eIF4A Inhibition Allows Translational Regulation of mRNAs Encoding Proteins Involved in Alzheimer's Disease

Alzheimer's disease (AD) is the main cause of dementia in our increasingly aging population. The debilitating cognitive and behavioral symptoms characteristic of AD make it an extremely distressing illness for patients and carers. Although drugs have been developed to treat AD symptoms and to slow disease progression, there is currently no cure. The incidence of AD is predicted to increase to over one hundred million by 2050, placing a heavy burden on communities and economies, and making the development of effective therapies an urgent priority. Two proteins are thought to have major contributory roles in AD: the microtubule associated protein tau, also known as MAPT; and the amyloid-beta peptide (A-beta), a cleavage product of amyloid precursor protein (APP). Oxidative stress is also implicated in AD pathology from an early stage. By targeting eIF4A, an RNA helicase involved in translation initiation, the synthesis of APP and tau, but not neuroprotective proteins, can be simultaneously and specifically reduced, representing a novel avenue for AD intervention. We also show that protection from oxidative stress is increased upon eIF4A inhibition. We demonstrate that the reduction of these proteins is not due to changes in mRNA levels or increased protein degradation, but is a consequence of translational repression conferred by inhibition of the helicase activity of eIF4A. Inhibition of eIF4A selectively and simultaneously modulates the synthesis of proteins involved in Alzheimer's disease: reducing A-beta and tau synthesis, while increasing proteins predicted to be neuroprotective

Homoeologous gene silencing in tissue cultured wheat callus

Abstract Background In contrast to diploids, most polyploid plant species, which include the hexaploid bread wheat, possess an additional layer of epigenetic complexity. Several studies have demonstrated that polyploids are affected by homoeologous gene silencing, a process in which sub-genomic genomic copies are selectively transcriptionally inactivated. This form of silencing can be tissue specific and may be linked to developmental or stress responses. Results Evidence was sought as to whether the frequency of homoeologous silencing in in vitro cultured wheat callus differ from that in differentiated organs, given that disorganized cells are associated with a globally lower level of DNA methylation. Using a reverse transcription PCR (RT-PCR) single strand conformation polymorphism (SSCP) platform to detect the pattern of expression of 20 homoeologous sets of single-copy genes known to be affected by this form of silencing in the root and/or leaf, we observed no silencing in any of the wheat callus tissue tested. Conclusion Our results suggest that much of the homoeologous silencing observed in differentiated tissues is probably under epigenetic control, rather than being linked to genomic instability arising from allopolyploidization. This study reinforces the notion of plasticity in the wheat epi-genome.</p

The human insulin receptor mRNA contains a functional internal ribosome entry segment.

Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5'-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective

PRAME Is a Golgi-Targeted Protein That Associates with the Elongin BC Complex and Is Upregulated by Interferon-Gamma and Bacterial PAMPs

Preferentially expressed antigen in melanoma (PRAME) has been described as a cancer-testis antigen and is associated with leukaemias and solid tumours. Here we show that PRAME gene transcription in leukaemic cell lines is rapidly induced by exposure of cells to bacterial PAMPs (pathogen associated molecular patterns) in combination with type 2 interferon (IFNγ). Treatment of HL60 cells with lipopolysaccharide or peptidoglycan in combination with IFNγ resulted in a rapid and transient induction of PRAME transcription, and increased association of PRAME transcripts with polysomes. Moreover, treatment with PAMPs/IFNγ also modulated the subcellular localisation of PRAME proteins in HL60 and U937 cells, resulting in targeting of cytoplasmic PRAME to the Golgi. Affinity purification studies revealed that PRAME associates with Elongin B and Elongin C, components of Cullin E3 ubiquitin ligase complexes. This occurs via direct interaction of PRAME with Elongin C, and PRAME colocalises with Elongins in the Golgi after PAMP/IFNγ treatment. PRAME was also found to co-immunoprecipitate core histones, consistent with its partial localisation to the nucleus, and was found to bind directly to histone H3 in vitro. Thus, PRAME is upregulated by signalling pathways that are activated in response to infection/inflammation, and its product may have dual functions as a histone-binding protein, and in directing ubiquitylation of target proteins for processing in the Golgi

