Mechanisms involved in the establishment of HCMV latency are poorly understood, however, work in our laboratory has demonstrated the ULb' encoded protein, pUL138, as the first viral determinant to function in the establishment of HCMV latency in CD34+ hematopoietic progenitor cells (HPCs). This work characterizes the transcripts that encode pUL138, identifies three novel ULb' proteins (pUL133, pUL135, and pUL136) and represents the first demonstration of an internal ribosome entry site (IRES) mediated expression of pUL138. pUL138 is encoded on three polycistronic transcripts of 3.6-, 2.7- and 1.4-kb in length. pUL133, pUL135 and truncated pUL136, are expressed on the 3.6-, 2.7- and 1.4-kb transcripts, respectively, in addition to pUL138. We demonstrate that pUL138 expression is inducible from the IRES on the 3.6- and 2.7-kb transcripts under conditions of cellular stress, whereas pUL138 expression from the 1.4-kb transcript is inhibited under these same conditions. Differential utilization of the UL138 transcripts and their respective encoded proteins may regulate the outcome of viral infection in a cell type or cell context dependent manner. The interaction of these proteins during HCMV latency is the focus of ongoing research. In addition, this work represents preliminary data regarding the type I interferon (IFN) response during HCMV during productive infection in MRC5 fibroblasts and during the establishment of HCMV latency in CD34+ HPCs.

No description available.

The purpose of this thesis was to examine changes in the expression of translation regulatory proteins following both an acute bout of endurance exercise and chronic muscle contractile activity. In experiment 1, female Sprague-Dawley rats ran for 2 h at 15 m/min followed by an increase in speed of 5 m/min every 5 min until volitional fatigue. Red gastrocnemius muscle was harvested from non-exercised animals (control), immediately following cessation of exercise (0 h) and after 3 hours of recovery (3 h). Compared to control, rpS6 mRNA was elevated (p < .05) at both 0 h (+32%) and 3 h (+47%). Both eIF2Bε (+127%) and mTOR mRNA (+44%) were higher than control at 3 h, while eIF4E decreased (-24%) immediately following exercise (p < .05). Phosphorylation of mTOR (+40%) and S6K1 (+266%) also increased immediately post-exercise (p < .05). In experiment 2, female Sprague-Dawley rats underwent chronic stimulation of the peroneal nerve continuously for 7 days. The red gastrocnemius muscle was removed 24 h following cessation of the stimulation. Chronic muscle stimulation up-regulated (P < .05) mTOR protein (+74%), rpS6 (+31%), and eIF2α (+44%, P < .07), and this was accompanied by an increase in cytochrome C (+31%). Phosphorylation of rpS6 (Ser235/Ser236) was increased (+51%, P < .05), while mTOR (Ser2448) and 4E-BP1 (Thr37/46) did not change. These experiments demonstrate that acute and chronic endurance contractile activity up-regulate the mTOR signalling pathway and mitochondrial content in murine skeletal muscle. This up-regulation of the mTOR pathway may increase translation efficiency and may also represent an important control point in exercise mediated mitochondrial biogenesis. / Thesis (Master, Kinesiology & Health Studies) -- Queen's University, 2012-10-02 13:35:04.072

