Tissue-specific variants of translation elongation factor eEF1A and their role in cancer

Eukaryotic translation elongation factor eEF1A exists in two closely related variant forms, eEF1A1 and eEF1A2, that are encoded by separate loci. The former is the second most abundant protein in the cell and is almost ubiquitously expressed but eEF1A2 expression is more limited and its presence was defined predominantly in neurons and muscle cells. Both perform equally well in translation elongation and are responsible for delivering aminoacylated tRNA to the A site of the ribosome in a GTP-dependent manner. Translation factor eEF1A2 was identified as an oncogene due to inappropriate expression being observed in the high proportion of ovarian, breast, lung, colon and pancreatic tumours. Additionally, its forced expression in rodent fibroblasts resulted in soft agar colony formation along with tumours when overexpressing cells were injected into nude mice. The mechanism by which eEF1A2 contributes to oncogenesis remains unclear. Gene amplification is not solely responsible for eEF1A2 upregulation and neither activating mutations nor methylation status changes are seen in tumours. Interestingly, no connection of eEF1A1 with any malignancy has been made. It is proposed that the oncogenic properties of eEF1A2 might be associated with its conventional role in translation or perhaps with non-canonical functions that differ from those of the eEF1A1 variant. The main objectives of this PhD project were to elucidate the differential functions of both variants of eEF1A in cancer and to investigate other possible mechanism of eEF1A2 upregulation. In order to compare the contribution of overexpressed eEF1A variants to cellular transformation, stable cell lines were generated in NIH-3T3 mouse fibroblasts and tested in a panel of in vitro transformation assays. Mammalian expression plasmids used for transfection contained each eEF1A variant coding sequence with or without its own 5‟UTR and each variant with the 5‟UTR from the other eEF1A form. Transient transfections with the same mammalian expression plasmids were performed to observe that incorporation of exogenous eEF1A1 and eEF1A2 resulted in a decrease of the endogenous eEF1A1 expression at the mRNA and protein level. The dynamic interplay between exogenous and endogenous variants occurred within the first 48 hours post transfection but Eef1a1 returned to the levels seen in controls as soon as the expression of any of the exogenous eEF1A forms started to decline. In contrast, in almost all tested stable cell lines, the levels of endogenous eEF1A1 remained unchanged, at both the mRNA and protein level. NIH-3T3 lines constitutively expressing eEF1A forms were subsequently subjected to various in vitro transformation assays. Stable cell lines of eEF1A1 coding sequence origin formed colonies and foci but with lower efficiency when compared to the eEF1A2 coding sequence variant. It was also shown that anchorage independent growth and foci formation were affected by incorporating either the eEF1A1 or eEF1A2 5‟UTR in front of either eEF1A1 or eEF1A2 coding sequence. There was no apparent increase in migration and invasion of the cell lines stably expressing eEF1A. No significant association between protein synthesis rate or increased overall eEF1A level and transformed phenotype in all tested stable cell lines was observed. Expression of eEF1A1 or eEF1A2 was also determined immunohistologically in panels of different tumour arrays. Moderate to high expression of eEF1A2 protein was observed in 43% of colorectal cancers analysed. The level of eEF1A2 expression appeared to be inversely correlated (P = 0.024) with metastasis in lymph nodes in one of the tested colorectal tumour arrays. Moreover, no substantial upregulation of eEF1A2 at the protein level was confirmed in hepatocellular carcinoma and malignant melanoma arrays. In contrast, eEF1A1 protein expression was mostly weak or absent in these malignancies

Stearoyl-CoA desaturase 1 activity determines the maintenance of DNMT1-mediated DNA methylation patterns in pancreatic β\beta-Cells

