A study published online in PNAS and led by Prof. YAO Yikun’s group from the Shanghai Institute of Nutrition and Health (SINH) of the Chinese Academy of Sciences identified and characterized the key factor of Activating Molecule in BECN1-regulated Autophagy Protein 1 (AMBRA1) in the FAS-mediated T cell death process, and revealed a new translation dependent mechanism by which AMBRA1 controls TCR signaling, T cell cycle, and T cell death. 

After antigen stimulation of the T cell receptor, T cells are rapidly activated and differentiate into effector T cells or memory T cells. Activated T cells quickly initiate glycolysis to generate energy and essential components for T cell proliferation, requiring the rapid synthesis of a large amount of protein. Therefore, in addition to transcriptional regulation, the regulation of translation at a more immediate level may also play a significant role in T cell activation. 

Following T cell activation, amino acid uptake increases rapidly, but mRNA levels do not show a corresponding increase, suggesting that the main driving factor for the increased protein synthesis in activated T cells is likely translation rather than transcription. Previous studies on T cell activation and activation-induced T cell death have largely focused on transcriptional regulation, with insufficient exploration of translation-level regulation in T cells. 

To uncover new regulatory factors mediating T cell death, utilizing a whole-genome CRISPR screening, researchers discovered that the scaffold protein AMBRA1 plays a crucial role in FAS-mediated T cell death. AMBRA1 is an evolutionarily conserved scaffold protein, and has been shown that it is a key regulatory factor in autophagy, E3 ubiquitin ligase activity, and cyclin proteins. However, the function of AMBRA1 and its translation-related regulatory mechanisms in lymphocytes are unknown. 

Then, researchers performed flow cytometry, Western blotting, and a series of functional rescue experiments, and verified that the knockout of AMBRA1 reduces FAS protein expression and inhibits FAS pathway-induced T cell apoptosis. Utilizing techniques including OPP incorporation combined with click chemistry, and sucrose gradient centrifugation to isolate ribosomes and their associated mRNA, they found that AMBRA1 promotes the translation of FAS mRNA, a regulatory effect crucial for the complete expression of FAS following T cell activation. 

Through RNA-seq and mass spectrometry, researchers found that TCR signaling significantly stimulates the expression of AMBRA1. Using TCR downstream pathway inhibitors and an AMBRA1 5' UTR reporter gene, they revealed the existence of a translation control circuit induced by TCR stimulation. This circuit can enhance the translation of AMBRA1 after T cell activation via the CD28–PI3K–mTORC1–eIF4F axis, regulating the expression of FAS and other immune-related genes. 

Moreover, using a series of protein interaction and translation level detection techniques, researchers revealed that AMBRA1 interacts with multiple ribosomal proteins and promotes the translation of various ribosome biogenesis-related proteins. Experiments including proteomic analyses demonstrated that in addition to FAS, the translation of many TCR signaling genes, such as CD69, CD28, PD1, and CTLA4, is also regulated by AMBRA1, suggesting that AMBRA1 has a broad impact on the translation of T cell signaling proteins. 

This study shows that the AMBRA1 gene plays a crucial role in the translational regulation of TCR activation and FAS-induced T cell death signals. It provides potential directions for future research on T cell translation and immunotherapies targeting translational regulation.