The body’s immune cells use a controlled burst of reactive oxygen species (ROS) to kill microbes, but a newly discovered protein called TMEM128 appears to act as a molecular brake on that process. When ROS production goes wrong, the consequences are severe. Too little ROS leaves people vulnerable to life-threatening infections, as seen in chronic granulomatous disease (CGD), while too much ROS drives autoimmune conditions such as lupus and rheumatoid arthritis. The protein EROS is already known to be essential for building the ROS-producing enzyme, but how EROS itself is controlled has remained a mystery. The researcher has now found that TMEM128 binds directly to EROS and physically blocks it from doing its job, revealing a previously unknown regulatory layer. This is fundamental science. The immediate goal is to understand how TMEM128 works at the molecular level, how its production is controlled, and what happens when it is missing in mice facing bacterial or viral infections. If the work succeeds, it could eventually point toward new ways to fine-tune ROS production in patients—boosting it to fight infection in CGD, or dampening it to calm autoimmune inflammation. Similar fundamental discoveries about ROS regulation have already led to clinical insights into immune disorders.
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Reactive oxygen species (ROS) are vital for host defence and immuno-regulation. Although reactive oxygen species such as hydrogen peroxide are often considered damaging to cells through oxidative stress, humans have several dedicated systems for the production of ROS. In the immune system, the phagocyte NADPH oxidase (NOX2) generates ROS. Its importance is underlined by the fact that mutations in the genes encoding subunits of this protein complex lead to chronic granulomatous disease (CGD)[1]. CGD is an inherited inborn error of immunity (IEI), characterised by severe recurrent opportunistic infections as well as autoinflammation and autoimmunity [2]. Furthermore, genome-wide association studies demonstrate that hypomorphic coding polymorphisms in genes encoding phagocyte NADPH oxidase subunits lead to an increased risk of several autoimmune diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis and inflammatory bowel disease [4][5] The propensity towards autoinflammatory manifestations in the absence of ROS arises because some ROS must be made for normal immunity, for example in the degradation of dead cells and the control of cell signalling. This latter effect is via the reversible oxidation of cysteine residues in proteins that are central to the immune response [6]. It is therefore crucial that the production of ROS is tightly regulated: both too much and too little are bad. The Thomas lab identified the Essential for Reactive Oxygen Species protein (EROS) as necessary for the generation of ROS. It is a chaperone for gp91phox, the key transmembrane protein of the phagocyte NADPH oxidase [3]. EROS is essential for host defence in both mouse and humans and homozygous mutations in EROS have been identified as a novel cause of CGD [7][8][9][13]. When EROS was first described it was a completely uncharacterised protein giving us very few clues to how its actions are regulated. My preliminary work, using multiple orthologous techniques, has identified TMEM128, another uncharacterised transmembrane protein, as forming a stable interaction with EROS. We have also demonstrated its functional relevance. TMEM128 can bind directly to EROS and physically sequester it away from gp91phox, thus regulating its ability to act as a chaperone for this protein. This is an exciting development because we believe that we have now found another crucial regulator of the ancient and conserved process of ROS production in the immune system. I will characterise the role of TMEM128 in regulating ROS production. I will focus on immune and endothelial cells as our preliminary data has shown high expression of TMEM128 (and EROS) in these cell types. I will use NanoBiT technology to dissect the nature of the interaction between TMEM128 and EROS by deciphering how this protein-protein interaction takes place. I will use our alpha-fold prediction of the EROS-TMEM128 complex, together with site-directed mutagenesis to understand the TMEM128-EROS interface. Yeast two hybrid and immunoprecipitation will also be used to further characterise the interaction. I will also determine what controls TMEM128 production physiologically. The functional relevance of TMEM128 will be addressed by knockout/knockdown using CRISPR-Cas9/siRNA and lentiviral overexpression followed by evaluation of NOX2 component expression and ROS production. Following this, I will phenotype TMEM128 knock-out mice and inoculate them with bacterial and viral challenges to understand the role of TMEM128 in host defence. I will therefore define the role of this novel ROS regulator in the immune system and vascular biology.
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