All bacteria initiate translation using formylated methionine, yet directly after translation, the formyl-group is removed. This sequence of addition and removal appears futile, yet every sequenced bacterial genome encodes the enzymes for formylation and deformylation, suggesting this process is essential. Puzzlingly, the process is absent from both Archaea and Eukaryotes, and moreover, bacterial mutants lacking both the formylase and deformylase activities are viable, albeit with a diminished growth rate. We created an Escherichia coli strain devoid of formylase and deformylase activity. This strain was then allowed to evolve over 1500 generations whereupon it reached wild-type growth rate, demonstrating that formylation can be completely dispensed with. This raises an additional question: if the formylation cycle is unnecessary, how did it emerge and why has it persisted? Our results show that the formylation-deformylation cycle could have evolved as a toxin-antitoxin pair (TA) with post-segregational killing (PSK) activity. TAs ‘addict’ cells to the plasmids that carry them by inducing PSK. We measured the stability of formylase-deformylase encoding plasmids and their ability to elicit PSK in our evolved E. coli strain. We report several lines of evidence consistent with the formylation-cycle having evolved from a plasmid-borne PSK element: 1) in the absence of deformylation, formyl-methionine on proteins is cytotoxic in bacteria 2) deformylation relieves the cytotoxicity of formyl-methionine, 3) the loss of a plasmid containing formylase and deformylase genes from evolved cells results in cessation of growth – a standard PSK phenotype. In addition, we introduced the E. coli formylase and deformylase genes into yeast and demonstrate that Met-tRNA formylation is not lethal, even in the absence of deformylation. This suggests PSK would be ineffectual in yeast, accounting for the absence of formylation from eukaryotic cytoplasmic translation. We also report the presence of formylase and deformylase genes in the two representative members of the archaeal Methanocopusculum genus. Moreover, we demonstrate that these genes have been acquired by a recent horizontal gene transfer from bacteria. Our results indicate that formylmethionine use in bacteria evolved, not through a direct functional benefit to cells, but through competition between infectious genetic elements.

abstract: Recombinant protein expression is essential to biotechnology and molecular medicine, but facile methods for obtaining significant quantities of folded and functional protein in mammalian cell culture have been lacking. Here I describe a novel 37-nucleotide in vitro selected sequence that promotes unusually high transgene expression in a vaccinia driven cytoplasmic expression system. Vectors carrying this sequence in a monocistronic reporter plasmid produce >1,000-fold more protein than equivalent vectors with conventional vaccinia promoters. Initial mechanistic studies indicate that high protein expression results from dual activity that impacts both transcription and translation. I suggest that this motif represents a powerful new tool in vaccinia-based protein expression and vaccine development technology. / Dissertation/Thesis / M.S. Biochemistry 2012

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-10T17:54:57Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-TamaraLima.pdf: 15100477 bytes, checksum: 79a87ae32652a2bd9c0dae960dc0a539 (MD5) / Made available in DSpace on 2017-04-10T17:54:57Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-TamaraLima.pdf: 15100477 bytes, checksum: 79a87ae32652a2bd9c0dae960dc0a539 (MD5) Previous issue date: 2012-09-21 / Em eucariotos,a proteína de ligação à cauda poli-A (PABP) se liga especificamente a seqüência de poli-adenosinas na extremidade 3’ dos mRNAs, atuando em múltiplas funções associadas ao metabolismo destes, incluindo biogênese, processamento, transporte núcleo-citoplasmático e degradação. A PABP também participa na síntese proteíca, via interações diretas com fatores de tradução, como o fator de iniciação eIF4G, parceiro da proteína de ligação ao capeIF4E. O objetivo desta Tese foi dar continuidade a caracterização de três homólogos da PABP identificados em Leishmania sp.(PABPs1,2 e 3), protozoários patogênicos de características biológicas diferenciadas. Em estudos prévios estas proteínas se mostraram abundantes e de expressão simultânea e a PABP1 se mostrou representada por isoformas associadas a sua fosforilação. Neste trabalho, foi observado que as três proteínas possuem localização citoplasmática, porém sob condições de inibição da transcrição, mas não de tradução ou processamento de mRNAs, as PABPs 2 e 3 migram para o núcleo. Esse comportamento está de acordo com ensaios de interação proteína-proteina, onde foi observada uma associação direta entre as PABPs 2 e 3, independente de RNA. A expressão de isoformas fosforiladas da PABP1 foi então analisada e observou-se que esta era afetada pela inibição dos processos de síntese de mRNAs. Investigando sua participação no processo de tradução, foram confirmadas interações específicas entre a PABP1 e um homólogo de eIF4G in vivo e interações inéditas entre esta e homólogos de eIF4E. As três proteínas se mostraram capazes de se associar com os polissomas de células em fase de crescimento exponencial, mas não de fase estacionária. Ao longo do trabalho foram mapeadosmotivos envolvidos com as interações observadas identificando características novas não descritas. Por fim, ensaios de co-imuno precipitação seguido de análise protéica por espectrometria de massa e de RNAs por RT-PCR confirmaram a presença de parceiros protéicos diferenciados para cada homólogo e mRNAs alvo para as PABPs 2 e 3 cuja tradução está associada a fase S do ciclo celular. Nossos resultados mostram que estas proteínas apresentam funções divergentes e reforça um papel mais geral da PABP1 na tradução. / In eukaryotes, the poly-A binding protein (PABP) binds specifically to a poly-adenosines sequence present in the 3' end of the mRNAs where performes multiple functions associated with their metabolism, including biogenesis, processing, nucleocytoplasmic transport, and degradation. The PABP also participates in protein synthesis, via direct interactions with translation factors such as the initiation factor eIF4G, partner of the cap binding protein eIF4E. The aim of this Thesis was to continue the characterization of the three PABP homologues identified in Leishmaniasp. (PABPs 1, 2 and 3), a pathogenic protozoan with differentiated biological characteristics. Previous studies have shown that these proteins are abundant and simultaneously expressed and that PABP1 is characterized by multiples isoformes associated to phosphorylation. In this study, it was observed that all three proteins have cytoplasmic localization, but under transcription inhibition conditions but not translation or mRNAs processing, the PABPs 2 and 3 migrate to the nucleus. This behavior is in agreement with a protein-protein interaction observed between PABPs 2 and 3, and this interaction isindependent of RNA. The expression of the phosphorylated isoforms of PABP1 was also analyzed and it was found that some of those isoforms were affected by the inhibition of the processessing of mRNAs sinthesis. When investigated their participation in thetranslation process were observed specific interactions between PABP1 and a homolog of eIF4G in vivoand a novel interaction between PABP 1 and the homologues of eIF4E. Throughout this study the motifs involved in the interactios mentioned above were mapped and new motifs not yet described were observed. All three proteins have been shown to be associated with polysomes in cells in in exponential phase, but not in stationary phase. Finally, co-immunoprecipitation assays followed by protein analysis by mass spectrometry and of RNAs by RT-PCR confirmed the presence of different protein partners and mRNAs targets to the PABPs 2 and 3 whose translation is associated with the S phase of cell cycle. Our results show that these proteins have different functions and reinforces a more general role of PABP1 in translation.

