Department of Stress Responce Science

1. Research on the role of GCN1

1. Research on the role of GCN1 --- Amino acid sensing and the regulation of cellular function

Amino acids exert many biological functions, serving as allosteric regulators and neurotransmitters, as constituents in proteins and as nutrients. mTOR is known as an important regulator of cellular homeostasis as a sensor of amino acid sufficiency. However, the research about the sensor of amino acid insufficiency (i.e. GCN2 pathway) has been less explored in mammals.
Protein translation is a highly energy demanding process. Therefore, it is important for the cells to repress overall protein translation and produce limited proteins which is necessary for cell survival during various stresses.
Upon exposure to stresses such as amino acid starvation (AAS), phosphorylation of translational initiation factor eIF2a represses general translation. At the same time, it increases the selective translation of cytoprotective proteins, such as ATF4, that transcriptionally activate gene expression involved in a stress response to promote cell survival. Among four eIF2? kinases, GCN2 responds to AAS and phosphorylates eIF2?. In yeast, Gcn1 interacts with both GCN2 and ribosome, and is required for Gcn2 activation by AAS. Upon AAS, uncharged tRNAs are increased and Gcn1 transfers the uncharged tRNA to Gcn2 at the ribosome.

We explored the function of GCN1 using two types of mutant mouse lines: Gcn1 knockout mice and Gcn1?RWDBD mice which lack GCN2 binding domain (1). Both mutant mice showed growth retardation, which was not observed in the Gcn2 KO mice. Gcn1 KO mice died at the intermediate stage of embryonic development because of severe growth retardation. Gcn1??RWDBD embryos showed mild growth retardation and malformation, and died soon after birth, most likely due to respiratory failure. Collectively, it was revealed that GCN1 contributes to normal embryogenesis in a GCN2-independent manner. We further generated mice embryonic fibroblasts from Gcn1?RWDBD embryos, and showed that GCN1 is necessary for response to AAS. Interestingly, GCN1 regulates not only the eIF2?-mediated stress response but also cell cycle and cell proliferation in a GCN2-independent manner. Taking these findings together, we propose that GCN1 integrates cellular energetic status including amino acid availability to enhance cell viability.
Kim et al. recently demonstrated that GCN1 suppresses a set of inflammatory cytokine expression in a GCN2-independent manner in response to the increased uncharged tRNA by glutamyl-prolyl-tRNA synthetase (EPRS) inhibitor halofuginone mainly in the human fibroblast-like synoviocytes (FLS) (2). Thus, the above-mentioned two researches demonstrated that GCN1 also acts in a GCN2-independent manner in mice and human. Indeed, recent studies revealed that GCN1 regulates apoptosis (3) and immunity (4) in a GCN2-independent manner, in C. elegans and Arabidopsis, respectively.
Our lab thus aims to clarify the role of GCN1 in physiology and diseases in mammals.

References
1. Ribosome binding protein GCN1 regulates the cell cycle and cell proliferation and is essential for the embryonic development of mice. Yamazaki H, Kasai S, Mimura J, Ye P, Inose-Maruyama A, Tanji K, Wakabayashi K, Mizuno S, Sugiyama F, Takahashi S, Sato T, Ozaki T, Cavener DR, Yamamoto M, Itoh K. PLoS Genet. 2020 Apr 23;16(4):e1008693.
2. Aminoacyl-tRNA synthetase inhibition activates a pathway that branches from the canonical amino acid response in mammalian cells. Kim Y, Sundrud MS, Zhou C, Edenius M, Zocco D, Powers K, Zhang M, Mazitschek R, Rao A, Yeo CY, Noss EH, Brenner MB, Whitman M, Keller TL. Proc Natl Acad Sci U S A. 2020 Apr 21;117(16):8900-8911.
3. Arabidopsis ILITHYIA protein is necessary for proper chloroplast biogenesis and root development independent of eIF2? phosphorylation. Faus I, Niñoles R, Kesari V, Llabata P, Tam E, Nebauer SG, Santiago J, Hauser MT, Gadea J. J Plant Physiol. 2018 May - Jun;224-225:173-182.
4. The translational regulators GCN-1 and ABCF-3 act together to promote apoptosis in C. elegans. Hirose T, Horvitz HR. PLoS Genet. 2014 Aug 7;10(8):e1004512.

2. Role of Nrf2 stress response pathway in physiology and diseases

2. Role of Nrf2 stress response pathway in physiology and diseases

Oxidative stress has been implicated in the pathogenesis of many age-related diseases such as Alzheimer's disease, age-related macular degeneration, and diabetes. Goal of this project is to develop a strategy for preventing age-related diseases targeting Nrf2-mediated cytoprotective mechanisms. Nrf2 is transcription factor that forms heterodimer with small Maf proteins to bind its target sequence antioxidant responsive element (ARE) (1, 2). Normally, Nrf2 is trapped to the E3 ligase adaptor Kelch-like ECH-associated protein 1 (Keap1) protein and subsequently degraded by the proteasome. However, when reactive cysteine residues of Keap1 protein are modified by oxidative stress or electrophilic substances, degradation of Nrf2 is inhibited, and accumulated Nrf2 translocates to the nucleus to bind to the ARE.

