Dr Alain BRUHAT

Research interests

Nutrition encompasses not only food absorption but also the metabolic fate and effect of nutrients on various physiological functions. To cope with the highly variable intake of nutrients, higher organisms, and particularly mammals, have developed complex mechanisms capable of buffering the effects of these fluctuations. In particular, certain nutritional (protein malnutrition, etc.) and/or pathological (burns, infections, cancer, etc.) situations result in significant variations in the blood concentration of amino acids (AAs). Given that some AAs (i) cannot be synthesized by the body (9 AAs are essential) and (ii) there is no dedicated storage system as is the case for lipids or glucose, the individual must adapt to a deficiency in essential AAs (EAAs). To do this, it must regulate certain physiological functions (growth, appetite, etc.). This regulation requires, on the one hand, detecting the AEA deficiency and, on the other hand, adjusting the expression level of a certain number of specific genes involved in defense processes and/or adaptation to EAA limitation. It is now well established that EAAs can regulate certain physiological functions (food intake, autophagy, etc.), notably by directly varying the expression of target genes via the activation of signaling pathways such as the GCN2-eIF2α-ATF4 pathway.

During my career, I initially contributed to the characterization and subsequent investigation of the role played by the GCN2–eIF2α–ATF4 signaling pathway in the adaptation process to essential amino acid (EAA) restriction in cultured mammalian cells. Following this, the strategic choices I made have enabled me to develop a more integrated research program, which is currently divided into two complementary components.

(1)   Study of the eIF2α-ATF4 pathway at the whole animal level in relation to nutritional and pathophysiological situations.

• Generation of a bioluminescent transgenic mouse line (CARE-LUC) to visualize the activation of the eIF2α-ATF4 pathway.

To visualize the activation of the eIF2α/ATF4 pathway in response to stress at the whole-animal level, we generated a transgenic mouse line called CARE-LUC that expresses the luciferase reporter gene (LUC) under the control of CARE sequences capable of binding the ATF4 transcription factor. The luciferase expressed in the different tissues catalyzes the oxidation of luciferin injected into the animal. The light emitted during this reaction is therefore proportional to the level of expression of the luciferase gene. Its detection thus makes it possible to visualize the activation of the eIF2α-ATF4 pathway as a function of stress or pathophysiological or pathological situations at the level of (i) the whole living animal by bioluminescent imaging, (ii) each organ or tissue by bioluminescent imaging and enzymatic assay, and (iii) each cell by immunohistochemistry using an anti-luciferase antibody (Chaveroux et al., Science Sci, 2015).

• Prevention of eIF2α-ATF4 pathway activation by cysteine supplementation during drug treatment.

Chronic treatment with paracetamol (PCM) induces a deficiency of cysteine, a sulfur-containing AA (SAA), and glutathione (GSH), causing adverse metabolic effects such as muscle atrophy. The eIF2α-ATF4 signaling pathway, activated in response to SAA deficiency, was studied in mice treated with PCM. In the liver, this treatment simultaneously activates GCN2 and PERK kinases, leading to the expression of ATF4 target genes. In skeletal muscle, activation of the pathway is associated with atrophy, independent of GCN2. Dietary cysteine supplementation prevents the effects of PCM, including reduced plasma levels of free cysteine, hepatic and muscle GSH, and loss of muscle mass. It also inhibits eIF2α-ATF4 pathway activation, highlighting the importance of ASA homeostasis in mitigating the side effects of chronic PCM treatment (Carraro et al., IJMS, 2022).

•   Role of the GCN2-ATF4 signaling pathway in the hepatic response to short-term sulfur amino acid (SAA) deficiency.

Our work on the importance of SAA homeostasis and the eIF2α-ATF4 pathway in managing the effects of paracetamol led us to explore the short-term role of GCN2 kinase in the liver, in response to the consumption of a SAA-deficient diet. It is now well established that dietary restriction of SAA, such as methionine and cysteine, significantly improves metabolic health and extends lifespan in rodents. These benefits include reduced body weight, decreased fat accumulation, increased insulin sensitivity, and lower circulating and tissue lipid levels. The eIF2α-ATF4 pathway is activated in response to this restriction, but the exact role of the GCN2 kinase in this process remains poorly understood. Our main results show that GCN2 plays a central role in the induction of an early transcriptional program, essential for the adaptive response to 3-hour SAA-deficiency. This program notably involves SAA metabolism and glutathione biosynthesis (Carraro et al., in preparation).

(2) Using the knowledge acquired about the GCN2-eIF2α-ATF4 pathway to develop a gene regulation system applicable to human gene therapy.

Our work on the GCN2/ATF4 signaling pathway, combined with our expertise in AA metabolism and protein nutrition, has led to the development of NUTRIREG, an innovative nutrition-controlled gene expression system for gene therapy (see Figure).

This system activates the expression of a therapeutic transgene via a diet deficient in an EAA. NUTRIREG is based on an artificial promoter sensitive to EAA deficiency and on a diet inducing this deficiency, thus activating the GCN2-ATF4 pathway. A proof of concept, published in 2016 (Chaveroux et al. Nat Biotech.), demonstrated its efficacy in mice. In 2019, the development of NUTRIREG led to the creation of the startup NUTRITHERAGENE (which became ON OFF Therapeutics at the end of 2024), and focuses on its application in oncology.

Furthermore, a clinical study, conducted in 2020, validated in humans a nutritional protocol to lower plasma leucine levels, a necessary condition for activating the transgene. This advance also led to the filing of a patent (#EP23306459, September 1, 2023).

