Mutational analysis of the alpha subunit of eIF2B provides insights into the role of eIF2B bodies in translational control and VWM disease.

Eukaryotic initiation factor 2B (eIF2B) serves as a vital control point within protein synthesis and regulates translation initiation in response to cellular stress. Mutations within eIF2B result in the fatal disease, leukoencephalopathy with vanishing white matter (VWM). Previous biochemical studies on VWM mutations have illustrated that changes in the activity of eIF2B poorly correlate with disease severity. This suggests that there may be additional characteristics of eIF2B contributing to VWM pathogenesis. Here, we investigated whether the localization of eIF2B to eIF2B bodies was integral for function and whether this localization could provide insight into the pathogenesis of VWM. We demonstrate that the regulatory subunit, eIF2Bα, is required for the assembly of eIF2B bodies in yeast and that loss of eIF2B bodies correlates with an inability of cells to regulate eIF2B activity. Mutational analysis of eIF2Bα showed that missense mutations that disrupt the regulation of eIF2B similarly disrupt the assembly of eIF2B bodies. In contrast, when eIF2Bα mutations that impact the catalytic activity of eIF2B were analyzed, eIF2B bodies were absent and instead eIF2B localized to small foci, termed microfoci. Fluorescence recovery after photobleaching analysis highlighted that within these microfoci, eIF2 shuttles more slowly indicating that formation of eIF2B bodies correlates with full eIF2B activity. When eIF2Bα VWM mutations were analyzed, a diverse impact on localization was observed, which did not seem to correlate with eIF2B activity. These findings provide key insights into how the eIF2B body assembles and suggest that the body is a fundamental part of the translational regulation via eIF2α phosphorylation.

Propagation of Parkinson's disease by extracellular vesicle production and secretion.

Parkinson's disease (PD) is a common neurodegenerative condition affecting a significant number of individuals globally, resulting in the presentation of debilitating motor and non-motor symptoms, including bradykinesia, resting tremor, as well as mood and sleep disorders. The pathology of PD has been observed to spread through the central nervous system resulting in progressive brain degeneration and a poor prognosis. Aggregated forms of the protein α-synuclein, particularly intermediary aggregates, referred to as oligomers, or preformed fibrils, have been implicated as the causative agent in the degeneration of neuronal processes, including the dysfunction of axonal transport, mitochondrial activity, and ultimately cellular death. Extracellular vesicles (EVs) have been strongly implicated in the propagation of PD pathology. Current observations suggest that aggregated α-synuclein is transported between neurons via small EVs in a series of exocytosis and endocytosis cellular processes leading to the observed spread of neurotoxicity and cellular death. Despite some understanding of the role of EVs in neurodegeneration, the exact mechanism by which these lipidic particles participate in the progression of Parkinson's pathology is not entirely understood. Here we review the current understanding of the role of EVs in the propagation of PD and explore their potential as a therapeutic target.

Mechanisms of translational regulation by a human eIF5-mimic protein.

The translation factor eIF5 is an important partner of eIF2, directly modulating its function in several critical steps. First, eIF5 binds eIF2/GTP/Met-tRNA(i)(Met) ternary complex (TC), promoting its recruitment to 40S ribosomal subunits. Secondly, its GTPase activating function promotes eIF2 dissociation for ribosomal subunit joining. Finally, eIF5 GDP dissociation inhibition (GDI) activity can antagonize eIF2 reactivation by competing with the eIF2 guanine exchange factor (GEF), eIF2B. The C-terminal domain (CTD) of eIF5, a W2-type HEAT domain, mediates its interaction with eIF2. Here, we characterize a related human protein containing MA3- and W2-type HEAT domains, previously termed BZW2 and renamed here as eIF5-mimic protein 1 (5MP1). Human 5MP1 interacts with eIF2 and eIF3 and inhibits general and gene-specific translation in mammalian systems. We further test whether 5MP1 is a mimic or competitor of the GEF catalytic subunit eIF2Bε or eIF5, using yeast as a model. Our results suggest that 5MP1 interacts with yeast eIF2 and promotes TC formation, but inhibits TC binding to the ribosome. Moreover, 5MP1 is not a GEF but a weak GDI for yeast eIF2. We propose that 5MP1 is a partial mimic and competitor of eIF5, interfering with the key steps by which eIF5 regulates eIF2 function.

Dysregulation of Stress-Induced Translational Control by Porphyromonas gingivalis in Host Cells.

