Tryptophan catabolism is a tolerogenic effector system in regulatory T cell function, yet the general mechanisms whereby tryptophan catabolism affects T cell responses remain unclear. We provide evidence that the short-term, combined effects of tryptophan deprivation and tryptophan catabolites result in GCN2 kinase-dependent down-regulation of the TCR ζ-chain in murine CD8+ T cells. TCR ζ down-regulation can be demonstrated in vivo and is associated with an impaired cytotoxic effector function in vitro. The longer-term effects of tryptophan catabolism include the emergence of a regulatory phenotype in naive CD4+CD25− T cells via TGF-β induction of the forkhead transcription factor Foxp3. Such converted cells appear to be CD25+, CD69−, CD45RBlow, CD62L+, CTLA-4+, BTLAlow and GITR+, and are capable of effective control of diabetogenic T cells when transferred in vivo. Thus, both tryptophan starvation and tryptophan catabolites contribute to establishing a regulatory environment affecting CD8+ as well as CD4+ T cell function, and not only is tryptophan catabolism an effector mechanism of tolerance, but it also results in GCN2-dependent generation of autoimmune-preventive regulatory T cells.

Phosphorylation of eukaryotic initiation factor 2␣ (eIF-2␣) is typically associated with stress responses and causes a reduction in protein synthesis. However, we found high phosphorylated eIF-2␣ (eIF-2␣[P]) levels in nonstressed pancreata of mice. Administration of glucose stimulated a rapid dephosphorylation of eIF-2␣. Among the four eIF-2␣ kinases present in mammals, PERK is most highly expressed in the pancreas, suggesting that it may be responsible for the high eIF-2␣[P] levels found therein. We describe a Perk knockout mutation in mice. Pancreata of Perk ؊/؊ mice are morphologically and functionally normal at birth, but the islets of Langerhans progressively degenerate, resulting in loss of insulin-secreting beta cells and development of diabetes mellitus, followed later by loss of glucagon-secreting alpha cells. The exocrine pancreas exhibits a reduction in the synthesis of several major digestive enzymes and succumbs to massive apoptosis after the fourth postnatal week. Perk ؊/؊ mice also exhibit skeletal dysplasias at birth and postnatal growth retardation. Skeletal defects include deficient mineralization, osteoporosis, and abnormal compact bone development. The skeletal and pancreatic defects are associated with defects in the rough endoplasmic reticulum of the major secretory cells that comprise the skeletal system and pancreas. The skeletal, pancreatic, and growth defects are similar to those seen in human Wolcott-Rallison syndrome.

Expression of long-lasting synaptic plasticity and long-term memory requires new protein synthesis, which can be repressed by phosphorylation of eukaryotic initiation factor 2α subunit (eIF2α). It was reported previously that eIF2α phosphorylation is elevated in the brains of Alzheimer’s disease (AD) patients and AD model mice. Therefore, we determined whether suppressing eIF2α kinases could alleviate synaptic plasticity and memory deficits in AD model mice. The genetic deletion of the eIF2α kinase PERK prevented enhanced eIF2α phosphorylation, as well as deficits in protein synthesis, synaptic plasticity, and spatial memory in APP/PS1 AD model mice. Similarly, deletion of another eIF2α kinase, GCN2, prevented impairments of synaptic plasticity and spatial memory defects displayed in the APP/PS1 mice. Our findings implicate aberrant eIF2α phosphorylation as a novel molecular mechanism underlying AD-related synaptic pathophysioloy and memory dysfunction and suggest that PERK and GCN2 are potential therapeutic targets for the treatment of individuals with AD.

The previously presented consensus sequence for eukaryotic translation initiation sites by Kozak (1) was derived substantially from vertebrate mRNA sequences. Drosophila nuclear genes exhibit a significantly different translation start consensus sequence. These differences probably do not represent mechanistic differences in translation initiation inasmuch as both taxa exhibit identical preferences and restrictions at the crucial -3 position. Using more conservative criteria for the assignment of consensus the following consensus sequences were derived: vertebrate--CANCAUG and Drosophila--jAA8AUG.