Two major projects leveraging this technology within the startup have been undertaken:

• Treatment of liver metastases from colorectal cancer: use of NUTRIREG to intermittently express a protein drug in the healthy liver. This project is being carried out in collaboration with the Digestive Surgery and Oncology Department of the Clermont-Ferrand University Hospital (M. Jary and D. Pezet) and the INSERM LNC-UMR1231 Research Center in Dijon (E. Limagne and F. Ghiringhelli).
• Cellular immunotherapy: intermittent expression of a protein of interest in T lymphocytes to increase therapeutic efficacy. This project is developed in partnership with the Cell Therapy and Clinical Hematology Department of the Clermont-Ferrand University Hospital (J.O. Bay, A. Dougé) and the CHELTER-CHU-UCA team (P.-O. Rouzaire). This work has led to the filing of a patent (#EP22306008, July 5, 2022) and the publication of two articles (Dougé et al., HLA 2024; Dougé et al., PlosONE 2025).

Most significant scientific publications

A. Dougé, G. Cueff, C. Keime, V. Carraro, C. Jousse, P. Rouzaire*, A. Bruhat* (2025). Transcriptomic analysis of human primary T cells after short-term leucine-deprivation and evaluation of kinase GCN2’s role in regulating differential gene expression. PLoS ONE, 20 (2), e0317505, https://dx.doi.org/10.1371/journal.pone.0317505, https://hal.inrae.fr/hal-05031012 * Co-derniers auteurs

A. Dougé, C. Vituret, V. Carraro, L. Parry, C. Coudy-Gandilhon, R. Lemal, L. Combaret, A.C. Maurin, J. Averous, C. Jousse, J.-O. Bay, P. Verrelle, P. Fafournoux, A. Bruhat*, P Rouzaire*. (2024). Temporal regulation of transgene expression controlled by amino acid availability in human T cells. HLA: Immune Response Genetics, Jan: 103 (1), e15252, https://dx.doi.org/10.1111/tan.15252, https://hal.inrae.fr/hal-04214017 * Co-derniers auteurs

V. Carraro, L. Combaret, C. Coudy-Gandilhon, L. Parry, J. Averous, A.-C. Maurin, C. Jousse, G. Voyard, P. Fafournoux, I. Papet, A. Bruhat (2022). Activation of the eIF2α-ATF4 Pathway by Chronic Paracetamol Treatment Is Prevented by Dietary Supplementation with Cysteine. International Journal of Molecular Sciences, 23 (13), 7196, https://dx.doi.org/10.3390/ijms23137196, https://hal.inrae.fr/hal-03741978

C. Chaveroux, A. Bruhat, V. Carraro, C. Jousse, J. Averous, A.-C. Maurin, L. Parry, F. Mesclon, Y. Muranishi, P. Cordelier, A. Meulle, P. Baril, A. Do Thi, P. Ravassard, J. Mallet, P. Fafournoux (2016). Regulating the expression of therapeutic transgenes by controlled intake of dietary essential amino acids. Nature Biotechnology, 34, 746-751, https://dx.doi.org/10.1038/nbt.3582, https://hal.inrae.fr/hal-01594469

C. Chaveroux, V. Carraro, L. Canaple, J. Averous, A.-C. Maurin, C. Jousse, Y. Muranishi, L. Parry, F. Mesclon, E. Gatti, J. Mallet, P. Ravassard, P. Fafournoux, A. Bruhat (2015). In vivo imaging of the spatiotemporal activity of the eIF2α-ATF4 signaling pathway: Insights into stress and related disorders. Science Signaling, 8 (374), rs5-rs5, https://dx.doi.org/10.1126/scisignal.aaa0549, https://hal.inrae.fr/hal-02459218

W. B'Chir, A.-C. Maurin, V. Carraro, J. Averous, C. Jousse, Y. Muranishi, L. Parry, G. Stepien, P. Fafournoux, A. Bruhat (2013). The eIF2 alpha/ATF4 pathway is essential for stress-induced autophagy gene expression. Nucleic Acids Research, 41 (16), 7683 - 7699, https://dx.doi.org/10.1093/nar/gkt563, https://hal.inrae.fr/hal-01056786

V. Carraro, A.-C. Maurin, S. Lambert-Langlais, J. Averous, C. Chaveroux, L. Parry, C. Jousse, D. Oerd, T. Oerd, P. Fafournoux, A. Bruhat (2010). Amino Acid Availability Controls TRB3 Transcription in Liver through the GCN2/eIF2 alpha/ATF4 Pathway. PLoS ONE, 5 (12), https://dx.doi.org/10.1371/journal.pone.0015716, https://hal.inrae.fr/hal-02660079

Y. Cherasse, A.-C. Maurin, C. Chaveroux, C. Jousse, V. Carraro, L. Parry, C. Deval, C. Chambon, P. Fafournoux, A. Bruhat (2007). The p300/CBP-associated factor (PCAF) is a cofactor of ATF4 for amino acid-regulated transcription of CHOP. Nucleic Acids Research, 35 (17), 5954-5965, https://dx.doi.org/10.1093/nar/gkm642, https://hal.inrae.fr/hal-0265840

Other links:

Linkedin : https://www.linkedin.com/in/alain-bruhat-2250b81a9

Researchgate : https://www.researchgate.net/profile/Alain-Bruhat?ev=hdr_xprf