Beatriz A. Castilho

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 Alzheimers 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.

Prion protein interaction with stress-inducible protein 1 enhances neuronal protein synthesis via mTOR

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases caused by the conversion of prion protein (PrPC) into an infectious isoform (PrPSc). How this event leads to pathology is not fully understood. Here we demonstrate that protein synthesis in neurons is enhanced via PrPC interaction with stress-inducible protein 1 (STI1). We also show that neuroprotection and neuritogenesis mediated by PrPC–STI1 engagement are dependent upon the increased protein synthesis mediated by PI3K-mTOR signaling. Strikingly, the translational stimulation mediated by PrPC–STI1 binding is corrupted in neuronal cell lines persistently infected with PrPSc, as well as in primary cultured hippocampal neurons acutely exposed to PrPSc. Consistent with this, high levels of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation were found in PrPSc-infected cells and in neurons acutely exposed to PrPSc. These data indicate that modulation of protein synthesis is critical for PrPC–STI1 neurotrophic functions, and point to the impairment of this process during PrPSc infection as a possible contributor to neurodegeneration.

Novel Membrane-Bound eIF2α Kinase in the Flagellar Pocket of Trypanosoma brucei

ABSTRACT Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2α) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations. Trypanosoma brucei, the agent of African trypanosomiasis, encodes three potential eIF2α kinases (TbeIF2K1 to -K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and in bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2α at Ser51. It also phosphorylates the highly unusual form of eIF2α found in trypanosomatids specifically at residue Thr169 that corresponds to Ser51 in other eukaryotes. T. brucei eIF2α, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2α kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting that it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in T. brucei, an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2α kinase described in unicellular eukaryotes.

Translation initiation at non-AUG codons mediated by weakened association of eukaryotic initiation factor (eIF) 2 subunits.

The heterotrimeric eukaryotic initiation factor (eIF) 2 binds the initiator methionyl-tRNA in a GTP-dependent mode and delivers it to the 40 S ribosomal subunit. In the present study, we have identified amino acid residues in eIF2beta required for binding to eIF2gamma in yeast. Alteration of six residues in the central region of eIF2beta abolished this interaction, as determined by GST-pull down and two-hybrid assays, and leads to cell lethality. Substitution of (131)Tyr and (132)Ser by alanine residues ((131)YS), although abolishing the binding to eIF2gamma in these assays, resulted in a functional but defective protein in vivo, imparting a temperature-sensitive growth phenotype to cells. A dramatically weakened association of this mutant protein with eIF2gamma in vivo was shown by co-immunoprecipitation. The (131)YS mutation in eIF2beta allows translation to initiate at non-AUG codons, as defined by the ability of cells carrying an initiator codon mutation in the HIS4 mRNA to grow in the absence of histidine. The combination of this mutation with the (264)Ser-->Tyr alteration, a previously isolated suppressor of initiator codon mutations which has been shown to increase the spontaneous GTP hydrolysis in the ternary complex, caused a recessive lethality, suggesting additive defects. Thus the impaired interaction of these two subunits represents a novel type of defect in eIF2 function, providing in vivo evidence that the strength of interaction between eIF2beta and eIF2gamma defines the correct usage of the AUG codon for translation initiation.

IMPACT Is a Developmentally Regulated Protein in Neurons That Opposes the Eukaryotic Initiation Factor 2α Kinase GCN2 in the modulation of Neurite Outgrowth

Background: IMPACT inhibits GCN2, a kinase that directs stress remedial responses by attenuating translation and controls feeding behavior and memory. Results: Neuronal IMPACT is developmentally up-regulated, promoting protein synthesis and neuritogenesis, opposing GCN2. Conclusion: GCN2 and IMPACT modulate an early step in neuronal differentiation. Significance: A neuron-specific developmental program is controlled by two evolutionarily conserved translational regulators. The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system.

Antibody response against Escherichia coli heat-stable enterotoxin expressed as fusions to flagellin

The heat-stable toxin (ST) produced by enterotoxigenic Escherichia coli strains causes diarrhoea by altering the fluid secretion in intestinal epithelial cells. Here, the effectiveness of a flagellin fusion protein of Salmonella containing a 19-amino-acid sequence derived from the ST sequence (FLA--ST) in generating antibodies capable of neutralizing the toxic activity of ST was evaluated. This fusion protein, and an alternative construction where two cysteine residues in the ST sequence were substituted by alanines (ST(mt)), were delivered to the immune system by three distinct strategies: (i) orally, using an attenuated Salmonella strain expressing FLA--ST; (ii) intraperitoneally, by injection of purified FLA--ST; (iii) orally, using attenuated Salmonella carrying a eukaryotic expression plasmid (pCDNA3) with the gene encoding FLA-ST. The results showed that the flagellin system can be used as a carrier to generate ST-neutralizing antibodies. However, it should be mentioned that humoral immune response against ST was only obtained when the mutated ST sequence was employed. FLA-ST was found to be non-immunogenic when delivered via the oral route with attenuated Salmonella strains. However, a flagellin antibody response was obtained by immunizing mice with Salmonella carrying pCDNA3/FLA-ST(mt). Oral immunization with Salmonella carrying the eukaryotic expression plasmid (pCDNA3/FLA--ST(mt)) seems to be a promising method to elicit an appropriate response against fusions to flagellin.

