Cellular needs are fulfilled by the combined activity of functional proteins. Consequently, cells are equipped with a complex proteostatic network that controls protein production in response to cellular requisites. Thereby, protein synthesis is induced or attenuated depending on the particular cellular conditions. One of the mechanisms to control protein synthesis is the phosphorylation of eIF2, which triggers the so-called Integrated Stress Response (ISR). Kinases that sense stresses induce the phosphorylation of eIF2, which, on the one hand, attenuates global rates of protein synthesis and, on the other hand, activates the expression of specific proteins that help to alleviate the stress. One of the proteins preferentially expressed during the ISR is PPP1R15A, a regulatory subunit of Protein Phosphatase 1 (PP1). The PP1/PPP1R15A holophosphatase dephosphorylates eIF2 and terminates the ISR once the stresses are resolved. Hence, eIF2 kinases and phosphatases work together to control levels of phosphorylated eIF2. Maintaining the right balance between the activity of these kinases and phosphatases is important, as is seen by the correlation between their perturbance and the appearance of certain cellular malfunctions or diseases. However, affecting this balance has been also suggested to have beneficial effects. For example, genetic interference with the PPP1R15A regulatory subunit is proposed to confer protection to mice and cells under ER-stress conditions. This observation led to the search for compounds with the ability to modulate the ISR, in particular, by acting on the eIF2 phosphatases. Three compounds (Salubrinal, Guanabenz and Sephin1) have been proposed as eIF2 phosphatase inhibitors with potential use as therapeutic tools in protein misfolding diseases. However, their precise mechanism of action and their direct effect on the enzyme remains an open question. This thesis focuses on the in vitro reconstitution of the eIF2 phosphatase, which served as a platform for characterizing the enzyme (in terms of its structure, activity and assembly) and for studying the proposed inhibitors. This report details key structural features of the PPP1R15/PP1 holophosphatase, the discovery of a cellular cofactor of the enzyme and the conclusions obtained after analysing the effect of its proposed inhibitors. It also includes the development of several in vitro assays, which could potentially be used to screen libraries of compounds in search for modulators of the enzyme.

The Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. Although mainly considered to be a protective mechanism, prolonged and severe ISR can result in cell death. The ISR is activated by diverse stress pathways converged on phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2) that inhibits the guanine nucleotide exchange activity of its partner eIF2B and attenuates overall rates of protein synthesis. Numerous mutations in eIF2B are linked to a fatal neurodegenerative disease of vanishing white matter. A new chemical inhibitor of the ISR (ISRIB), a bis-O-arylglycolamide, can reverse the attenuation of mRNA translation by phosphorylated eIF2 protecting mice from prion-induced neurodegeneration and traumatic brain injury. The work presented in this dissertation describes identification of mammalian eIF2B as a cellular target of ISRIB by implementing biochemical, biophysical, structural and chemogenetic methods. The herein reported cryo-electron microscopy-based structure of eIF2B uncovers a novel allosteric site on the translation factor capturing the ISRIB-binding pocket at the interface between its β and δ regulatory subunits. The extensive CRISPR/ Cas9-based screen for ISRIB-resistant and analogue-sensitive phenotypes revealed residues on the eIF2B dimer interface important for ISRIB binding. Based on the results reported in this dissertation along with the similar findings of others the potential molecular basis of ISRIB action, and its implication for the regulation of eIF2B's activity is broadly discussed. The identification of the ISRIB binding pocket away from the known interaction sites between eIF2B and eIF2 is also put into the context of a possible molecular mechanism of eIF2B's guanine exchange inhibition by phosphorylated eIF2. The work described in this dissertation provides new insight into the translational regulation and points to the importance of fine-tuning the activity of translation factors by small chemical molecules.