Porphyromonas gingivalis contributes to the chronic oral disease periodontitis, triggering the activation of host inflammatory responses, inducing cellular stresses such as oxidation. During stress, host cells can activate the Integrated Stress Response (ISR), a pathway which determines cellular fate, by either downregulating protein synthesis and initiating a stress-response gene expression program, or by initiating programmed cell death. Recent studies have implicated the ISR within both host antimicrobial defenses and the pathomechanism of certain microbes. In this study, using a combination of immunofluorescence confocal microscopy and immunoblotting, the molecular mechanisms by which P. gingivalis infection alters translation attenuation during oxidative stress-induced activation of the ISR in oral epithelial cells were investigated. P. gingivalis infection alone did not result in ISR activation. In contrast, infection coupled with stress caused differential stress granule formation and composition. Infection heightened stress-induced translational repression independently of core ISR mediators. Heightened translational repression during stress was observed with both P. gingivalis-conditioned media and outer membrane vesicles, implicating a secretory factor in this exacerbated translational repression. The effects of gingipain inhibitors and gingipain-deficient P. gingivalis mutants confirmed these pathogen-specific proteases as the effector of exacerbated translational repression. Gingipains are known to degrade the mammalian target of rapamycin (mTOR) and the findings of this study implicate the gingipain-mTOR axis as the effector of host translational dysregulation during stress.

Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies.

Cytoplasmic RNA granules serve key functions in the control of messenger RNA (mRNA) fate in eukaryotic cells. For instance, in yeast, severe stress induces mRNA relocalization to sites of degradation or storage called processing bodies (P-bodies). In this study, we show that the translation repression associated with glucose starvation causes the key translational mediators of mRNA recognition, eIF4E, eIF4G, and Pab1p, to resediment away from ribosomal fractions. These mediators then accumulate in P-bodies and in previously unrecognized cytoplasmic bodies, which we define as EGP-bodies. Our kinetic studies highlight the fundamental difference between EGP- and P-bodies and reflect the complex dynamics surrounding reconfiguration of the mRNA pool under stress conditions. An absence of key mRNA decay factors from EGP-bodies points toward an mRNA storage function for these bodies. Overall, this study highlights new potential control points in both the regulation of mRNA fate and the global control of translation initiation.

Inhibition of translation initiation following glucose depletion in yeast facilitates a rationalization of mRNA content.

Glucose is the preferred carbon source for most eukaryotes and so it is important that cells can sense and react rapidly to fluctuations in glucose levels. It is becoming increasingly clear that the regulation of gene expression at the post-transcriptional level is important in the adaptation to changes in glucose levels, possibly as this could engender more rapid alterations in the concentrations of key proteins, such as metabolic enzymes. Following the removal of glucose from yeast cells a rapid inhibition of translation is observed. As a consequence, mRNPs (messenger ribonucleoproteins) relocalize into cytoplasmic granules known as P-bodies (processing bodies) and EGP-bodies. mRNA decay components localize into P-bodies, and thus these assemblies are likely to represent sites where mRNAs are targeted for degradation. In contrast, EGP-bodies lack any decay components and contain the eukaryotic translation initiation factors eIF4E, eIF4G and Pab1p, as well as other RNA-binding proteins. Therefore EGP-bodies probably constitute sites where mRNAs are earmarked for storage. So, it is possible that cells distinguish between transcripts and target them to either P-bodies or EGP-bodies depending on their functional value. The localization of mRNAs into these granules following glucose starvation may serve to preserve mRNAs that are involved in the diauxic shift to ethanol growth and entry into stationary phase, as well as to degrade mRNAs that are solely involved in glucose fermentation.

Dynamic cycling of eIF2 through a large eIF2B-containing cytoplasmic body: implications for translation control.

The eukaryotic translation initiation factor 2B (eIF2B) provides a fundamental controlled point in the pathway of protein synthesis. eIF2B is the heteropentameric guanine nucleotide exchange factor that converts eIF2, from an inactive guanosine diphosphate-bound complex to eIF2-guanosine triphosphate. This reaction is controlled in response to a variety of cellular stresses to allow the rapid reprogramming of cellular gene expression. Here we demonstrate that in contrast to other translation initiation factors, eIF2B and eIF2 colocalize to a specific cytoplasmic locus. The dynamic nature of this locus is revealed through fluorescence recovery after photobleaching analysis. Indeed eIF2 shuttles into these foci whereas eIF2B remains largely resident. Three different strategies to decrease the guanine nucleotide exchange function of eIF2B all inhibit eIF2 shuttling into the foci. These results implicate a defined cytoplasmic center of eIF2B in the exchange of guanine nucleotides on the eIF2 translation initiation factor. A focused core of eIF2B guanine nucleotide exchange might allow either greater activity or control of this elementary conserved step in the translation pathway.

Self-selecting peer groups formed within the laboratory environment have a lasting effect on individual student attainment and working practices.

