Collybistin and gephyrin are novel components of the eukaryotic translation initiation factor 3 complex

Abstract Background Collybistin (CB), a neuron-specific guanine nucleotide exchange factor, has been implicated in targeting gephyrin-GABAA receptors clusters to inhibitory postsynaptic sites. However, little is known about additional CB partners and functions. Findings Here, we identified the p40 subunit of the eukaryotic translation initiation factor 3 (eIF3H) as a novel binding partner of CB, documenting the interaction in yeast, non-neuronal cell lines, and the brain. In addition, we demonstrated that gephyrin also interacts with eIF3H in non-neuronal cells and forms a complex with eIF3 in the brain. Conclusions Together, our results suggest, for the first time, that CB and gephyrin associate with the translation initiation machinery, and lend further support to the previous evidence that gephyrin may act as a regulator of synaptic protein synthesis.

Potential Contribution of Translational Factors to Triiodo-L-Thyronine-Induced Insulin Synthesis by Pancreatic Beta Cells

Background: Thyroid hormones (THs) are known to regulate protein synthesis by acting at the transcriptional level and inducing the expression of many genes. However, little is known about their role in protein expression at the post-transcriptional level, even though studies have shown enhancement of protein synthesis associated with mTOR/p70S6K activation after triiodo-l-thyronine (T3) administration. On the other hand, the effects of TH on translation initiation and polypeptidic chain elongation factors, being essential for activating protein synthesis, have been poorly explored. Therefore, considering that preliminary studies from our laboratory have demonstrated an increase in insulin content in INS-1E cells in response to T3 treatment, the aim of the present study was to investigate if proteins of translational nature might be involved in this effect. Methods: INS-1E cells were maintained in the presence or absence of T3 (10(-6) or 10(-8) M) for 12 hours. Thereafter, insulin concentration in the culture medium was determined by radioimmunoassay, and the cells were processed for Western blot detection of insulin, eukaryotic initiation factor 2 (eIF2), p-eIF2, eIF5A, EF1A, eIF4E binding protein (4E-BP), p-4E-BP, p70S6K, and p-p70S6K. Results: It was found that, in parallel with increased insulin generation, T3 induced p70S6K phosphorylation and the expression of the translational factors eIF2, eIF5A, and eukaryotic elongation factor 1 alpha (eEF1A). In contrast, total and phosphorylated 4E-BP, as well as total p70S6K and p-eIF2 content, remained unchanged after T3 treatment. Conclusions: Considering that (i) p70S6K induces S6 phosphorylation of the 40S ribosomal subunit, an essential condition for protein synthesis; (ii) eIF2 is essential for the initiation of messenger RNA translation process; and (iii) eIF5A and eEF1A play a central role in the elongation of the polypeptidic chain during the transcripts decoding, the data presented here lead us to suppose that a part of T3-induced insulin expression in INS-1E cells depends on the protein synthesis activation at the post-transcriptional level, as these proteins of the translational machinery were shown to be regulated by T3.

Leucine Is Essential for Attenuating Fetal Growth Restriction Caused by a Protein-Restricted Diet in Rats

Certain amino acids, such as leucine (Leu) are not only substrates for protein synthesis but also are important regulators of protein metabolism. Moreover, it is known that alterations in intrauterine growth favor the development of chronic diseases in adulthood. Therefore, we investigated the role of Leu in combination with other BCAA on effects that are induced by maternal protein restriction on fetal growth. Wistar rats were divided into 4 groups according to the diet provided during pregnancy: control (C; 20% casein); V+I [5% casein + 2% L-valine (Val) + 2% L-isoleucine (Ile)1; KYT 15% casein + 1.8% L-lysine (Lys) + 1.2% L-tyrosine (Tyr) + 1% L-threonine (Thr)1; and BCAA (5% casein + 1.8% L-Leu + 1.2% L-Val + 1% L-Ile). Maternal protein restriction reduced the growth and organ weight of the offspring of dams receiving the V+I and KYT diets compared with the C group. Supplementation with BCAA reversed this growth deficit, minimizing the difference or restoring the mass of organs and carcass fat, the liver and muscle protein, and the RNA concentrations compared with newborns in the C group (P < 0.05). These effects could be explained by the activation of the mTOR signaling pathway, because phosphorylation of 4E-BP1 in the liver of offspring of the BCAA group was greater than that in the C, V+I, and KYT groups. The present results identify a critical role for Leu in association with other BCAA in the activation of the mTOR signaling pathway for the control of altered intrauterine growth induced by a maternal low-protein diet. J. Nutr. 142: 924-930, 2012.

Influence of HAART on Alternative Reading Frame Immune Responses over the Course of HIV-1 Infection

Background: Translational errors can result in bypassing of the main viral protein reading frames and the production of alternate reading frame (ARF) or cryptic peptides. Within HIV, there are many such ARFs in both sense and the antisense directions of transcription. These ARFs have the potential to generate immunogenic peptides called cryptic epitopes (CE). Both antiretroviral drug therapy and the immune system exert a mutational pressure on HIV-1. Immune pressure exerted by ARF CD8(+) T cells on the virus has already been observed in vitro. HAART has also been described to select HIV-1 variants for drug escape mutations. Since the mutational pressure exerted on one location of the HIV-1 genome can potentially affect the 3 reading frames, we hypothesized that ARF responses would be affected by this drug pressure in vivo. Methodology/Principal findings: In this study we identified new ARFs derived from sense and antisense transcription of HIV-1. Many of these ARFs are detectable in circulating viral proteins. They are predominantly found in the HIV-1 env nucleotide region. We measured T cell responses to 199 HIV-1 CE encoded within 13 sense and 34 antisense HIV-1 ARFs. We were able to observe that these ARF responses are more frequent and of greater magnitude in chronically infected individuals compared to acutely infected patients, and in patients on HAART, the breadth of ARF responses increased. Conclusions/Significance: These results have implications for vaccine design and unveil the existence of potential new epitopes that could be included as vaccine targets.