GCN2 in Viral Defence and the Subversive Tactics Employed by Viruses.

The recent SARS-CoV-2 pandemic and associated COVID19 disease illustrates the important role of viral defence mechanisms in ensuring survival and recovery of the host or patient. Viruses absolutely depend on the host's protein synthesis machinery to replicate, meaning that impeding translation is a powerful way to counteract viruses. One major approach used by cells to obstruct protein synthesis is to phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). Mammals possess four different eIF2α-kinases: PKR, HRI, PEK/PERK, and GCN2. While PKR is currently considered the principal eIF2α-kinase involved in viral defence, the other eIF2α-kinases have also been found to play significant roles. Unsurprisingly, viruses have developed mechanisms to counteract the actions of eIF2α-kinases, or even to exploit them to their benefit. While some of these virulence factors are specific to one eIF2α-kinase, such as GCN2, others target all eIF2α-kinases. This review critically evaluates the current knowledge of viral mechanisms targeting the eIF2α-kinase GCN2. A detailed and in-depth understanding of the molecular mechanisms by which viruses evade host defence mechanisms will help to inform the development of powerful anti-viral measures.

Yeast as a Model to Understand Actin-Mediated Cellular Functions in Mammals-Illustrated with Four Actin Cytoskeleton Proteins.

The budding yeast Saccharomyces cerevisiae has an actin cytoskeleton that comprises a set of protein components analogous to those found in the actin cytoskeletons of higher eukaryotes. Furthermore, the actin cytoskeletons of S. cerevisiae and of higher eukaryotes have some similar physiological roles. The genetic tractability of budding yeast and the availability of a stable haploid cell type facilitates the application of molecular genetic approaches to assign functions to the various actin cytoskeleton components. This has provided information that is in general complementary to that provided by studies of the equivalent proteins of higher eukaryotes and hence has enabled a more complete view of the role of these proteins. Several human functional homologues of yeast actin effectors are implicated in diseases. A better understanding of the molecular mechanisms underpinning the functions of these proteins is critical to develop improved therapeutic strategies. In this article we chose as examples four evolutionarily conserved proteins that associate with the actin cytoskeleton: 1) yeast Hof1p/mammalian PSTPIP1, 2) yeast Rvs167p/mammalian BIN1, 3) yeast eEF1A/eEF1A1 and eEF1A2 and 4) yeast Yih1p/mammalian IMPACT. We compare the knowledge on the functions of these actin cytoskeleton-associated proteins that has arisen from studies of their homologues in yeast with information that has been obtained from in vivo studies using live animals or in vitro studies using cultured animal cell lines.

A Rapid Extraction Method for mammalian cell cultures, suitable for quantitative immunoblotting analysis of proteins, including phosphorylated GCN2 and eIF2α.

Many studies require the detection and relative quantitation of proteins from cell culture samples using immunoblotting. Limiting factors are the cost of protease inhibitors, the time required to break cells and generate samples, as well as the high risk of protein loss during cell breakage procedures. In addition, a common problem is the viscosity of lysed samples due to the released genomic DNA. As a consequence, the DNA needs to be broken down prior to denaturing polyacrylamide protein gel electrophoresis (SDS-PAGE), e.g. by passing the sample through a syringe gauge needle, sonication, or DNase treatment. In a quest to find a more cost-effective, fast, and yet robust procedure, we found that cell lysis, protein denaturation, and DNA fragmentation can be done in only two steps: harvesting followed by a simple non-laborious 2nd step. Similarly to many pre-existing cell breakage procedures, in our Rapid Protein Extraction (RPE) method, proteins liberated from cells are immediately exposed to a denaturing environment. However, advantages of our method are: •No breaking buffer is needed, instead proteins are liberated directly into the denaturing protein loading buffer used for SDS-PAGE. Consequently, our RPE method does not require any expensive inhibitors.•The RPE method does not involve post-lysis centrifugation steps; instead all cell material is dissolved during the 2nd step, the mixing-heat-treatment step which is new to this method. This prevents potential protein loss that may occur during centrifugation. In addition, this 2nd step simultaneously shears the genomic DNA, making an additional step for DNA fragmentation unnecessary.•The generated samples are suitable for high-quality quantitative immunoblotting. With our RPE method we successfully quantified the phosphorylated forms of protein kinase GCN2 and its substrate eIF2α. In fact, the western signals were stronger and with less background, as compared to samples generated with a pre-existing method.

Cost-effective and rapid lysis of Saccharomyces cerevisiae cells for quantitative western blot analysis of proteins, including phosphorylated eIF2α.

