Interaction of human tRNA-dihydrouridine synthase-2 with interferon-induced protein kinase PKR

PKR is an interferon (IFN)-induced protein kinase, which is involved in regulation of antiviral innate immunity, stress signaling, cell proliferation and programmed cell death. Although a low amount of PKR is expressed ubiquitously in all cell types in the absence of IFNs, PKR expression is induced at transcriptional level by IFN. PKR's enzymatic activity is activated by its binding to one of its activators. Double-stranded (ds) RNA, protein activator PACT and heparin are the three known activators of PKR. Activation of PKR in cells leads to a general block in protein synthesis due to phosphorylation of eIF2α on serine 51 by PKR. PKR activation is regulated very tightly in mammalian cells and a prolonged activation of PKR leads to apoptosis. Thus, positive and negative regulation of PKR activation is important for cell viability and function. The studies presented here describe human dihydrouridine synthase-2 (hDUS2) as a novel regulator of PKR. We originally identified hDUS2 as a protein interacting with PACT in a yeast two-hybrid screen. Further characterization revealed that hDUS2 also interacts with PKR through its dsRNA binding/dimerization domain and inhibits its kinase activity. Our results suggest that hDUS2 may act as a novel inhibitor of PKR in cells

Control of gene expression at the level of translation initiation

Protein synthesis is controlled at the level of translation initiation. Cells rapidly respond to environmental changes by disassembly of polysomes and recruitment of specific mRNAs from inactive ribonucleoprotein particles into polysomes active in translation. Recent insights have elucidated specific protein and RNA sequence interactions that are required to mobilize translation of selective mRNAs. The specificity of translational control provides a unique target to inhibit synthesis of specific polypeptides to control infectious disease as well as to control aberrant cell growth. In addition, greater understanding of the factors that limit protein synthesis is enabling the design of novel strategies to optimize protein expression and engineer host cells for enhanced growth and protein synthesis capacity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31300/1/0000206.pd

Control of eIF2 alpha kinases by tyrosine phosphorylation : implications for gene translation and anti-viral signaling

Control of mRNA translation is one of the major regulatory events in eukaryotic gene expression. Recent research has established the existence of a protein kinase family in mammalian cells, whose members phosphorylate the alpha (alpha) subunit of the eukaryotic initiation factor eIF2 (eIF2alpha) at serine 51 and regulate mRNA translation under various stress conditions. The interferon (IFN)-inducible double-stranded (ds) RNA-activated protein kinase PKR is the prototype of this family. Stress conditions activate PKR by autophosphorylation which leads to inhibition of global protein (including viral protein) synthesis and the apoptosis of infected cells. PKR has been well-characterized as a serine/threonine kinase. However, the tyrosine kinase property of PKR and its functional activity remains undetermined. This study demonstrates that human PKR possesses tyrosine kinase activity and undergoes autophosphorylation at tyrosine (Tyr) residues 101, 162 and 293 in vitro and in vivo. Phosphorylation at these tyrosine residues enhances dsRNA binding-efficiency as well as the dimerization of PKR, which in turn favours the full-scale kinase activation and its substrate phosphorylation. Biologically, tyrosine phosphorylated PKR mediates the anti-viral and cellular anti-proliferation activity of the enzyme through its ability to regulate protein synthesis. In addition, the IFNs modulate PKR at both the transcriptional and posttranslational level. Specifically, tyrosine phosphorylation of PKR is inducible in response to stimulations with IFNs. The Janus kinases (Jaks), a group of cytoplasmic tyrosine kinases, are the upstream enzymes which phosphorylate PKR at Tyr101 and Tyr293 in vitro and in vivo. Moreover, induction of PKR tyrosine phosphorylation by IFNs presents a critical missing link between IFN signaling and the translational machinery which contributes to the early effect of IFNs in inhibiting viral protein synthesis. Such a prompt reaction might allow cells to induce IFN responsive-genes and further fortify the antiviral state of the host