Evidence That Eukaryotic Translation Elongation Factor 1A (eEF1A) Binds the Gcn2 Protein C Terminus and Inhibits Gcn2 Activity

The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His6-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His6-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.

Salicylates Trigger Protein Synthesis Inhibition in a Protein Kinase R-like Endoplasmic Reticulum Kinase-dependent Manner

The non-steroidal anti-inflammatory drug aspirin and its metabolite, sodium salicylate, have profound effects on cellular protein synthesis and cell physiology. However, the underlying mechanism by which they cause these responses remains unclear. We show here that salicylates induce phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in the inhibition of mRNA translation in cells. Exposure of cells to acetyl salicylic acid resulted in strong activation of eIF2α stress-activated protein kinase R-like endoplasmic reticulum kinase (PERK). Analysis of fibroblasts with a targeted deletion of the perk gene revealed that PERK is indispensable for triggering the phosphorylation of eIF2α as well as the inhibition of protein synthesis induced by salicylates. Although salicylate treatment did not trigger activation of inositol-requiring enzyme 1, there was an increased expression of the pro-apoptotic transcription factor CHOP-(gadd153), a downstream event to eIF2α phosphorylation known to mediate endoplasmic reticulum stress-mediated responses. Thus, salicylates selectively trigger an endoplasmic reticulum stress-responsive signaling pathway initiated through activation of PERK to induce their cellular effects.

Phosphorylation of the α subunit of translation initiation factor-2 by PKR mediates protein synthesis inhibition in the mouse brain during status epilepticus

In response to different cellular stresses, a family of protein kinases phosphorylates eIF2alpha (alpha subunit of eukaryotic initiation factor-2), contributing to regulation of both general and genespecific translation proposed to alleviate cellular injury or alternatively induce apoptosis. Recently, we reported eIF2alpha(P) (phosphorylated eIF2alpha) in the brain during SE (status epilepticus) induced by pilocarpine in mice, an animal model of TLE (temporal lobe epilepsy) [Carnevalli, Pereira, Longo, Jaqueta, Avedissian, Mello and Castilho (2004) Neurosci. Lett. 357, 191-194]. We show in the present study that one eIF2alpha kinase family member, PKR (double-stranded-RNA-dependent protein kinase), is activated in the cortex and hippocampus at 30 min of SE, reflecting the levels of eIF2alpha(P) in these areas. In PKR-deficient animals subjected to SE, eIF2alpha phosphorylation was clearly evident coincident with activation of a secondary eIF2alpha kinase, PEK/PERK (pancreatic eIF2alpha kinase/RNA-dependent-protein-kinase-like endoplasmic reticulum kinase), denoting a compensatory mechanism between the two kinases. The extent of eIF2alpha phosphorylation correlated with the inhibition of protein synthesis in the brain, as determined from polysome profiles. We also found that C57BL/6 mice, which enter SE upon pilocarpine administration but are more resistant to seizure-induced neuronal degeneration, showed very low levels of eIF2alpha(P) and no inhibition of protein synthesis during SE. These results taken together suggest that PKR-mediated phosphorylation of eIF2alpha contributes to inhibition of protein synthesis in the brain during SE and that sustained high levels of eIF2alpha phosphorylation may facilitate ensuing cell death in the most affected areas of the brain in TLE.

Distribution of the protein IMPACT, an inhibitor of GCN2, in the mouse, rat, and marmoset brain

IMPACT is an inhibitor of GCN2, a kinase that phosphorylates the alpha subunit of the translation initiation factor 2 (eIF2α). GCN2 has been implicated in regulating feeding behavior and learning and memory in mice. IMPACT is highly abundant in the brain, suggesting its relevance in the control of GCN2 activation in the central nervous system. We describe here the distribution of IMPACT in the brain of rodents (mice and rats) and of a primate (marmoset) using highly specific antibodies raised against the mouse IMPACT protein. Neurons expressing high levels of IMPACT were found in most areas of the brain. In the hippocampal formation the lack of IMPACT in the dentate gyrus granule cells was striking. The hypothalamus is exceptionally rich in neurons expressing high levels of IMPACT, particularly in the suprachiasmatic nucleus. The only exception to this pattern was the ventromedial nucleus. The thalamic neurons are mostly devoid of IMPACT, with the exception of the paraventricular, reuniens and reticular nuclei, and intergeniculate leaf. The brainstem displayed high levels of IMPACT. For the marmoset, IMPACT expression in the brain is not as prominent when compared to other organs. In the marmoset brain the pattern of IMPACT expression was similar to rodents in most areas, except for the very strong labeling of the Purkinje cells, the lack of IMPACT‐positive neurons in the nucleus reuniens, and weak labeling of interneurons in the hippocampus. GCN1, the activator of GCN2 to which IMPACT binds, is widely distributed in all neuronal populations, and all IMPACT‐positive cells were also GCN1‐positive. The data presented herein suggest that IMPACT may be involved in biochemical homeostatic mechanisms that would prevent GCN2 activation and therefore ATF4 (CREB‐2) synthesis in neurons. J. Comp. Neurol. 507:1811–1830, 2008.