Signaling Functions of Reactive Oxygen Species (ROS Signal)
Grants-in-Aid for Scientific Research on Innovative Areas (Research in a Proposed Research Area), MEXT, Japan
Message from the Principal Investigator | Outline of the Programmed Project Research |
Subject and Scientist of Each Programmed Research Project
Message from the Principal Investigator
Reactive oxygen species (ROS) have been believed to be toxic substances that induce nonspecific destructive alterations in biological molecules. However, as a result of evolving ROS toxicology, it is now thought that ROS may play important roles in regulation of physiological cell signal transductions. This new concept of ROS signaling is the central theme of this programmed project, which is supported by Grants-in-Aid for Scientific Research on Innovative Areas (Research in a Proposed Research Area), MEXT, Japan. The specific title of our project is “Signaling Functions of Reactive Oxygen Species (ROS Signal)”. It started in 2008 and continues for 5 years, until 2012. The mission of this Proposed Research Area is to clarify ROS signaling functions and their mechanisms in diverse physiological and pathophysiological phenomena in terms of chemical biology, which is an emerging scientific discipline comprising the integrated fields of chemistry and biology. Comprehensive understanding of these molecular mechanisms, which conduct ROS cell signals through receptors to effector molecules at molecular, cellular, and organismic levels, will contribute to the remarkable innovative progress occurring in cell signaling research. Several breakthroughs achieved by the many researchers in this field have expanded the frontiers in basic research and clinical medicine in such topics as infection, cancer biology, metabolic syndromes, ageing, and even stem cell research. More important, this ROS signaling research has the potential to promote progress in various life science fields including agricultural and medical sciences.
Takaaki Akaike, MD, PhD
Chief Scientist/Principal Investigator
Outline of the Programmed Project Research
Background of the Research
Aerobes conduct high-level vital activities by means of energy metabolism that uses the chemical reactivity of molecular oxygen (oxidation-reduction: redox activity). ROS are reduced derivatives of molecular oxygen (e.g., superoxide anion O2-, hydrogen peroxide H2O2), which are produced during energy metabolism and infection defense processes in cells and tissues. ROS have been thought to be noxious agents that mediate oxygen toxicity (ROS toxicity theory). Indeed, ROS were suggested to be involved in the pathogenesis of various diseases. These disorders include infections; inflammations; cancers; lifestyle-related diseases and metabolic diseases such as arteriosclerosis and diabetes mellitus; and neurological diseases such as Alzheimer’s disease. However, clinical application of antioxidant agents for treatment and prevention of these diseases has not yet achieved the expected results.
Several isoforms of ROS-producing enzymes, called Nox, the NADPH oxidase (and Duox), have been recognized. Expressed by many types of cells, these isoforms have not only antimicrobial actions but also other biological functions in a wide range of cells and tissues including vasculature, phagocytes and immune system, and epithelial and endocrine tissues. H2O2 has been suggested to act as a signal that mediates vascular dilatation. Furthermore, the toxicity of nitric oxide (NO) is known to be augmented by reacting with ROS. However, NO functions as a master cell signaling molecule involved in diverse biological phenomena.
From these evidences, the concept of ROS toxicity causing nonspecifically injures biomolecules has changed drastically. Thus, in a wide variety of life science fields, the recognition of ‘the physiological cell signaling functions of ROS’ has greatly advanced.
Aims of This Area
In this innovative scientific research area, we explore the signaling functions of ROS as a new paradigm evolving from the changing concept of ROS toxicity. The most innovative results are expected from the present ROS project in this emerging field of chemical biology.
The following elements, based on the three steps in the ROS signaling process, were constructed and promoted as a practical strategy for programmed project-type research: (a) generation of ROS signals, (b) regulation of the ROS sensing function, and (c) regulation of ROS effector molecules.