Within the present study, we investigate the lasting effect of laboratory peer group interactions on the end of year attainment of Biosciences and Chemistry students. By asking students to identify who they primarily work with within the laboratory environment and evaluating the interactions through cluster analysis, we identified two main categories of laboratory peer groups: the first long-lived well-established pairings of two students, 'swans', who work together for all or the majority of the laboratory sessions; and the second dynamic fluid groups, 'dolphins', of between three and nine students who work with each other interchangeably. Statistical analysis is presented, which demonstrates that individuals within each laboratory peer group were likely to achieve a similar average mark at the end of the first year of study on the course. We identified the driving factors for the formation of these groups as friendship and perceived work ethic. There is a preference for high-achieving students to work with other high-achieving students and lower-achieving to group around a shared social background. Targeted interventions, in which pairings were selected by the tutor at the onset of the study, altered the ratio from long-lived pairs to more dynamic groups and increased students' willingness to work with others outside of their group but did not change the drivers of group formation or resulting pattern of achievement. We conclude with recommendations around group working within the laboratory environment.

Cellular eIF2B subunit localization: implications for the integrated stress response and its control by small molecule drugs.

Eukaryotic initiation factor 2 (eIF2) is a G protein critical for translation. It is tightly regulated in the integrated stress response (ISR) via phosphorylation of eIF2α and the subsequent control of eukaryotic initiation factor 2B (eIF2B), a multisubunit guanine nucleotide exchange factor. Through studying the localization of eIF2B subunits, we identified cytoplasmic eIF2B bodies in mammalian cells. We highlight a relationship between body size and the eIF2B subunits localizing to them; larger bodies contain all subunits and smaller bodies contain predominantly catalytic subunits. eIF2 localizes to eIF2B bodies and shuttles within these bodies in a manner that correlates with eIF2B activity. On stress, eIF2α-P localizes predominately to larger bodies and results in a decreased shuttling of eIF2. Interestingly, drugs that inhibit the ISR can rescue eIF2 shuttling in a manner correlating to levels of eIF2α-P. In contrast, smaller bodies show increased eIF2 shuttling in response to stress, which is accompanied by the localization of eIF2Bδ to these bodies, suggesting the formation of a novel trimeric complex of eIF2B. This response is mimicked by ISR-inhibiting drugs, providing insight into their potential mechanism of action. This study provides evidence that the composition and function of mammalian eIF2B bodies are regulated by the ISR and the drugs that control it.

An approach to studying the localization and dynamics of eukaryotic translation factors in live yeast cells.

The discovery of Green Fluorescent Protein (GFP) and the development of technology that allows specific proteins to be tagged with GFP has fundamentally altered the types of question that can be asked using cell biological methods. It is now possible not only to study where a protein is within a cell, but also feasible to study the precise dynamics of protein movement within living cells. We have exploited these technical developments and applied them to the study of translation initiation factors in yeast, focusing particularly on the key regulated guanine nucleotide exchange step involving eIF2B and eIF2. This chapter summarizes current methodologies for the tagging and visualization of GFP-tagged proteins involved in translation initiation in live yeast cells.

A sequence element downstream of the yeast HTB1 gene contributes to mRNA 3' processing and cell cycle regulation.

Histone mRNAs accumulate in the S phase and are rapidly degraded as cells progress into the G(2) phase of the cell cycle. In Saccharomyces cerevisiae, fusion of the 3' untranslated region and downstream sequences of the yeast histone gene HTB1 to a neomycin phosphotransferase open reading frame is sufficient to confer cell cycle regulation on the resulting chimera gene (neo-HTB1). We have identified a sequence element, designated the distal downstream element (DDE), that influences both the 3'-end cleavage site selection and the cell cycle regulation of the neo-HTB1 mRNA. Mutations in the DDE, which is located approximately 110 nucleotides downstream of the HTB1 gene, lead to a delay in the accumulation of the neo-HTB1 mRNA in the S phase and a lack of mRNA turnover in the G(2) phase. The DDE is transcribed as part of the primary transcript and binds a protein factor(s). Maximum binding is observed in the S phase of the cell cycle, and mutations that affect the turnover of the HTB1 mRNA alter the binding activity. While located in the same general region, mutations that affect 3'-end cleavage site selection act independently from those that alter the cell cycle regulation.

Granules harboring translationally active mRNAs provide a platform for P-body formation following stress.

The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems.

Glucose depletion inhibits translation initiation via eIF4A loss and subsequent 48S preinitiation complex accumulation, while the pentose phosphate pathway is coordinately up-regulated.