Eukaryotic initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor (GEF) that converts inactive eIF2 GDP-bound binary complexes into active eIF2 GTP-bound complexes that can bind initiator t-RNA molecules and ribosomes to begin translation. eIF2B is functionally divided into two subcomplexes: the catalytic core comprised of eIF2B epsilon and eIF2B gamma, and the regulatory core comprised of eIF2B alpha, eIF2B beta and eIF2B delta. While the catalytic subunits are responsible for exerting GEF activity, the regulatory subunits recognize eIF2 and respond to eIF2 alpha phosphorylation. Cellular stress, such as virus infection, inhibits host protein synthesis by activating specific kinases that are capable of phosphorylating the alpha subunit of eIF2, which can then sequester eIF2B to stall guanine nucleotide exchange by a currently unresolved mechanism. Importantly, we demonstrate that loss of eIF2B alpha or expression of a variant of the human eIF2B alpha subunit harboring a single point mutation (T41A) is sufficient to neutralize the consequences of eIF2 alpha phosphorylation, and render primary MEFs significantly more susceptible to vesicular stomatitis virus infection. To extend this analysis, we further exhibit the vital function of eIF2B alpha in protein synthesis through phenotypic studies in yeast. Here, we report that this subunit can sufficiently substitute for its yeast counterpart, GCN3, and reproduce similar growth phenotypes under normal and amino acid deprived conditions. In addition, the human eIF2B alpha-T41A variant was unable derepress GCN4 translation in response to an inhibitor of amino acid biosynthesis in yeast, an activity that requires sensitivity to phosphorylation of the yeast eIF2 alpha homolog, SUI2. Previously, we have demonstrated that vesicular stomatitis virus can infect and replicate to high levels in tumor cells. Moreover, these cells appear to contain defects in eIF2 alpha-mediated translational control, plausibly due to disregulation of eIF2B activity, which overcomes the inhibitory effects of eIF2 alpha phosphorylation. Our data suggest a role for eIF2B, specifically eIF2B alpha, in suppression of translation following virus infection, and imply that this complex may contribute to oncogenic transformation. These results emphasize the importance of eIF2B alpha in mediating eIF2 kinase translation inhibitory activity and may provide insight into the complex nature of viral oncolysis and cellular transformation.

Indiana University-Purdue University Indianapolis (IUPUI) / Human skin, consisting of the outer epidermis and inner dermis, serves as a barrier that protects the body from an onslaught of environmental stresses. Keratinocytes in the stratified epidermis undergo sequential differentiation that consists of multiple layers of cells differing in structure and function. Therefore, keratinocytes must not only combat environmental stress, but need to undergo massive changes in gene expression and morphology to form a proper barrier. One mode by which cells cope with stress and differentiation is through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α-P), which causes global inhibition of protein synthesis coincident with preferential translation of select gene transcripts. Translational repression allows stressed cells to conserve energy and prioritize pro-survival processes to alleviate stress damage. Since eIF2α kinases are each activated by distinct types of stress, this pathway is referred to as the Integrated Stress Response (ISR). We sought to identify the roles of the ISR in the keratinocyte response to the stresses associated with differentiation and ultraviolet B (UVB) irradiation. In this thesis, we show that both general and gene-specific translational control in the ISR are activated following differentiation or UVB irradiation of human keratinocytes. ISR deficiency through genetic modifications or pharmacological interventions caused severe divergence from the appropriate keratinocyte response to differentiation or UVB. Differentiation genes were selectively translated by eIF2α-P, and inhibition of the ISR diminished their induction during differentiation. Furthermore, loss of the eIF2α kinase GCN2 (EIF2AK4) adversely affected the ability of keratinocytes to stratify in three dimensional cultures. Our analysis also revealed a non-canonical ISR response following UVB irradiation, in which downstream factors ATF4 (CREB2) and CHOP (DDIT3/GADD153) were poorly expressed due to repressed transcription, despite preferential translation in response to eIF2α-P. The ISR was cytoprotective during UVB and we found that eIF2α-P was required for a UVB induced G1 arrest, cell fate determination, and DNA repair via a mechanism involving translational control of human CDKN1A (p21 protein) transcript variant 4 mRNA. Collectively, this thesis describes novel roles for the ISR in keratinocyte differentiation and response to UVB, emphasizing the utility of targeting translational control in skin disease therapy.