The rising demand for powerful oncolytic virotherapy agents has led to the identification of Maraba virus, one of the most potent oncolytic viruses from Rhabdoviridae family which displays high selectivity for killing malignant cells and low cytotoxicity in normal cells. Although the virus is readied to be used for clinical trials, the interactions between the virus and the host cells is still unclear. Using a newly developed interferon-sensitive mutant Maraba virus (MG1), we have identified two key regulators of global translation (4E-BP1 and eIF2α) responsible for the inhibition of protein synthesis in the infected cells. Despite the translational arrest upon viral stress, we showed an up-regulation of anti-apoptotic Bcl-xL protein that provides a survival benefit for the host cell, yet facilitates effective viral propagation. Given the fact that eIF5B canonically regulates 60S ribosome subunit end joining, and is able to replace the role of eIF2 in delivering initiator tRNA to the 40S ribosome subunit upon the phosphorylation of eIF2α, we have tested whether eIF5B mediates the translation of target mRNAs during MG1 infection. Our results show that the inhibition of eIF5B significantly down-regulates the level of Bcl-xL steady-state mRNA, thus indirectly attenuates viral propagation.

Internal ribosome entry site (IRES)-mediated translation is critical for the cell’s ability to respond to stress. Understanding how RNA binding proteins (IRES trans-acting factors; ITAFs) regulate IRESes is crucial to elucidating the mechanism of alternative translation initiation. Furthermore, determining how these ITAFs are regulated is central to understanding their functions in diseased states. I have identified the tumour suppressor programmed cell death 4 (PDCD4) as a novel ITAF of the XIAP and Bcl-xL IRES elements. I demonstrate that under normal conditions, PDCD4 acts to inhibit translation from these IRES elements by preventing formation of the 48S translation initiation complex. Furthermore, I show that in response to treatment with the pro-survival fibroblast growthfactor-2 (FGF-2), S6 kinase 2 (S6K2) phosphorylates PDCD4 leading to its degradation and the subsequent de-repression of XIAP and Bcl-xL translation. Importantly, I demonstrate the clinical significance of this regulation in glioblastoma multiforme (GBM) tumours where the loss of PDCD4 expression correlates with an increase in Bcl-xL protein and poor patient outcome. Additionally, re-expression of PDCD4 down-regulates Bcl-xL and decreases cell viability, and direct inhibition of Bcl-xL by a small molecule antagonist ABT-737 sensitizes GBM cells to the chemotherapeutic doxorubicin. Finally, I demonstrate that PDCD4 can be regulated at multiple levels. Importantly, I identify the RNA binding protein HuR as a regulator of microRNA (miR) -21 induced silencing of PDCD4. I show that HuR can bind the PDCD4 3'UTR and prevent miR-21 binding, and that a loss of PDCD4 expression following H2O2 treatment is mediated via miR-21. These results provide novel insight into the role of PDCD4 as a tumour suppressor and highlight the importance of ITAFs in cancer progression.