a. Generation of ROS Signals
Different from many other signaling molecules, ROS are simple, low-molecular-weight inorganic compounds that are also chemically reactive and thus mostly unstable in biological systems. The specificity of the signaling functions depends on the ROS production system and the chemical reactivity, especially as related to a spatiotemporal property, which defines the molecular reaction environment. Therefore, to clarify the production and sensing mechanisms of ROS signals, it is essential to analyze the structure and function of ROS-producing enzymes (e.g., Nox), which mediate the signaling function in various cells and tissues, and the localization and spatiotemporal dynamics of ROS production in cells. Identifying the signaling ligand in the ROS production system is thus extremely important. The exact spatiotemporal regulation mechanisms of ROS signaling and sensing remain totally unknown, however. Accordingly, in this element of programmed project-type research, we will analyze ROS production mechanisms with Nox or mitochondria, so that we can understand the total picture of the spatiotemporal generation of ROS signals. In particular, we will explore the interaction of signal production and sensor-effector molecules at cellular and individual organism levels, by means of our original detection and imaging techniques with specific fluorescent probes to localize sites of ROS production in cells.
b. Regulation of the ROS Sensing Function
The ROS signal is an unstable primary signal that transforms to a more stable secondary signal. During this process, effective chemical sensors of ROS (e.g., nucleic acids, nucleotides, lipids, and reactive protein residues) seem to be present in cells with a wide range of repertoires. For example, during the interaction of ROS/NO with various sensors, such as nucleic acids, lipids, and protein sulfhydryls, stable secondary signaling molecules (e.g., 8-nitro-cGMP and nitro-fatty acids) are produced. Also, a sensor protein with cysteine (Cys) sulfhydryls directly or indirectly mediates the receptor function for ROS signaling, because of high redox activity. Identification and analysis of these sensor molecules are very important, therefore, to understand the sensing specificity of the ROS signaling system. Thus, in this element of programmed research, we will attempt to identify ROS sensor molecules and clarify their chemical sensing mechanisms. We will also analyze structures and functions of sensor and effector proteins modified by the ROS signal and its secondary signaling molecules (e.g., 8-nitro-cGMP). More important, we will explore new ROS sensors yet to be identified. By using our original chemical probes for detection and imaging, we will clarify the overall picture of various ROS sensing functions in transmission of cell signals.
c. Regulation of ROS Effector molecules
The biological functions of effectors, being directly affected by ROS or indirectly mediated by secondary signaling molecules of ROS, can be induced by ROS signal-caused structural changes of sensor proteins, which in some cases simultaneously act as effectors. For example, phosphorylation and transcription signaling pathways are regulated via structural changes occurring in sensor-effector proteins (i.e., specific redox-sensitive protein kinases and phosphatases) and transcription factors. These structural changes are caused by chemical modification by ROS (e.g., oxidation, nitrosylation, alkylation, and guanylation of Cys sulfhydryls). Clarifying the molecular mechanisms of various sensor-effector relationships with ROS is an important theme of this area. Specifically, we will investigate cell response mechanisms (cell proliferation and cell death) mediated by ROS signaling, with a focus on particular sensor-effector proteins involved in intracellular phosphorylation signal transduction, transcriptional regulation, endoplasmic reticulum stress, and neuronal and vascular signal transduction.
Subjects and Scientists for Each Programmed Research Project
Section A01: Mechanisms of ROS Signal Formation
| A01-1 | Regulatory mechanisms of ROS-producing, Nox-family NADPH oxidases |
| PI | Hideki Sumimoto (Department of Biochemistry, Kyushu University Graduate School of Medical Sciences) |
| Outline | ROS have been thought to be signal-transducing molecules. This study will clarify mechanisms by which ROS-producing enzymes of the NADPH oxidase (Nox) family are regulated at many levels, from the atom to the cell. |
| A01-2 | Development of novel fluorescent probes for ROS and related species |
| PI | Yasuteru Urano (Graduate School of Pharmaceutical Sciences, The University of Tokyo) |
| Outline | This programmed study aims to develop fluorescent probes that can detect ROS and various electrophilic intermediates and their protein modifications in living cells or in specific organelles, which will be useful for clarifying signaling functions of ROS. |
| A01-3 | Regulation of mitochondrial ROS signaling in the cardiovascular stress response |
