A role for neuroglobin in the inhibition of cytochrome c-mediated apoptosome formation

Abstract

The mitochondrial pathway of apoptosis is an important mediator of cell death in many human diseases. A key event in this pathway is permeabilisation of the outer mitochondrial membrane and subsequent release of the heme-protein cytochrome c from mitochondria into the cytosol. Thereafter, cytosolic cytochrome c mediates the formation of the apoptosome complex, which cleaves the initiator caspase 9, thereby activating the caspase signalling cascade. Apoptosome formation is thought to require cytochrome c which has its heme iron in the ferric form. Neuroglobin, a recently discovered globin, protects various cell lines and tissues from apoptosis. Previously, neuroglobin has been shown to efficiently reduce the heme iron of cytochrome c in vitro. We hypothesise that neuroglobin prevents apoptosome assembly by reducing cytochrome c. The work carried out in this thesis aimed to investigate the properties of the neuroglobin-cytochrome c complex and identify its role in the regulation of apoptosome formation. Surface Plasmon Resonance experiments have been used to explore the nature of the complex and have revealed that neuroglobin binds to cytochrome c with weak affinity (KD = 27-56 μM), forming a transient protein complex that is mediated largely by electrostatic interactions. A putative specific complex structure has been predicted by computer modelling. The interacting residues on the neuroglobin interface in the putative complex have been mutated and changes in binding affinity were measured with SPR. The results from these studies are consistent with the neuroglobin-cytochrome c interaction occurring via an encounter complex mechanism. Furthermore, cell-free in vitro assay systems have been developed to measure caspase activation and apoptosome formation induced by exogenous cytochrome c. In keeping with previous studies, ferric cytochrome c efficiently initiated apoptosome formation, while ferrous cytochrome c exhibited very little of this activity. Ferrous neuroglobin was capable of efficiently inhibiting apoptosome formation initiated by ferric cytochrome c, whereas ferric neuroglobin was significantly less effective. Taken together, these experiments suggest a role for ferrous neuroglobin in the prevention of apoptosome formation.