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

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No description available.

Les cellules dendritiques (DCs) sont les plus efficaces cellules présentatrices d'antigène. La détection de motifs pathogènes, tel que lipopolysaccharides bactériens et ARNs double-brins (ARNdb) viraux, par les DCs provoque leur maturation et induit de nombreux changements morphologiques et biochimiques permettant aux DCs d'acquérir leurs puissants fonctions activatrices des cellules T. Dans ce travail, les réponses des DCs à l'ARNdb ont été analysées. Nous avons montré que, en réponse à au poly I:C, un analogue synthétique des ARNdb, les DCs montent une réponse de stress intégré spécifique au cours de laquelle le facteur de transcription ATF4 et le cofacteur de la phosphatase 1, GADD34, sont exprimés. Les DCs activées par le poly I:C présentent un profil de transcrits similaire à ce qui est produit au cours d'une ‘unfolded protein response'. GADD34 est important pour contrebalancer la phosphorylation du facteur d'initiation de la synthèse protéique eIF2α par la kinase PKR au cours de l'activation des DCs. Contrairement aux fibroblastes embryonnaires murins, les DCs résistent à l'inhibition de la synthèse des protéines induite en réponse à la stimulation avec poly I:C. Néanmoins, l'expression de GADD34 n'a pas un impact majeur sur la synthèse protéique globale. Par contre, GADD34 a été démontré être absolument nécessaire à la production d'interféron du type I et d'IL-6 par les fibroblastes et les DCs en réponse à l'ARNdb. Cette observation a des implications importantes en liant la détection des pathogènes avec les voies intégrés de réponse au stress. / Dendritic cells (DCs) are the most important antigen presenting cells. In response to inflammatory stimulation, DCs display a distinct pattern of differentiation that exhibits specific mechanisms to control the immune response. In this work the responses to dsRNA were analyzed. We have shown that in response to a mimic of dsRNA, polyriboinosinic:polyribocytidylic acid (poly I:C), DCs mount a specific integrated stress response during which the transcription factor ATF4 and the growth arrest and DNA damage-inducible protein 34 (GADD34), a phosphatase 1 (PP1) cofactor, are expressed. GADD34 is important to counteract phosphorylation of eIF2α by PKR. In contrast to murine embryonic fibroblasts (MEFs), DCs resist to protein synthesis inhibition induced in response to cytosolic dsRNA. Nevertheless, GADD34 expression does not have a major impact on global protein synthesis. Importantly, GADD34 was shown to be absolutely required for type I-IFN and IL-6 production by MEFs and DCs in response to dsRNA. This observation has important implications in linking pathogen detection with the integrated stress response pathways. The importance of this link is further underlined by the extreme susceptibility of GADD34-deficient fibroblasts and neonate mice to Chikungunya virus infection.