INVESTIGATION OF HOW ENDOPLASMIC RETICULUM STRESS CAUSES INSULIN RESISTANCE AND NEUROINFLAMMATION

Endoplasmic reticulum (ER) stress is caused by the accumulation of mis/unfolded proteins in the ER. ER stress signalling pathways termed the unfolded protein response are employed to alleviate ER stress through increasing the folding capacity and decreasing the folding demand of the ER as well as removing mis/unfolded proteins. However, ER stress signalling pathways induce diverse cellular changes beyond changes to the ER. This study aims to further investigate some of these ER stress-mediated events. ER stress can cause activation of JNK. Prolonged ER stress-mediated JNK activation is reported to promote apoptosis whilst both acute and long-lasting JNK activation is proposed to cause insulin resistance. To begin with it is reported in this thesis that acute ER stress-induced JNK activation, which is dependent on IRE1α and TRAF2, promotes survival. In contrast to other studies, this thesis provides evidence that acute ER stress-mediated JNK activation does not inhibit insulin signalling during ER stress in several cell lines. However, prolonged ER stress, in four different cell lines, does inhibit insulin signalling in a JNK independent manner. This study argues that ER-stress-induced insulin resistance during prolonged ER stress involves inhibition of trafficking of newly synthesised insulin receptors through the secretory pathway to the plasma membrane. Finally ER stress can activate inflammatory signalling pathways other than JNK and thus ER stress may promote inflammation. Neuroinflammation and ER stress are reported in Parkinson’s disease (PD) yet a link between them has so far not been investigated. Using a cellular model of PD, it is reported in this thesis that ER stress has the potential to activate neuroinflammation in PD

New mechanisms modulating S100A8 gene expression

S100A8 is a highly-expressed calcium-binding protein in neutrophils and activated macrophages, and has proposed roles in myeloid cell differentiation and host defense. Functions of S100A8 are not fully understood, partly because of difficulties in generating S100A8 knockout mice. Attempts to silence S100A8 gene expression in activated macrophages and fibroblasts using RNA interference (RNAi) technology were unsuccessful. Despite establishing validated small interfering RNA (siRNA) systems, enzymaticallysynthesized siRNA targeted to S100A8 suppressed mRNA levels by only 40% in fibroblasts activated with FGF-2+heparin, whereas chemically-synthesized siRNAs suppressed S100A8 driven by an S100A8-expression vector by ~75% in fibroblasts. Suppression of the gene in activated macrophages/fibroblasts was low, and some enzymatically-synthesized siRNAs to S100A8, and unrelated siRNA to GAPDH, induced/enhanced S100A8 expression in macrophages. This indicated that S100A8 may be upregulated by type-1 interferon (IFN). IFN-β enhanced expression, but did not directly induce S100A8. Poly (I:C), a synthetic dsRNA, directly induced S100A8 through IL-10 and IFN-dependent pathways. Induction by dsRNA was dependent on RNA-dependent protein kinase (PKR), but not cyclooxygenase-2, suggesting divergent pathways in LPS- and dsRNA-induced responses. New mechanisms of S100A8 gene regulation are presented, that suggest functions in anti-viral defense. S100A8 expression was confirmed in lungs from influenza virus-infected mice and from a patient with severe acute respiratory syndrome (SARS). Multiple pathways via mitochondria mediated S100A8 induction in LPS-activated macrophages; Generation of reactive oxygen species via the mitochondrial electron transport chain and de novo synthesis of ATP may be involved. This pathway also regulated IL-10 production, possibly via PKR. Extracellular ATP and its metabolites enhanced S100A8 induction. Results support involvement of cell stress, such as transfection, in S100A8 expression. A breast tumor cell line (MCF-7) in which the S100A8 gene was silenced, was established using micro RNA technology; S100A8 induction by oncostatin M was reduced by >90% in stably-transfected cells. This did not alter MCF-7 growth. The new approach to investigate the role of S100A8 in a human tumor cell line may assist in exploring its functions and lead to new studies concerning its role in cancer

The role of eukaryotic initiation factor 2 alpha phosphorylation pathway in translational control and virus-mediated oncongenesis /

Two important steps of translation initiation include the recognition of the mRNA cap structure by eIF4E and the recycling of erF2. Each step is thought to be regulated independently through the interaction of eIF4E with 4E-BPs and the phosphorylation of the a subunit of eIF2 at serine 51. Phosphorylation of eIF2alpha by dsRNA-dependent protein kinase PKR inhibits protein synthesis in cells subjected to virus infection; therefore, most viruses have evolved mechanism to overcome the deleterious effects of PKR. The human papillomavirus (HPV) E6 oncoprotein contributes to virus-induced pathogenicity through multiple mechanisms including the inhibition of apoptosis and the blockade of interferon action. This study demonstrates a novel function of PKR providing a link between the two mechanisms of regulation of translation initiation. Activation of PKR induces the PI3K-PKB/Akt and FRAP/mTOR pathways leading to S6 and 4E-BP1 phosphorylation upon stress conditions and in response to growth stimuli. Induction of the PI3K pathway antagonizes the apoptotic effects of PKR activation, but does not intervene with its translational inhibitory activity. Investigating functional interaction of HPV E6 and PKR, we determined that BPV-18 E6 protein synthesis is regulated by eIF2alpha phosphorylation. On the other hand, E6 oncoprotein is able to rescue cells from PKR-mediated inhibition of protein synthesis and induction of apoptosis by promoting eIF2alpha dephosphorylation through physical association with GADD34/PP1 holophosphatase complex. These findings demonstrate, for the first time, the ability of PKR to activate a growth-stimulatory pathway; PI3K. Furthermore, it demonstrates role of oncogenic E6 in antagonizing signaling pathways induced by PKR including eIF2alpha phosphorylation and PI3K pathway