The common method for liberating proteins from Saccharomyces cerevisiae cells involves mechanical cell disruption using glass beads and buffer containing inhibitors (protease, phosphatase and/or kinase inhibitors), followed by centrifugation to remove cell debris. This procedure requires the use of costly inhibitors and is laborious, in particular when many samples need to be processed. Also, enzymatic reactions can still occur during harvesting and cell breakage. As a result low-abundance and labile proteins may be degraded, and enzymes such as kinases and phosphatases may still modify proteins during and after cell lysis. We believe that our rapid sample preparation method helps overcome the above issues and offers the following advantages: (a) it is cost-effective, as no inhibitors and breaking buffer are needed; (b) cell breakage is fast (about 15 min) since it only involves a few steps; (c) the use of formaldehyde inactivates endogenous proteases prior to cell lysis, dramatically reducing the risk of protein degradation; (d) centrifugation steps only occur prior to cell lysis, circumventing the problem of losing protein complexes, in particular if cells were treated with formaldehyde intended to stabilize and capture large protein complexes; and (e) since formaldehyde has the potential to instantly terminate protein activity, this method also allows the study of enzymes in live cells, i.e. in their true physiological environment, such as the short-term effect of a drug on enzyme activity. Taken together, the rapid sample preparation procedure provides a more accurate snapshot of the cell's protein content at the time of harvesting. Copyright © 2017 John Wiley & Sons, Ltd.

Protein SUMOylation is Involved in Cell-cycle Progression and Cell Morphology in Giardia lamblia.

The unicellular protozoa Giardia lamblia is a food- and waterborne parasite that causes giardiasis. This illness is manifested as acute and self-limited diarrhea and can evolve to long-term complications. Successful establishment of infection by Giardia trophozoites requires adhesion to host cells and colonization of the small intestine, where parasites multiply by mitotic division. The tight binding of trophozoites to host cells occurs by means of the ventral adhesive disc, a spiral array of microtubules and associated proteins such as giardins. In this work we show that knock down of the Small Ubiquitin-like MOdifier (SUMO) results in less adhesive trophzoites, decreased cell proliferation and deep morphological alterations, including at the ventral disc. Consistent with the reduced proliferation, SUMO knocked-down trophozoites were arrested in G1 and in S phases of the cell cycle. Mass spectrometry analysis of anti-SUMO immunoprecipitates was performed to identify SUMO substrates possibly involved in these events. Among the identified SUMOylation targets, α-tubulin was further validated by Western blot and confirmed to be a SUMO target in Giardia trophozoites.

Protein SUMOylation is involved in cell-cycle progression and cell morphology in giardia lamblia

The unicellular protozoa Giardia lamblia is a food- and waterborne parasite that causes giardiasis. This illness is manifested as acute and self-limited diarrhea and can evolve to long-term complications. Successful establishment of infection by Giardia trophozoites requires adhesion to host cells and colonization of the small intestine, where parasites multiply by mitotic division. The tight binding of trophozoites to host cells occurs by means of the ventral adhesive disc, a spiral array of microtubules and associated proteins such as giardins. In this work we show that knock down of the Small Ubiquitin-like MOdifier (SUMO) results in less adhesive trophzoites, decreased cell proliferation and deep morphological alterations, including at the ventral disc. Consistent with the reduced proliferation, SUMO knocked-down trophozoites were arrested in G1 and in S phases of the cell cycle. Mass spectrometry analysis of anti-SUMO immunoprecipitates was performed to identify SUMO substrates possibly involved in these events. Among the identified SUMOylation targets, -tubulin was further validated by Western blot and confirmed to be a SUMO target in Giardia trophozoites.

Perturbations in actin dynamics reconfigure protein complexes that modulate GCN2 activity and promote an eIF2 response.

Genetic and pharmacological interventions in yeast and mammalian cells have suggested a cross-talk between the actin cytoskeleton and protein synthesis. Regulation of the activity of the translation initiation factor 2 (eIF2) is a paramount mechanism for cells to rapidly adjust the rate of protein synthesis and to trigger reprogramming of gene expression in response to internal and external cues. Here, we show that disruption of F-actin in mammalian cells inhibits translation in a GCN2-dependent manner, correlating with increased levels of uncharged tRNA. GCN2 activation increased phosphorylation of its substrate eIF2α and the induction of the integrated stress response master regulator, ATF4. GCN2 activation by latrunculin-B is dependent on GCN1 and inhibited by IMPACT. Our data suggest that GCN2 occurs in two different complexes, GCN2-eEF1A and GCN2-GCN1. Depolymerization of F-actin shifts GCN2 to favor the complex with GCN1, concomitant with GCN1 being released from its binding to IMPACT, which is sequestered by G-actin. These events might further contribute to GCN2 activation. Our findings indicate that GCN2 is an important sensor of the state of the actin cytoskeleton.