Metabolic stress, such as lipotoxicity, affects the DNA methylation profile in pancreatic β-cells and thus contributes to β-cell failure and the progression of type 2 diabetes (T2D). Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme that is involved in monounsaturated fatty acid synthesis, which protects pancreatic β-cells against lipotoxicity. The present study found that SCD1 is also required for the establishment and maintenance of DNA methylation patterns in β-cells. We showed that SCD1 inhibition/deficiency caused DNA hypomethylation and changed the methyl group distribution within chromosomes in β-cells. Lower levels of DNA methylation in SCD1-deficient β-cells were followed by lower levels of DNA methyltransferase 1 (DNMT1). We also found that the downregulation of SCD1 in pancreatic β-cells led to the activation of adenosine monophosphate-activated protein kinase (AMPK) and an increase in the activity of the NAD-dependent deacetylase sirtuin-1 (SIRT1). Furthermore, the physical association between DNMT1 and SIRT1 stimulated the deacetylation of DNMT1 under conditions of SCD1 inhibition/downregulation, suggesting a mechanism by which SCD1 exerts control over DNMT1. We also found that SCD1-deficient β-cells that were treated with compound c, an inhibitor of AMPK, were characterized by higher levels of both global DNA methylation and DNMT1 protein expression compared with untreated cells. Therefore, we found that activation of the AMPK/SIRT1 signaling pathway mediates the effect of SCD1 inhibition/deficiency on DNA methylation status in pancreatic β-cells. Altogether, these findings suggest that SCD1 is a gatekeeper that protects β-cells against the lipid-derived loss of DNA methylation and provide mechanistic insights into the mechanism by which SCD1 regulates DNA methylation patterns in β-cells and T2D-relevant tissues

Characterisation of Translation Elongation Factor eEF1B Subunit Expression in Mammalian Cells and Tissues and Co-Localisation with eEF1A2

Translation elongation is the stage of protein synthesis in which the translation factor eEF1A plays a pivotal role that is dependent on GTP exchange. In vertebrates, eEF1A can exist as two separately encoded tissue-specific isoforms, eEF1A1, which is almost ubiquitously expressed, and eEF1A2, which is confined to neurons and muscle. The GTP exchange factor for eEF1A1 is a complex called eEF1B made up of subunits eEF1Bα, eEF1Bδ and eEF1Bγ. Previous studies have cast doubt on the ability of eEF1B to interact with eEF1A2, suggesting that this isoform might use a different GTP exchange factor. We show that eEF1B subunits are all widely expressed to varying degrees in different cell lines and tissues, and at different stages of development. We show that ablation of any of the subunits in human cell lines has a small but significant impact on cell viability and cycling. Finally, we show that both eEF1A1 and eEF1A2 colocalise with all eEF1B subunits, in such close proximity that they are highly likely to be in a complex

Fat and Sugar—A Dangerous Duet. A Comparative Review on Metabolic Remodeling in Rodent Models of Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is a common disease in Western society and ranges from steatosis to steatohepatitis to end-stage liver disease such as cirrhosis and hepatocellular carcinoma. The molecular mechanisms that are involved in the progression of steatosis to more severe liver damage in patients are not fully understood. A deeper investigation of NAFLD pathogenesis is possible due to the many different animal models developed recently. In this review, we present a comparative overview of the most common dietary NAFLD rodent models with respect to their metabolic phenotype and morphological manifestation. Moreover, we describe similarities and controversies concerning the effect of NAFLD-inducing diets on mitochondria as well as mitochondria-derived oxidative stress in the progression of NAFLD

Differential regulation of serum microRNA expression by HNF1β and HNF1α transcription factors.

We aimed to identify microRNAs (miRNAs) under transcriptional control of the HNF1β transcription factor, and investigate whether its effect manifests in serum.This article is freely available via Open Access. Click on the 'Additional Link' above to access the full-text via the publisher's site.Published (Open Access

Our invisible addicts. First report of the older persons’ substance misuse working group of the Royal College of Psychiatrists.

Both alcohol and illicit drugs are among the top ten risk factors for mortality and morbidity in Europe and substance misuse by older people is now a growing public health problem. Between 2001 and 2031, there is projected to be a 50% increase in the number of older people in the UK. The percentage of men and women drinking more than the weekly recommended limits has also risen, by 60% in men and 100% in women between 1990 and 2006 (NHS Information Centre, 2009a). Given the likely impact of these two factors on health and social care services, there is now a pressing need to address substance misuse in older people