Cellular stress can globally inhibit translation initiation, and glucose removal from yeast causes one of the most dramatic effects in terms of rapidity and scale. Here we show that the same rapid inhibition occurs during yeast growth as glucose levels diminish. We characterize this novel regulation showing that it involves alterations within the 48S preinitiation complex. In particular, the interaction between eIF4A and eIF4G is destabilized, leading to a temporary stabilization of the eIF3-eIF4G interaction on the 48S complex. Under such conditions, specific mRNAs that are important for the adaptation to the new conditions must continue to be translated. We have determined which mRNAs remain translated early after glucose starvation. These experiments enable us to provide a physiological context for this translational regulation by ascribing defined functions that are translationally maintained or up-regulated. Overrepresented in this class of mRNA are those involved in carbohydrate metabolism, including several mRNAs from the pentose phosphate pathway. Our data support a hypothesis that a concerted preemptive activation of the pentose phosphate pathway, which targets both mRNA transcription and translation, is important for the transition from fermentative to respiratory growth in yeast.

Fusel alcohols regulate translation initiation by inhibiting eIF2B to reduce ternary complex in a mechanism that may involve altering the integrity and dynamics of the eIF2B body.

Recycling of eIF2-GDP to the GTP-bound form constitutes a core essential, regulated step in eukaryotic translation. This reaction is mediated by eIF2B, a heteropentameric factor with important links to human disease. eIF2 in the GTP-bound form binds to methionyl initiator tRNA to form a ternary complex, and the levels of this ternary complex can be a critical determinant of the rate of protein synthesis. Here we show that eIF2B serves as the target for translation inhibition by various fusel alcohols in yeast. Fusel alcohols are endpoint metabolites from amino acid catabolism, which signal nitrogen scarcity. We show that the inhibition of eIF2B leads to reduced ternary complex levels and that different eIF2B subunit mutants alter fusel alcohol sensitivity. A DNA tiling array strategy was developed that overcame difficulties in the identification of these mutants where the phenotypic distinctions were too subtle for classical complementation cloning. Fusel alcohols also lead to eIF2alpha dephosphorylation in a Sit4p-dependent manner. In yeast, eIF2B occupies a large cytoplasmic body where guanine nucleotide exchange on eIF2 can occur and be regulated. Fusel alcohols impact on both the movement and dynamics of this 2B body. Overall, these results confirm that the guanine nucleotide exchange factor, eIF2B, is targeted by fusel alcohols. Moreover, they highlight a potential connection between the movement or integrity of the 2B body and eIF2B regulation.

Medea SUMOylation restricts the signaling range of the Dpp morphogen in the Drosophila embryo.

Morphogens are secreted signaling molecules that form concentration gradients and control cell fate in developing tissues. During development, it is essential that morphogen range is strictly regulated in order for correct cell type specification to occur. One of the best characterized morphogens is Drosophila Decapentaplegic (Dpp), a BMP signaling molecule that patterns the dorsal ectoderm of the embryo by activating the Mad and Medea (Med) transcription factors. We demonstrate that there is a spatial and temporal expansion of the expression patterns of Dpp target genes in SUMO pathway mutant embryos. We identify Med as the primary SUMOylation target in the Dpp pathway, and show that failure to SUMOylate Med leads to the increased Dpp signaling range observed in the SUMO pathway mutant embryos. Med is SUMO modified in the nucleus, and we provide evidence that SUMOylation triggers Med nuclear export. Hence, Med SUMOylation provides a mechanism by which nuclei can continue to monitor the presence of extracellular Dpp signal to activate target gene expression for an appropriate duration. Overall, our results identify an unusual strategy for regulating morphogen range that, rather than impacting on the morphogen itself, targets an intracellular transducer.

Localization of the translational guanine nucleotide exchange factor eIF2B: a common theme for GEFs?

The eukaryotic initiation factor 2B (eIF2B) serves an essential recycling function in protein synthesis. As the guanine nucleotide exchange factor for eIF2, it recycles eIF2 from a GDP to a GTP bound form that is competent for translation initiation. Stress-dependent controls target this eIF2B-recycling step allowing a reprogramming of the global gene expression profile. In addition, a human disease, leukoencephalopathy with vanishing white matter (VWM), is caused by mutations in the eIF2B subunit genes. Recently, we have found that the eIF2B guanine nucleotide exchange factor resides in a specific cytoplasmic focus in the yeast, Saccharmoyces cerevisiae. eIF2B is a resident feature of this focus, whereas eIF2 shuttles to and fro. Moreover, the in vivo rate of eIF2 shuttling correlates with changes in guanine nucleotide exchange activity implicating this large cytoplasmic focus as a site of guanine nucleotide exchange. In this perspective, we discuss these findings in the wider context of the assortment of guanine nucleotide exchange factors.