IMPACT is a GCN2 inhibitor that limits lifespan in Caenorhabditis elegans.

BACKGROUND: The General Control Nonderepressible 2 (GCN2) kinase is a conserved member of the integrated stress response (ISR) pathway that represses protein translation and helps cells to adapt to conditions of nutrient shortage. As such, GCN2 is required for longevity and stress resistance induced by dietary restriction (DR). IMPACT is an ancient protein that inhibits GCN2. RESULTS: Here, we tested whether IMPACT down-regulation mimics the effects of DR in C. elegans. Knockdown of the C. elegans IMPACT homolog impt-1 activated the ISR pathway and increased lifespan and stress resistance of worms in a gcn-2-dependent manner. Impt-1 knockdown exacerbated DR-induced longevity and required several DR-activated transcription factors to extend lifespan, among them SKN-1 and DAF-16, which were induced during larval development and adulthood, respectively, in response to impt-1 RNAi. CONCLUSIONS: IMPACT inhibits the ISR pathway, thus limiting the activation of stress response factors that are beneficial during aging and required under DR.

The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast.

The Saccharomyces cerevisiae protein Yih1, when overexpressed, inhibits the eIF2 alpha kinase Gcn2 by competing for Gcn1 binding. However, deletion of YIH1 has no detectable effect on Gcn2 activity, suggesting that Yih1 is not a general inhibitor of Gcn2, and has no phenotypic defect identified so far. Thus, its physiological role is largely unknown. Here, we show that Yih1 is involved in the cell cycle. Yeast lacking Yih1 displays morphological patterns and DNA content indicative of a delay in the G2/M phases of the cell cycle, and this phenotype is independent of Gcn1 and Gcn2. Accordingly, the levels of phosphorylated eIF2α, which show a cell cycle-dependent fluctuation, are not altered in cells devoid of Yih1. We present several lines of evidence indicating that Yih1 is in a complex with Cdc28. Yih1 pulls down endogenous Cdc28 in vivo and this interaction is enhanced when Cdc28 is active, suggesting that Yih1 modulates the function of Cdc28 in specific stages of the cell cycle. We also demonstrate, by Bimolecular Fluorescence Complementation, that endogenous Yih1 and Cdc28 interact with each other, confirming Yih1 as a bona fide Cdc28 binding partner. Amino acid substitutions within helix H2 of the RWD domain of Yih1 enhance Yih1-Cdc28 association. Overexpression of this mutant, but not of wild type Yih1, leads to a phenotype similar to that of YIH1 deletion, supporting the view that Yih1 is involved through Cdc28 in the regulation of the cell cycle. We further show that IMPACT, the mammalian homologue of Yih1, interacts with CDK1, the mammalian counterpart of Cdc28, indicating that the involvement with the cell cycle is conserved. Together, these data provide insights into the cellular function of Yih1/IMPACT, and provide the basis for future studies on the role of this protein in the cell cycle.

Extracellular enolase of Candida albicans is involved in colonization of mammalian intestinal epithelium.

Enolase is secreted by Candida albicans and is present in its biofilms although its extracellular function is unknown. Here we show that extracellular enolase mediates the colonization of small intestine mucosa by C. albicans. Assays using intestinal mucosa disks show that C. albicans adhesion is inhibited, in a dose dependent mode, either by pretreatment of intestinal epithelium mucosa disks with recombinant C. albicans enolase (70% at 0.5 mg/ml enolase) or by pretreatment of C. albicans yeasts with anti-enolase antibodies (48% with 20 µg antiserum). Also using flow cytometry, immunoblots of conditioned media and confocal microscopy we demonstrate that enolase is present in biofilms and that the extracellular enolase is not an artifact due to cell lysis, but must represent functional secretion of a stable form. This is the first direct evidence that C. albicans' extracellular enolase mediates colonization on its primary translocation site. Also, because enolase is encoded by a single locus in C. albicans, its dual role peptide, as glycolytic enzyme and extracellular peptide, is a remarkable example of gene sharing in fungi.

Keeping the eIF2 alpha kinase Gcn2 in check.