A comparative assessment of the utility of PCR-based marker systems in pearl millet

A set of 22 pearl millet inbred lines including the parents of eleven mapping populations, was screened with 627 markers including 100 pearl millet genomic SSRs (gSSRs), 60 pearl millet ESTSSRs (eSSRs), 410 intron sequence haplotypes (ISHs), and 57 exon sequence haplotypes (ESHs). In all, 267 (59%) of themarkers were informative for at least one of the 11 mapping populations, which segregate for traits like drought and salinity tolerance; host plant resistance to downymildew, rust and blast; fertility restoration and sterility and maintenance of cytoplasmic male sterility etc. An average of 116 polymorphic markers was identified per mapping population. The average PIC values and number of profiles (P) per polymorphic marker were: gSSRs (PIC = 0.62, P = 6.1), ISHs (PIC = 0.39, P = 2.6), eSSRs (PIC = 0.36, P = 3.1) and ESHs (PIC = 0.35, P = 3.1). A high correlation (r[0.97, P\0.05) was observed between the patterns of diversity exposed by the different marker systems. The polymorphic markers identified are suitable for the de novo construction, or the supplementation of pearl millet linkagemaps. The genetic relationships identified among the panel of inbred lines may be useful in designing strategies to improve the use of available genetic variation in the context of pearl millet breeding

An internal ribosome entry site in the 5′ untranslated region of epidermal growth factor receptor allows hypoxic expression

The expression of epidermal growth factor receptor (EGFR/ERBB1/HER1) is implicated in the progress of numerous cancers, a feature that has been exploited in the development of EGFR antibodies and EGFR tyrosine kinase inhibitors as anti-cancer drugs. However, EGFR also has important normal cellular functions, leading to serious side effects when EGFR is inhibited. One damaging characteristic of many oncogenes is the ability to be expressed in the hypoxic conditions associated with the tumour interior. It has previously been demonstrated that expression of EGFR is maintained in hypoxic conditions via an unknown mechanism of translational control, despite global translation rates generally being attenuated under hypoxic conditions. In this report, we demonstrate that the human EGFR 5′ untranslated region (UTR) sequence can initiate the expression of a downstream open reading frame via an internal ribosome entry site (IRES). We show that this effect is not due to either cryptic promoter activity or splicing events. We have investigated the requirement of the EGFR IRES for eukaryotic initiation factor 4A (eIF4A), which is an RNA helicase responsible for processing RNA secondary structure as part of translation initiation. Treatment with hippuristanol (a potent inhibitor of eIF4A) caused a decrease in EGFR 5′ UTR-driven reporter activity and also a reduction in EGFR protein level. Importantly, we show that expression of a reporter gene under the control of the EGFR IRES is maintained under hypoxic conditions despite a fall in global translation rates

Human papillomavirus, high-grade intraepithelial neoplasia and killer immunoglogulin-like receptors: a Western Australian cohort study

Background: Human papillomavirus (HPV) is the causative agent in cervical cancer and HPV genotypes 16 and 18 cause the majority of these cancers. Natural killer (NK) cells destroy virally infected and tumour cells via killer immunoglobulin-like receptors (KIR) that recognize decreased MHC class I expression. These NK cells may contribute to clearance of HPV infected and/or dysplastic cells, however since KIR controls NK cell activity, KIR gene variation may determine outcome of infection.Methods: KIR gene frequencies were compared between 147 patients with a history of high-grade cervical intraepithelial neoplasia (CIN) and a control population of 187, to determine if any KIR genes are associated with high-grade CIN. In addition a comparison was also made between cases of high grade CIN derived from 30 patients infected with HPV 16/18 and 29 patients infected with non-16/18 HPV to determine if KIR variation contributes to the disproportional carcinogenesis derived from HPV 16/18 infection.Results: High-grade CIN was weakly associated with the absence of KIR2DL2 and KIR2DS2 (p = 0.046 and 0.049 respectively, OR 0.6; 95% CI 0.4 – 0.9) but this association was lost after correction for multi-gene statistical analysis.No difference in KIR gene frequencies was found between high-grade CIN caused by HPV 16/18 and non-16/18.Conclusion: No strong association between KIR genes, high-grade CIN and HPV genotype was found in the Western Australian population

