by which these effects are regulated have not yet been established, this data provides evidence to show that the translation and actin binding functions of eEF1A are independent of each other as well as being suggestive of a role for eEF1A in cell motility as supported by the observation that overexpression of eEF1A protein tends to be associated with the cancer cells that are metastatic.

Alternative exon usage in the single CPT1 gene of Drosophila generates functional diversity in the kinetic properties of the enzyme:differential expression of alternatively spliced variants in drosophila tissues

The Drosophila melanogaster genome contains only one CPT1 gene (Jackson, V. N., Cameron, J. M., Zammit, V. A., and Price, N. T. (1999) Biochem. J. 341, 483-489). We have now extended our original observation to all insect genomes that have been sequenced, suggesting that a single CPT1 gene is a universal feature of insect genomes. We hypothesized that insects may be able to generate kinetically distinct variants by alternative splicing of their single CPT1 gene. Analysis of the insect genomes revealed that (a) the single CPT1 gene in each and every insect genome contains two alternative exons and (ii) in all cases, the putative alternative splicing site occurs within a small region corresponding to 21 amino acid residues that are known to be essential for the binding of substrates and of malonyl-CoA in mammalian CPT1A.Weperformed PCR analyses of mRNA from different Drosophila tissues; both of the anticipated splice variants of CPT1mRNAwere found to be expressed in all of the tissues tested (both in larvae and adults), with the expression level for one of the splice variants being significantly different between flight muscle and the fat body of adult Drosophila. Heterologous expression of the full-length cDNAs corresponding to the two putative variants of Drosophila CPT1 in the yeast Pichia pastoris revealed two important differences between the properties of the two variants: (i) their affinity (K 0.5) for one of the substrates, palmitoyl-CoA, differed by 5-fold, and (ii) the sensitivity to inhibition by malonyl-CoA at fixed, higher palmitoyl-CoA concentrations was 2-fold different and associated with different kinetics of inhibition. These data indicate that alternative splicing that specifically affects a structurally crucial region of the protein is an important mechanism through which functional diversity of CPT1 kinetics is generated from the single gene that occurs in insects. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

The administration route is decisive for the ability of the vaccine adjuvant CAF09 to induce antigen-specific CD8+ T-cell responses:the immunological consequences of the biodistribution profile

A prerequisite for vaccine-mediated induction of CD8+ T-cell responses is the targeting of dendritic cell (DC) subsets specifically capable of cross-presenting antigen epitopes to CD8+ T cells. Administration of a number of cationic adjuvants via the intraperitoneal (i.p.) route has been shown to result in strong CD8+ T-cell responses, whereas immunization via e.g. the intramuscular (i.m.) or subcutaneous (s.c.) routes often stimulate weak CD8+ T-cell responses. The hypothesis for this is that self-drainage of the adjuvant/antigen to the lymphoid organs, which takes place upon i.p. immunization, is required for the subsequent activation of cross-presenting lymphoid organ-resident CD8α+ DCs. In contrast, s.c. or i.m. immunization usually results in the formation of a depot at the site of injection (SOI), which hinders the self-drainage and targeting of the vaccine to cross-presenting CD8α+ DCs. We investigated this hypothesis by correlating the biodistribution pattern and the adjuvanticity of the strong CD8+ T-cell inducing liposomal cationic adjuvant formulation 09 (CAF09), which is composed of dimethyldioctadecylammonium bromide/monomycoloyl glycerol liposomes with polyinosinic:polycytidylic acid electrostatically adsorbed to the surface. Biodistribution studies with radiolabeled CAF09 and a surface-adsorbed model antigen [ovalbumin (OVA)] showed that a significantly larger fraction of the vaccine dose localized in the draining lymph nodes (dLNs) and the spleen 6 h after i.p. immunization, as compared to after i.m. immunization. Studies with fluorescently labelled OVA + CAF09 demonstrated a preferential association of OVA + CAF09 to DCs/monocytes, as compared to macrophages and B cells, following i.p. immunization. Administration of OVA + CAF09 via the i.p. route did also result in DC activation, whereas no DC activation could be measured within the same period with unadjuvanted OVA and OVA + CAF09 administered via the s.c. or i.m. routes. In the dLNs, the highest level of activated, cross-presenting CD8α+ DCs was detected at 24 h post immunization, whereas an influx of activated, migrating and cross-presenting CD103+ DCs to the dLNs could be measured after 48 h. This suggests that the CD8α+ DCs are activated by self-draining OVA + CAF09 in the lymphoid organs, whereas the CD103+ DCs are stimulated by the OVA + CAF09 at the SOI. These results support the hypothesis that the self-drainage of OVA + CAF09 to the draining LNs is required for the activation of CD8α+ DCs, while the migratory CD103+ DCs may play a role in sustaining the subsequent induction of strong CD8+ T-cell responses.