Proteomic analysis of GTP cyclohydrolase 1 multiprotein complexes in cultured normal adult human astrocytes under both basal and cytokine‐activated conditions

GTP cyclohydrolase 1 (GCH1) is the rate‐limiting enzyme of a metabolic pathway synthesizing tetrahydrobiopterin (BH₄), the cofactor dimerizing and activating inducible nitric oxide synthase (NOS‐2). GCH1 protein expression and enzyme activity are minimal in cultured, phenotypically stable, untreated normal adult human astrocytes (NAHA), but are strongly induced, together with NOS‐2, by a mixture of three proinflammatory cytokines (IL‐1β, TNF‐α, and IFN‐γ – the CM‐trio) released by microglia under brain‐damaging conditions. The resulting hyper‐production of NO severely harms neurons. In this study, using MALDI‐TOF/MS, PMF, Western immunoblotting (WB), and antibody microarrays we identified several proteins coimmunoprecipitating with GCH1. Under basal conditions, GCH1 was associated with various adaptor/regulator molecules involved in G‐protein‐coupled receptors signalling, protein serine/threonine phosphatase 2Cβ (PP2Cβ), and serine–threonine kinases like Ca²⁺ calmodulin kinases (CaMKs), casein kinases (CKs), cAMP‐dependent kinases (PKAs), and mitogen‐activated protein kinases (MAPKs). Exposure to the three cytokines' mixture (CM‐trio) significantly changed, within the 48–72 h required for the induction and activation of GCH1, the levels and identities of some of the 0 h‐associated proteins: after 72 h CK‐IIα tended to dissociate from, whereas MAPK12 and JNK3 were strongly associated with fully active GCH1. These findings provide a first enticing glimpse into the intricate mechanisms regulating GCH1 activation by proinflammatory cytokines in NAHA, and may have therapeutic implications.