The protein kinase Gcn2 is present in virtually all eukaryotes and is of increasing interest due to its involvement in a large array of crucial biological processes. Some of these are universally conserved from yeast to humans, such as coping with nutrient starvation and oxidative stress. In mammals, Gcn2 is important for e.g. long-term memory formation, feeding behaviour and immune system regulation. Gcn2 has been also implicated in diseases such as cancer and Alzheimer's disease. Studies on Gcn2 have been conducted most extensively in Saccharomyces cerevisiae, where the mechanism of its activation by amino acid starvation has been revealed in most detail. Uncharged tRNAs stimulate Gcn2 which subsequently phosphorylates its substrate, eIF2α, leading to reduced global protein synthesis and simultaneously to increased translation of specific mRNAs, e.g. those coding for Gcn4 in yeast and ATF4 in mammals. Both proteins are transcription factors that regulate the expression of a myriad of genes, thereby enabling the cell to initiate a survival response to the initial activating cue. Given that Gcn2 participates in many diverse processes, Gcn2 itself must be tightly controlled. Indeed, Gcn2 is regulated by a vast network of proteins and RNAs, the list of which is still growing. Deciphering molecular mechanisms underlying Gcn2 regulation by effectors and inhibitors is fundamental for understanding how the cell keeps Gcn2 in check ensuring normal organismal function, and how Gcn2-associated diseases may develop or may be treated. This review provides a critical evaluation of the current knowledge on mechanisms controlling Gcn2 activation or activity.

Examining marginal sequence similarities between bacterial type III secretion system components and Trypanosoma cruzi surface proteins: horizontal gene transfer or convergent evolution?

The cell invasion mechanism of Trypanosoma cruzi has similarities with some intracellular bacterial taxa especially regarding calcium mobilization. This mechanism is not observed in other trypanosomatids, suggesting that the molecules involved in this type of cell invasion were a product of (1) acquisition by horizontal gene transfer (HGT); (2) secondary loss in the other trypanosomatid lineages of the mechanism inherited since the bifurcation Bacteria-Neomura (1.9 billion to 900 million years ago); or (3) de novo evolution from non-homologous proteins via convergent evolution. Similar to T. cruzi, several bacterial genera require increased host cell cytosolic calcium for intracellular invasion. Among intracellular bacteria, the mechanism of host cell invasion of genus Salmonella is the most similar to T. cruzi. The invasion of Salmonella occurs by contact with the host's cell surface and is mediated by the type III secretion system (T3SS) that promotes the contact-dependent translocation of effector proteins directly into host's cell cytoplasm. Here we provide evidence of distant sequence similarities and structurally conserved domains between T. cruzi and Salmonella spp T3SS proteins. Exhaustive database searches were directed to a wide range of intracellular bacteria and trypanosomatids, exploring sequence patterns for comparison of structural similarities and Bayesian phylogenies. Based on our data we hypothesize that T. cruzi acquired genes for calcium mobilization mediated invasion by ancient HGT from ancestral Salmonella lineages.

Differential infectivity by the oral route of Trypanosoma cruzi lineages derived from Y strain.

BACKGROUND: Diversity of T. cruzi strains is a central problem in Chagas disease research because of its correlation with the wide range of clinical manifestations and the biogeographical parasite distribution. The role played by parasite microdiversity in Chagas disease epidemiology is still debatable. Also awaits clarification whether such diversity is associated with the outcome of oral T. cruzi infection, responsible for frequent outbreaks of acute Chagas disease. METHODS AND FINDINGS: We addressed the impact of microdiversity in oral T. cruzi infection, by comparative analysis of two strains, Y30 and Y82, both derived from Y strain, a widely used experimental model. Network genealogies of four nuclear genes (SSU rDNA, actin, DHFR-TS, EF1α) revealed that Y30 is closely related to Discrete Typing Unit TcII while Y82 is more closely related to TcVI, a group containing hybrid strains. Nevertheless, excepting one A-G transition at position 1463, Y30 and Y82 SSU rDNAs were identical. Y82 strain, expressing the surface molecule gp82, infected mice orally more efficiently than Y30, which expresses a related gp30 molecule. Both molecules are involved in lysosome exocytosis-dependent host cell invasion, but exhibit differential gastric mucin-binding capacity, a property critical for parasite migration toward the gastric mucosal epithelium. Upon oral infection of mice, the number of Y30 and Y82 parasites in gastric epithelial cells differed widely. CONCLUSIONS: We conclude that metacyclic forms of gp82-expressing Y82 strain, closely related to TcVI, are better adapted than Y30 strain (TcII) to traverse the stomach mucous layer and establish oral route infection. The efficiency to infect target cell is the same because gp82 and gp30 strains have similar invasion-promoting properties. Unknown is whether differences in Y30 and Y82 are natural parasite adaptations or a product of lab-induced evolution by differential selection along the 60 years elapsed since the Y strain isolation.