Protein synthesis is principally regulated at the initiation stage in which eIF4F complex plays an important role. The eIF4F complex contains three subunits - eIF4A, eIF4E and eIF4G. The eIF4E is cap binding protein, the eIF4A is RNA dependent helicase which unwinds secondary structures at mRNA and scaffolding eIF4G protein. The interaction with other translation initiation factors is important for protein synthesis. The goal of my thesis was to create a new Saccharomyces cerevisiae yeast strain with the human eIF4F factor. Firstly I replaced yeast eIF4E protein with human eIF4E protein. I used a cre/loxP recombination to prepare yeast strains with deleted genes eIF4GI (huΔ4G1) and eIF4GII (huΔ4G2). Characterization of the new yeast strains showed that the human eIF4E protein replaced yeast ortholog factor better than the eIF4E protein from yeast Candida albicans. First experiments showed putative role of the eIF4GII protein during the cell growth under the temperature and osmotic stress. Key words: translation initiation, eIF4E, eIF4G, Saccharomyces cerevisiae

Eukaryotic translation initiation factor 2 (eIF2) is a heterotrimeric G-protein that plays a critical role in protein synthesis regulation. eIF2-GTP binds Met-tRNAi to form the eIF2-GTP:Met-tRNAi ternary complex (TC), that is recruited to the 40S ribosomal subunit. Following GTP hydrolysis, eIF2-GDP is recycled back to TC by its guanine nucleotide exchange factor (GEF), eIF2B. Phosphorylation of the eIF2α subunit in response to various cellular stresses converts eIF2 into a competitive inhibitor of eIF2B, triggering the integrated stress response. Dysregulation of eIF2B activity is associated with a number of pathologies, including neurodegenerative diseases, metabolic disorders, and cancer. However, despite decades of research, the underlying molecular mechanisms remain unknown. This is due in large part to the absence of a structural understanding of the eIF2B assembly and of the eIF2B:eIF2 interaction. Common methods, such as yeast genetics, have been unable to unambiguously determine these mechanisms. Meanwhile, expanded interest in the integrated stress response has uncovered a diverse array of pathologies for which therapeutic modulation of the eIF2B:eIF2 interaction may ameliorate or overcome disease states. In this dissertation, a combination of structural and biochemical techniques is employed to elucidate the mechanisms of eIF2B action and its regulation by eIF2α phosphorylation. The aim is to provide a direct, unambiguous, structural understanding of eIF2B assembly and of eIF2B’s interactions with phosphorylated and unphosphorylated eIF2α. The work described here was among the first to challenge the widely held notion of a pentameric eIF2B assembly, as eventually confirmed by the recent publication of eIF2B’s crystal structure. The work further aims to overturn another long-standing assumption regarding the nature of inhibition of eIF2B activity: that competitive inhibition is mediated by a “direct effect” of the negatively charged phosphate group on the eIF2α:eIF2B interaction. Instead, we present evidence for an “indirect effect,” whereby phosphorylation disrupts a novel intramolecular interface within eIF2α, exposing an eIF2α surface that binds eIF2B and is responsible for inhibition of eIF2B. In the end, we combine a structural model of the eIF2B:eIF2 complex with our novel mechanism of inhibition, placing them within the larger thermodynamic context of eIF2-GDP recycling by eIF2B. / 2017-09-08T00:00:00Z