Low NKp30, NKp46 and NKG2D expression and reduced cytotoxic activity on NK cells in cervical cancer and precursor lesions

<p>Abstract</p> <p>Background</p> <p>Persistent high risk HPV infection can lead to cervical cancer, the second most common malignant tumor in women worldwide. NK cells play a crucial role against tumors and virus-infected cells through a fine balance between activating and inhibitory receptors. Expression of triggering receptors NKp30, NKp44, NKp46 and NKG2D on NK cells correlates with cytolytic activity against tumor cells, but these receptors have not been studied in cervical cancer and precursor lesions. The aim of the present work was to study NKp30, NKp46, NKG2D, NKp80 and 2B4 expression in NK cells from patients with cervical cancer and precursor lesions, in the context of HPV infection.</p> <p>Methods</p> <p>NKp30, NKp46, NKG2D, NKp80 and 2B4 expression was analyzed by flow cytometry on NK cells from 59 patients with cervical cancer and squamous intraepithelial lesions. NK cell cytotoxicity was evaluated in a 4 hour CFSE/7-AAD flow cytometry assay. HPV types were identified by PCR assays.</p> <p>Results</p> <p>We report here for the first time that NK cell-activating receptors NKp30 and NKp46 are significantly down-regulated in cervical cancer and high grade squamous intraepithelial lesion (HGSIL) patients. NCRs down-regulation correlated with low cytolytic activity, HPV-16 infection and clinical stage. NKG2D was also down-regulated in cervical cancer patients.</p> <p>Conclusion</p> <p>Our results suggest that NKp30, NKp46 and NKG2D down-regulation represent an evasion mechanism associated to low NK cell activity, HPV-16 infection and cervical cancer progression.</p

Characterization of the Endothelial Cell Cytoskeleton following HLA Class I Ligation

Vascular endothelial cells (ECs) are a target of antibody-mediated allograft rejection. In vitro, when the HLA class I molecules on the surface of ECs are ligated by anti-HLA class I antibodies, cell proliferation and survival pathways are activated and this is thought to contribute to the development of antibody-mediated rejection. Crosslinking of HLA class I molecules by anti-HLA antibodies also triggers reorganization of the cytoskeleton, which induces the formation of F-actin stress fibers. HLA class I induced stress fiber formation is not well understood.The present study examines the protein composition of the cytoskeleton fraction of ECs treated with HLA class I antibodies and compares it to other agonists known to induce alterations of the cytoskeleton in endothelial cells. Analysis by tandem mass spectrometry revealed unique cytoskeleton proteomes for each treatment group. Using annotation tools a candidate list was created that revealed 12 proteins, which were unique to the HLA class I stimulated group. Eleven of the candidate proteins were phosphoproteins and exploration of their predicted kinases provided clues as to how these proteins may contribute to the understanding of HLA class I induced antibody-mediated rejection. Three of the candidates, eukaryotic initiation factor 4A1 (eIF4A1), Tropomyosin alpha 4-chain (TPM4) and DDX3X, were further characterized by Western blot and found to be associated with the cytoskeleton. Confocal microscopy analysis showed that class I ligation stimulated increased eIF4A1 co-localization with F-actin and paxillin.Colocalization of eIF4A1 with F-actin and paxillin following HLA class I ligation suggests that this candidate protein could be a target for understanding the mechanism(s) of class I mediated antibody-mediated rejection. This proteomic approach for analyzing the cytoskeleton of ECs can be applied to other agonists and various cells types as a method for uncovering novel regulators of cytoskeleton changes