The finding challenges a longstanding assumption in immunology: that iNOS controls immune cell behaviour primarily through nitric oxide production. The study shows that the physical shape of iNOS – stabilised by its cofactor, tetrahydrobiopterin (BH4) – is what drives the interaction with IRG1, independently of whether iNOS is producing nitric oxide at all.
The researchers used co-immunoprecipitation and mass spectrometry to confirm that iNOS is a direct binding partner of IRG1 in living cells, with computational modelling and molecular dynamics simulations used to predict and validate the structure of the interaction. Surface plasmon resonance confirmed that the binding is stable and high-affinity in both mouse and human models, and that it does not occur with the related protein eNOS – pointing to a specific, evolutionarily conserved function.
In cells lacking iNOS, IRG1 produced more than 15 times more itaconate compared with normal cells following immune stimulation. Critically, iNOS mutants unable to produce nitric oxide still suppressed IRG1 – what mattered was whether iNOS could adopt the correct shape, determined by BH4 binding. Disrupting that binding abolished the effect entirely.
The work also showed that in the absence of iNOS, IRG1 associated with a different set of partner proteins involved in glycolysis and cell metabolism – suggesting iNOS effectively sequesters IRG1 away from those roles, with wider consequences for how immune cells manage energy during inflammation.
A protein long understood to drive inflammation by producing nitric oxide has a second, previously unknown role – it physically binds to another key protein inside cells to directly modulate the immune response. The discovery, published in Nature Metabolism, could open new routes to treating conditions such as cardiovascular disease, arthritis, Crohn’s and other inflammatory diseases.
When the immune system detects infection or injury, it triggers inflammation to fight back. That response is essential, but it must be carefully controlled. If it runs too hard for too long, it causes the tissue damage that underlies many chronic diseases. Understanding the molecular switches that regulate inflammation – and finding new ways to target them – is one of the biggest challenges in modern medicine.