A. Elucidation of Nrf2 transcriptional network To elucidate the transcriptional network mediated by Nrf2, we previously performed yeast two hybrid screening using the C-terminus of Nrf2 that contains basic leucine zipper region of Nrf2. Interestingly, most positive clones encoded ATF4 (unpublished data, see also ref 3). Nrf2 and ATF4 are activated by the proteasome inhibition, cooperatively activates xCT expression which increases the intracellular cysteine and glutathione thereby confers resistance to proteasome inhibitor-mediated cytotoxicity (4). We showed that xCT expression is regulated by ARE and ATF4 binding motif amino acid response element (AARE) in the promoter region. In addition, ATF4 also binds to ARE in the 2nd intron in an Nrf2-depedent manner, most probably making loop formation between the intron and the promoter. We surmise that these interaction in the promoter is important for the cooperative activation of xCT gene transcription. Another cooperation between Nrf2 and ATF4 is observed using carnosic acid that is a major polyphenol contained in the herb rosemary (5). Carnosic acid only activates Nrf2 at its lower concentration, but at its higher concentration also activates ATF4 and invokes huge induction of Nrf2 target genes such as xCT, ?-glutamyl cysteine ligase and nerve growth factor and the induction is dependent both on Nrf2 and ATF4. We also interested in the interaction of Nrf2 pathway and the mitochondria, both are involved in the redox homeostasis during aging.

B. Elucidation of the Role of Nrf2 in the vasculature and the heart Nrf2 is activated by not only by oxidative stress but also by vascular shear stress and regulates vascular redox homeostasis by inducing expression of antioxidant proteins and enzymes (6). Nrf2 activity in the endothelium is regulated by laminar shear stress such that Nrf2 is high in the straight blood vessel, but low in the curvature or branching points of the blood vessels. Nrf2 and downstream antioxidant genes are thought to function as safeguards against various oxidative stress in general tissues, and of note, Nrf2 activity decreases with age. Nrf2 ameliorates endothelial dysfunction in diabetes and oxidative stress and increases the amount of bioavailable nitric oxide (NO), which is a major vasodilator. As the heart consists of postmitotic cells that utilize ATP produced mainly by mitochondrial oxidative phosphorylation (OXPHOS), cardiomyocytes appear to be equipped with highly sophisticated mitochondrial quality control mechanisms. Consistent with these findings, it has been reported that Nrf2 in the heart is regulated via a specific translational mechanism and that Nrf2 activation confers cardioprotective effects in various disease models. We will explore the role of Nrf2 in the heart.

C. Elucidation of the Role of Nrf2 in age-related diseases In contrast to the well-established activation mechanisms in response to extrinsic and endogenously generated electrophiles, it is not well understood whether Nrf2 responds to endogenous reactive oxygen species (ROS) generated in the mitochondria and whether Nrf2 is involved in the regulation of redox homeostasis of the organelle (5). As mitochondrial function and ROS generation are impaired with aging, it is important to understand the relationship of mitochondrial perturbation and Nrf2 activity, especially as they relate to the mitochondrial ROS. Furthermore, mitochondrial ROS and mitochondrial dysfunction play important roles in diverse age-related diseases and in physiology (7). We will explore the mechanisms of crosstalk between Nrf2 and mitochondria and their potential role in aging and age-related heart disease.

References
1. Discovery of the negative regulator of Nrf2, Keap1: a historical overview. Itoh K, Mimura J, Yamamoto M. Antioxid Redox Signal. 2010 Dec 1;13(11):1665-78.
2. Emerging Regulatory Role of Nrf2 in Iron, Heme, and Hemoglobin Metabolism in Physiology and Disease. Kasai S, Mimura J, Ozaki T, Itoh K. Front Vet Sci. 2018 Oct 10;5:242.
3. Nrf2- and ATF4-dependent upregulation of xCT modulates the sensitivity of T24 bladder carcinoma cells to proteasome inhibition. Ye P, Mimura J, Okada T, Sato H, Liu T, Maruyama A, Ohyama C, Itoh K. Mol Cell Biol. 2014 Sep 15;34(18):3421-34.
4. Concomitant Nrf2- and ATF4-activation by Carnosic Acid Cooperatively Induces Expression of Cytoprotective Genes. Mimura J, Inose-Maruyama A, Taniuchi S, Kosaka K, Yoshida H, Yamazaki H, Kasai S, Harada N, Kaufman RJ, Oyadomari S, Itoh K. Int J Mol Sci. 2019 Apr 5;20(7). pii: E1706.
5. Regulation of Nrf2 by Mitochondrial Reactive Oxygen Species in Physiology and Pathology. Kasai S, Shimizu S, Tatara Y, Mimura J, Itoh K. Biomolecules. 2020 Feb 17;10(2). pii: E320.
6. Emerging evidence for crosstalk between Nrf2 and mitochondria in physiological homeostasis and in heart disease. Tsushima M, Liu J, Hirao W, Yamazaki H, Tomita H, Itoh K. Arch Pharm Res. 2020 Mar;43(3):286-296.
7. Role of Mitochondrial Reactive Oxygen Species in the Activation of Cellular Signals, Molecules, and Function. Indo HP, Hawkins CL, Nakanishi I, Matsumoto KI, Matsui H, Suenaga S, Davies MJ, St Clair DK, Ozawa T, Majima HJ. Handb Exp Pharmacol. 2017;240:439-456.