| PI | Hiroyuki Tsutsui (Department of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine) |
| Outline | This study will explore the role of mitochondrial ROS signaling in the cardiovascular stress response and its regulatory mechanisms. This project can provide fundamental information for developing novel therapeutic strategies for cardiovascular diseases based on the new paradigm. |
Section A02: ROS Sensing and Signaling Mechanisms
| A02-1 | ROS signal sensing via unique post-translational modification |
| PI | Takaaki Akaike (Graduate School of Medical Sciences, Kumamoto University) |
| Outline | A new cyclic nucleotide, 8-nitro-cGMP, which is a secondary signal of ROS and NO, forms in biological systems and undergoes a unique post-translational modification, called protein S-guanylation. This programmed project will utilize “S-guanylation of protein proteomics” to understand ROS sensing mechanisms and to discover new signal sensors of ROS. |
| A02-2 | Probing ROS signaling with small molecules |
| PI | Hirokazu Arimoto (Graduate School of Life Sciences, Tohoku University) |
| Outline | This project aims to identify targets of protein S-guanylation (Akaike, Arimoto et al. 2007), which senses ROS signals. The repair processes of guanylated proteins will also be investigated. For these purposes, fluorescent probes based on nitroguanosine will be synthesized and utilized in various analyses including live-cell imaging experiments. |
| A02-3 | Chemical biology of ROS ligand-receptor regulation |
| PI | Koji Uchida (Graduate School of Bioagricultural Sciences, Nagoya University) |
| Outline | This programmed study will clarify the target protein for low-molecular-weight electrophilic ligands mediating ROS signaling. The target protein thereby identified will be further characterized in terms of its activation mechanisms of signal transductions. |
| A02-4 | Explication of the regulatory mechanism of S-nitrosylation signaling |
| PI | Akio Matsumoto (Department of Pharmacology Graduate School of Medicine, Chiba University) |
| Outline | The major focus of this project is to elucidate the effects of NO, particularly through S-nitrosylation of the target protein, transmitted through the plasma membrane of juxtaposed cells. The following three projects are included under this theme: (1) The mechanism of NO signal transmission through the plasma membrane (2) The regulatory mechanism of NO bioactivity in cells (3) Pathophysiological involvement of anomalous S-nitrosylation signaling |
Section A03: Mechanisms of ROS Effector Regulation
| A03-1 | Roles of ROS as endothelium-derived relaxing factors |
| PI | Hiroaki Shimokawa (Department of Cardiovascular Medicine Tohoku University, Graduate School of Medicine) |
| Outline | The three major endothelium-derived relaxing factors (EDRFs) are prostacyclin, NO, and endothelium-derived hyperpolarizing factor (EDHF). We found that endothelium-derived H2O2 is an EDHF in animals and humans. In this project, we aim to examine the roles of NO and H2O2 as EDRFs and to elucidate their roles as signaling molecules. |
| A03-2 | Transcriptional regulation of ROS signaling |
| PI | Ken Itoh (Department of Stress Response Science, Hirosaki University Graduate School of Medicine) |
| Outline | In this programmed study, transcriptional regulation of ROS signaling will be explored at the nuclear level of cells, with a focus on Nrf2, a key redox-reactive transcription factor. The pathological consequence of such intranuclear ROS signaling will also be analyzed to establish a molecular basis for developing an innovative strategy for various disease treatments. |
| A03-3 | Nitrosative stress and signaling in the pathogenesis of neurodegenerative diseases |
| PI | Takashi Uehara (Department of Pharmacology, Graduate school of Pharmaceutical Sciences, Hokkaido University) |
| Outline | This project aims to identify novel S-nitrosylated proteins by using various protein microarray-based screening procedures. We are now clarifying the pathophysiological role of protein S-nitrosylation and S-oxidation related to the onset of neurodegenerative diseases. |
| A03-4 | ROS-sensitive protein phosphatase and its signal transduction |
| PI | Hideaki Kamata (Department of Molecular Medical Science., Hiroshima University) |
| Outline | Certain phosphates with redox-sensitive Cys residues function as major effectors in transmission of ROS signaling involving phosphorylation signals. This study will thus aim to clarify the molecular mechanisms of phosphate regulation by ROS, with a primary focus on sulfhydryl modification of the phosphatase by ROS. |
| A03-5 | ROS sensor-effector regulation via Cys modifications |
| PI | Motohiro Nishida (Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University) |
| Outline | ROS signaling is precisely regulated by the functional association of the ROS production system with sensor proteins and effectors. This study will analyze the molecular basis of the functional modification, with a special focus on ROS-sensitive sensor molecules, which regulate angiotensin receptor expression. |