Characterisation of the contribution of the kinase and RNase activities of Ire1α to activation of apoptotic JNK signalling

The unfolded protein response (UPR) is a highly conserved mechanism by which all eukaryotes respond to endoplasmic reticulum (ER) stress. In higher eukaryotes this response is mediated by three ER transmembrane stress sensors: activating transcription factor 6 (ATF6/), PKR-like ER kinase (PERK) and inositol requiring 1 (IRE1/). IRE1 is the most highly conserved of the three ER stress sensors and is also the only sensor to mediated UPR signalling via two different enzymatic domains. It is currently believed that during prolonged ER stress, the RNase domain of IRE1α provides cytoprotection via XBP1 splicing, whilst the kinase domain initiates proapoptotic JNK signalling via interaction with the adaptor protein TRAF2. However, characterising how these domains contribute to cell fate decisions is complicated by the fact that traditional models use ER stress mimetic drugs, which activate all three branches of the UPR and thus make it difficult to attribute downstream events to individual effectors. Therefore, the aim of the research presented in this thesis was to produce a model that allowed isolated activation of IRE1α in order to determine the contribution of its kinase and RNase activities to proapoptotic JNK signalling, without input from other upstream effectors. Using the Fv2E-IRE1α system, the data presented in this thesis provides novel insight into the mechanism by which IRE1α instigates proapoptotic JNK signalling by suggesting that a functional kinase domain is not required for IRE1α to interact with TRAF2 and that endoribonuclease function may be required for downstream JNK activation in humans. Furthermore, evidence is also provided to suggest that, whilst kinase activity is not required for interaction with TRAF2, it is required for downstream JNK activation. This gives rise to the possibility that, contrary to current knowledge, the IRE1α kinase domain has the capacity to phosphorylate proteins other than IRE1α

Characterization of the human Mov34 and PACT proteins and analyses of their interaction with dengue virus RNA