A malária é causada por parasitas Plasmodium falciparum, e embora vários aspectos ainda sejam desconhecidos, é sabido que a regulação do ciclo intraeritrocítico é crítica para a compreensão do ciclo celular e patogênese. A melatonina modula o ciclo de P. falciparum promovendo a sincronização, mas, o mecanismo de transdução de sinal é parcialmente caracterizado, envolvendo variações citosólicas de cálcio, AMPc e ativação da PKA. Modificações pós-traducionais participam na via de sinalização, e diversas proteínas quinase podem estar envolvidas na sinalização por melatonina. eIF2α fosforilado é capaz de ativar a tradução de mRNAs em resposta a situações desfavoráveis. O genoma de P. falciparum codifica três quinases cujo substrato é eIF2α: PfeIK1, PfeIK2 e PfPK4. Investigamos o papel da PfeIK1 na via de transdução de sinal de melatonina usando cepas nocaute para PfeIK1. Além disso, os efeitos de metabólitos da degradação do heme sobre a fosforilação de eIF2α. Sugerimos que o mecanismo de fosforilação e defosforilação de eIF2α possam ser relevantes para a resposta do parasita a hemina ou biliverdina. Nossos dados indicam a PfeIK1, juntamente com a PfK7 e PKA, como quinases-chaves no controle do desenvolvimento durante o ciclo intraeritrocítico. / Malaria is caused by Plasmodium falciparum parasites, and although some aspects are still unknown, its established that the intraerythrocytic cycle regulation is critic for understanding the cell cycle and pathogenesis of the parasite. Melatonin modulates the cycle of P. falciparum promoting synchronization; however, the signal transduction mechanism is partially characterized, and it contains cytosolic variations of calcium, AMPc and PKA activation. Post-translational modifications participate in this signal pathway, and several kinase proteins may be involved in melatonin signaling pathway. Phosphorylated eIF2α is able to activate mRNAs translation in stress conditions. The genome of P. falciparum encodes three kinases whose substrate is eIF2α: PfeIK1, PfeIK2 e PfPK4. We investigate the role of PfeIK1 in melatonin signaling pathway by using knockout strains for PfeIK1. Furthermore, we investigate the effects of heme degradation metabolities in eIF2α phosphorylation. We suggest that the phosphorylation and dephosphorylation mechanisms of eIF2α may be relevant for parasite response to heme and billiverdin. Our data indicates PfeIK1, PfK7 and PKA as key kinases for the development control during intraerythrocytic cycle.

A malária é causada por parasitas Plasmodium falciparum, e embora vários aspectos ainda sejam desconhecidos, é sabido que a regulação do ciclo intraeritrocítico é crítica para a compreensão do ciclo celular e patogênese. A melatonina modula o ciclo de P. falciparum promovendo a sincronização, mas, o mecanismo de transdução de sinal é parcialmente caracterizado, envolvendo variações citosólicas de cálcio, AMPc e ativação da PKA. Modificações pós-traducionais participam na via de sinalização, e diversas proteínas quinase podem estar envolvidas na sinalização por melatonina. eIF2α fosforilado é capaz de ativar a tradução de mRNAs em resposta a situações desfavoráveis. O genoma de P. falciparum codifica três quinases cujo substrato é eIF2α: PfeIK1, PfeIK2 e PfPK4. Investigamos o papel da PfeIK1 na via de transdução de sinal de melatonina usando cepas nocaute para PfeIK1. Além disso, os efeitos de metabólitos da degradação do heme sobre a fosforilação de eIF2α. Sugerimos que o mecanismo de fosforilação e defosforilação de eIF2α possam ser relevantes para a resposta do parasita a hemina ou biliverdina. Nossos dados indicam a PfeIK1, juntamente com a PfK7 e PKA, como quinases-chaves no controle do desenvolvimento durante o ciclo intraeritrocítico. / Malaria is caused by Plasmodium falciparum parasites, and although some aspects are still unknown, its established that the intraerythrocytic cycle regulation is critic for understanding the cell cycle and pathogenesis of the parasite. Melatonin modulates the cycle of P. falciparum promoting synchronization; however, the signal transduction mechanism is partially characterized, and it contains cytosolic variations of calcium, AMPc and PKA activation. Post-translational modifications participate in this signal pathway, and several kinase proteins may be involved in melatonin signaling pathway. Phosphorylated eIF2α is able to activate mRNAs translation in stress conditions. The genome of P. falciparum encodes three kinases whose substrate is eIF2α: PfeIK1, PfeIK2 e PfPK4. We investigate the role of PfeIK1 in melatonin signaling pathway by using knockout strains for PfeIK1. Furthermore, we investigate the effects of heme degradation metabolities in eIF2α phosphorylation. We suggest that the phosphorylation and dephosphorylation mechanisms of eIF2α may be relevant for parasite response to heme and billiverdin. Our data indicates PfeIK1, PfK7 and PKA as key kinases for the development control during intraerythrocytic cycle.