In response to environmental stress, cells induce a program of gene expression designed to remedy cellular damage or, alternatively, induce apoptosis. In this report, we explore the role of a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) in coordinating stress gene responses. We find that expression of activating transcription factor 3 (ATF3), a member of the ATF/CREB subfamily of basic-region leucine zipper (bZIP) proteins, is induced in response to endoplasmic reticulum (ER) stress or amino acid starvation by a mechanism requiring eIF2 kinases PEK (Perk or EIF2AK3) and GCN2 (EIF2AK4), respectively. Increased expression of ATF3 protein occurs early in response to stress by a mechanism requiring the related bZIP transcriptional regulator ATF4. ATF3 contributes to induction of the CHOP transcriptional factor in response to amino acid starvation, and loss of ATF3 function significantly lowers stress-induced expression of GADD34, an eIF2 protein phosphatase regulatory subunit implicated in feedback control of the eIF2 kinase stress response. Overexpression of ATF3 in mouse embryo fibroblasts partially bypasses the requirement for PEK for induction of GADD34 in response to ER stress, further supporting the idea that ATF3 functions directly or indirectly as a transcriptional activator of genes targeted by the eIF2 kinase stress pathway. These results indicate that ATF3 has an integral role in the coordinate gene expression induced by eIF2 kinases. Given that ATF3 is induced by a very large number of environmental insults, this study supports involvement of eIF2 kinases in the coordination of gene expression in response to a more diverse set of stress conditions than previously proposed.

Sequences flanking translational initiation and termination sites have been compiled and statistically analyzed for various eukaryotic taxonomic groups. A few key similarities between taxonomic groups support conserved mechanisms of initiation and termination. However, a high degree of sequence variation at these sites within and between various eukaryotic groups suggest that translation may be modulated for many mRNAs. Multipositional analysis of di-, tri-, and quadrinucleotide sequences flanking start/stop sites indicate significant biases. In particular, strong tri-nucleotide biases are observed at the -3, -2, and -1 positions upstream of the start codon. These biases and the interspecific variation in nucleotide preferences at these three positions have lead us to propose a revised model of the interaction of the 18S ribosomal RNA with the mRNA at the site of translation initiation. Unusually strong biases against the CG dinucleotide immediately downstream of termination codons suggest that they may lead to faulty termination and/or failure of the ribosome to disassociate from the mRNA.

Nuclear factor B (NF-B) serves to coordinate the transcription of genes in response to diverse environmental stresses. In this report we show that phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (eIF2) is fundamental to the process by which many stress signals activate NF-B. Phosphorylation of this translation factor is carried out by a family of protein kinases that each respond to distinct stress conditions. During impaired protein folding and assembly in the endoplasmic reticulum (ER), phosphorylation of eIF2␣ by PEK (Perk or EIF2AK3) is essential for induction of NF-B transcriptional activity. The mechanism by which NF-B is activated during ER stress entails the release, but not the degradation, of the inhibitory protein IB. During amino acid deprivation, phosphorylation of eIF2␣ by GCN2 (EIF2AK4) signals the activation of NF-B. Furthermore, inhibition of general translation or transcription by cycloheximide and actinomycin D, respectively, elicits the eIF2␣ phosphorylation required for induction of NF-B. Together, these studies suggest that eIF2␣ kinases monitor and are activated by a range of stress conditions that affect transcription and protein synthesis and assembly, and the resulting eIF␣ phosphorylation is central to activation of the NF-B. The absence of NF-B-mediated transcription and its antiapoptotic function provides an explanation for why eIF2␣ kinase deficiency in diseases such as Wolcott-Rallison syndrome leads to cellular apoptosis and disease.

The GCN2 eIF2␣ kinase is essential for activation of the general amino acid control pathway in yeast when one or more amino acids become limiting for growth. GCN2's function in mammals is unknown, but must differ, since mammals, unlike yeast, can synthesize only half of the standard 20 amino acids. To investigate the function of mammalian GCN2, we have generated a Gcn2 ؊/؊ knockout strain of mice. Gcn2 ؊/؊ mice are viable, fertile, and exhibit no phenotypic abnormalities under standard growth conditions. However, prenatal and neonatal mortalities are significantly increased in Gcn2 ؊/؊ mice whose mothers were reared on leucine-, tryptophan-, or glycine-deficient diets during gestation. Leucine deprivation produced the most pronounced effect, with a 63% reduction in the expected number of viable neonatal mice. Cultured embryonic stem cells derived from Gcn2 ؊/؊ mice failed to show the normal induction of eIF2␣ phosphorylation in cells deprived of leucine. To assess the biochemical effects of the loss of GCN2 in the whole animal, liver perfusion experiments were conducted. Histidine limitation in the presence of histidinol induced a twofold increase in the phosphorylation of eIF2␣ and a concomitant reduction in eIF2B activity in perfused livers from wild-type mice, but no changes in livers from Gcn2 ؊/؊ mice.