Orientador: Nilson Ivo Tonin ZanchinTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O combate à dengue atualmente está limitado praticamente aos esforços de eliminação do mosquito transmissor, o Aedes aegypti, porém esta estratégia não tem se mostrado eficiente. O desenvolvimento de novos instrumentos de combate à dengue requer, portanto, maior conhecimento sobre a biologia do vírus com relação à sua interação com seus hospedeiros. O genoma do vírus é constituído por um RNA simples-fita de polaridade positiva e possui duas regiões não traduzidas (5¿ e 3¿ UTR). A região 5¿UTR viral possui organização similar à dos mRNAs eucarióticos, diferentemente da região 3¿UTR que é longa e não possui cauda de poli(A). Em vez disso, na região 3¿UTR encontram-se estruturas conservadas entre os diferentes Flavivirus, dentre elas a estrutura 3¿ stem-loop (3¿SL) que é indispensável para a replicação do RNA viral. O objetivo do nosso estudo foi identificar novas proteínas humanas capazes de interagir com a estrutura 3¿SL do RNA do vírus da dengue. Dados da literatura descrevem que a proteína Mov34 de camundongo interage com 3¿SL do vírus da encefalite japonesa. Devido à alta similaridade entre as proteínas ortólogas humana e de camundongo, bem como das respectivas estruturas 3¿SL dos vírus da dengue e da encefalite japonesa, foi testada a interação entre a Mov34 humana com o 3¿SL do vírus da dengue. Porém, em nenhuma das condições testadas foi possível obter evidência de interação da Mov34 humana com 3¿SL dos vírus da dengue e da encefalite japonesa. Para a identificação de novas proteínas que são capazes de interagir com a estrutura 3¿SL do RNA do vírus da dengue foi utilizado o ensaio de triplo-híbrido de levedura. A proteína humana PACT, conhecida como proteína celular ativadora de PKR, foi isolada neste ensaio utilizando 3¿SL como isca. PKR é uma quinase ativada por PACT ou RNA dupla-fita. A ativação de PKR leva a um estado antiviral adquirido pela fosforilação do fator de iniciação da tradução eIF2a e conseqüente inibição da tradução. Além disso, PKR está envolvida em outras vias de transdução de sinal e na resposta celular à proteínas desenoveladas. A ação antiviral de PACT é evidenciada pela ação de proteínas dos vírus influenza A e herpes simplex tipo 1 que inibem a ativação de PKR por PACT e por RNA dupla-fita. A interação direta de PACT com 3¿SL do RNA do vírus da dengue foi confirmada por ensaio de UVcrosslinking PACT possui três domínios de interação com RNA dupla-fita, sendo que os dois domínios N-terminais são responsáveis pela sua interação com o 3¿SL. Foi identificada uma região específica do 3¿SL, o stem-loop superior, onde PACT interage com maior afinidade. Além disso, foi mostrado que PACT endógena de células HEK293 é capaz de interagir com o 3¿SL biotinilado. Para caracterizar a função desta interação durante a infecção viral, foi desenvolvida uma linhagem celular com inibição da expressão de PACT através da técnica de RNA de interferência. Com esta linhagem poderemos analisar a importância da interação entre PACT e o RNA do vírus da dengue quanto à ativação e/ou inibição de PKR durante a infecção viralAbstract: The combat to the dengue virus is basically limited to the efforts in eliminating the transmitter mosquito, the Aedes aegypti. But this strategy is not very efficient. The development of new instruments of combat to dengue virus requires improved knowledge about the virus biology and its relation to hosts. The dengue virus genome is a single-stranded RNA of positive polarity flanked by a 5¿ untranslated region (UTR) of ~100 bases and a highly structured 3¿ UTR of ~450 bases. As many other viruses, dengue encodes the enzymes required for its genome replication, but relies completely on the host translational machinery to synthesize its proteins. The essential difference between host cellular mRNAs and dengue virus genome RNA involves the 3¿UTR, which instead of a polyadenylate tail contains highly conserved structural elements, including the 3' stem-loop (3¿SL), located at the 3' terminus of the 3'UTR of many flaviviruses that is essential for their replication. The aim of this study is to identify new human proteins capable of interacting with dengue virus RNA 3¿SL structure. Literature data describe that the murine Mov34 protein interacts with Japanese encephalitis virus 3¿SL. Giving the high similarity between the human and murine ortholog proteins, as well as the conservation of the Flavivivirus RNA 3¿SL structure, we tested the interaction between the human Mov34 and the dengue virus 3¿SL. However, no interaction was detected under the conditions used in this work. In addition, the yeast three-hybrid system was used to screen for novel proteins that interact with the dengue virus 3¿SL. Human PACT, known as the cellular protein activator of PKR, was identified as a putative 3¿SL-interacting protein. PKR is an interferon-inducible, PACT or double-stranded RNA activated protein kinase. Activated PKR phosphorylates the translation initiation factor eIF2a, inhibiting translation of cellular and viral RNAs, leading to a cellular antiviral state. PACT and doublestranded RNA activation of PKR is inhibited by influenza A and herpes simplex type 1 virus proteins during viral infection, indicating that PACT plays a role in the cellular antiviral state. Direct interaction between PACT and 3¿SL was confirmed by UV-crosslinking assays. PACT contains three doublestranded RNA interaction motifs, but only the two N-terminal motifs are responsible for 3¿SL interaction. A 3¿SL specific region, the top stem-loop, was identified to interact with PACT with higher affinity. Furthermore, HEK293 cells endogenous PACT interacts with biotin-labeled 3¿SL. To further characterize PACT-3¿SL interaction during dengue virus infection, a cell line with low expression of PACT was developed using the RNA interference technique. This cell line will be used to determine the propagation rate of dengue virus which is expected to reveal the importance of PACT either for the cell antiviral state or for dengue virus proliferationDoutoradoGenetica Animal e EvoluçãoDoutor em Genetica e Biologia Molecula

Novel Therapeutic Concepts in Targeting Glioma

Novel Therapeutic Concepts for Targeting Glioma offers a comprehensive collection of current information and the upcoming possibilities for designing new therapies for Glioma by an array of experts ranging from Cell Biologists to Oncologists and Neurosurgeons. A variety of topics cover therapeutic strategies based on Cell Signaling, Gene Therapy, Drug Therapy and Surgical methods providing the reader with a unique opportunity to expand and advance his knowledge of the field