The signal transduction mechanisms of immune receptors and their clinical applications have always been a cutting-edge topic in biomedicine. They help us understand the most fundamental immune responses and are key to developing innovative immunotherapies. Current immunotherapies are largely based on signal regulation strategies of immune receptors, such as immune checkpoint blockade therapy (Nobel Prize in 2018), CAR-T and TCR-T cell therapies, etc.
On November 11, 2024, the team of Shi Xiaoshan from the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, and Xu Chenqi from the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, published a prospective article titled "Charge-based immunoreceptor signalling in health and disease" in the journal Nature Reviews Immunology. The article delves into a widely present signaling motif - the basic-residue-rich sequence (BRS). The presence of membrane-proximal positively charged residues in transmembrane proteins is a well-known conclusion from classical biochemistry textbooks. However, the signaling mechanisms, physiological and pathological functions, and application prospects of these sequences have not been systematically summarized. This article clearly defines the BRS signaling motif, summarizes its membrane-proximal signal transduction mechanisms, discusses the correlation between BRS mutations in immune receptors and human diseases, and explores the potential of BRS in innovative immunotherapies.
Article online screenshot
Paper link: https://www.nature.com/articles/s41577-024-01105-6
Figure 1 The signaling transduction mechanism and translational application of immune receptors mediated by BRS. a, BRS (blue) is widely present in the intracellular membrane-proximal region of various immune receptors; b, BRS regulates the signal and function of immune receptors through a spatiotemporally dynamic membrane electrostatic network; c, BRS mutations are closely related to human diseases; d, the design of synthetic receptors using BRS has great translational application prospects.
Definition and Universality of Basic-residue-rich Sequence (BRS)
BRS is usually 10 amino acids long, carries two or more net positive charges, and is often located in the intracellular membrane-proximal region, but can also be distributed in the distal region. BRS is usually an intrinsically disordered region (IDR), but can form secondary structures after interacting with other molecules such as acidic phospholipids. To date, BRS in various immune receptors such as antigen receptors (T cell receptors, B cell receptors), co-stimulatory receptors, co-inhibitory receptors, NK cell receptors, Fc receptors, and cytokine receptors have been experimentally reported. Not only immune receptors, but nearly 70% of human single-transmembrane proteins carry BRS in their intracellular membrane-proximal regions.
Membrane-proximal Electrostatic Regulation Network Mediated by BRS
Experiments have proven that BRS can interact electrostatically with negatively charged or π-electron-carrying lipid and protein molecules in the cell membrane and membrane-proximal region, and environmental factors can further regulate these interactions, forming a spatiotemporally dynamic membrane electrostatic network. The known members of the network include: BRS, acidic phospholipids (such as phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)), steroid molecules (such as cholesterol, hydroxycholesterol), positively charged metal ions (such as calcium ions), and membrane proteins or membrane-proximal proteins rich in negative charges or π-electrons (such as LCK, p85, LAG3, PLCγ1), etc.
Through the membrane electrostatic network, BRS regulates processes such as phosphorylation, ubiquitination, liquid-liquid phase separation, and mechanical signal transduction of immune receptors. Specifically, BRS-lipid electrostatic interactions can shield the phosphorylation and ubiquitination sites of immune receptors within the membrane, thereby limiting the basal signal and ubiquitination of immune receptors; at the same time, these interactions can pre-assemble immune receptors with signaling molecules PI(4,5)P2, ensuring that immune receptors are in a "ready-to-go" state. Research has found that the interaction between BRS and PI(4,5)P2 is crucial for the mechanical response of immune receptors. After ligand binding to the receptor, it may induce conformational changes in the receptor and changes in the membrane environment, causing BRS and surrounding sequences to dissociate from the membrane; then BRS quickly recruits functional proteins through electrostatic interactions and may form signal bodies with liquid-liquid phase separation characteristics, mediating the triggering and amplification of immune receptor signals. In addition, after BRS dissociates from the membrane, it will also induce the exposure of ubiquitination sites, thus producing ubiquitin-based degradation or signal regulation.
1. Correlation between BRS Mutations and Diseases
Examples of BRS-related mutations causing human diseases have been widely reported. The UniProt database already includes multiple pathogenic BRS mutations. These mutations result in the loss or gain of basic residues, thereby changing the signal transduction process. For example, the pathogenic mechanisms of IL-23R R381Q and IGHG1 G396R have been studied in detail. The loss of positive charge in IL-23R R381Q mutation leads to weakened IL-23R signaling, reducing immune response and thus lowering the risk of inflammation, but increasing the risk of infection. In contrast, the gain of positive charge in IGHG1 G396R mutation leads to enhanced IgG BCR signaling, enhancing immune response and thus increasing the risk of autoimmunity, but reducing the risk of infection, and at the same time showing better immune treatment response in cancer patients.
2. Prospects for Translational Application of BRS
The translational research on BRS focuses on two aspects: the signal regulation of BRS in natural immune receptors and the design of synthetic immune receptors using BRS. For example, the steroid metabolite 7α-hydroxycholesterol can weaken the arrangement density of cell membrane lipid molecules, helping the BRS of TCR signal subunit CD3ε to bind better with the membrane, thereby inhibiting the phosphorylation of TCR. This mechanism has been used in the preparation of TCR-T cells, by inhibiting the basal signal of TCR to increase the proportion of memory cells and improve the longevity of immunotherapy. On the other hand, BRS has been used in CAR-T cell therapy. The E-CAR molecule formed by adding the CD3ε signal domain to the second-generation CAR molecule has better signal transduction ability. BRS can mediate the production of liquid-liquid phase separation through cation-π bonds, helping cells form more mature and efficient immune synapses, thereby enhancing the antigen sensitivity and longevity of E-CAR-T cells. It is worth mentioning that BRS is also crucial for the efficient signal transduction of another synthetic receptor, SNIPR (synthetic intramembrane proteolysis receptor).
3. Future Directions
There is a rich library of BRS in membrane proteins. However, at the current stage, the understanding and application of BRS signal transduction mechanisms are still very limited, and there are a series of important issues that need to be addressed in the future. For example, can BRS be divided into multiple subclasses? Do different subclasses have different signal regulation patterns? How do various BRS mutations lead to human diseases? How to rationally manipulate BRS signals or rationally design synthetic receptors containing BRS? The answers to these questions will greatly enhance our understanding of the immune system and help the development of immunotherapies.
Shi Xiaoshan, a researcher and doctoral supervisor, is the first author and co-corresponding author of this article. Xu Chenqi, a researcher, is the corresponding author of this article. The research was supported by the Ministry of Science and Technology, the Shanghai Science and Technology Commission, the Chinese Academy of Sciences, and the Shenzhen Institute of Synthetic Biology.
Shi Xiaoshan, a researcher and doctoral supervisor, focuses on developing and utilizing quantitative mass spectrometry and other technologies to systematically analyze the signal transduction mechanisms of immune cells and rationally design new synthetic immunotherapy methods. Relevant research results have been published in journals such as Nature and Cell, and have been selected for "National Natural Science Award", "Top Ten Advances in Chinese Life Sciences", "Important Medical Progress in China", etc. The research group has long been recruiting postdoctoral fellows in synthetic biology, immunology, cell biology, molecular biology, biochemistry, and other related majors. Welcome to join the team.
Contact email: xs.shi@siat.ac.cn
