Abstract
Neuroglobin (NGB) is an O2-binding globin mainly expressed in the central and peripheral nervous systems and cerebrospinal fluid. Previously, it was demonstrated that NGB overexpression protects cells from hypoxia-induced death. To investigate processes promoted by NGB overexpression, we used a cellular model of neuroblastoma stably overexpressing an NGB-FLAG construct. We used a proteomic approach to identify the specific profile following NGB overexpression. To evaluate the role of NGB overexpression in increasing energetic metabolism, we measured oxygen consumption rate (OCR) and the extracellular acidification rate through Seahorse XF technology. The effect on autophagy induction was evaluated by analyzing SQSTM1/p62 and LC3-II expression. Proteomic analysis revealed several differentially regulated proteins, involved in oxidative phosphorylation and integral mitochondrial proteins linked to energy metabolism. The analysis of mitochondrial metabolism demonstrated that NGB overexpression increases mitochondrial ATP production. Indeed, NGB overexpression enhances bioenergetic metabolism, increasing OCR and oxygen consumption. Analysis of autophagy induction revealed an increase of LC3-II together with a significant decrease of SQSTM1/p62, and NGB-LC3-II association during autophagosome formation. These results highlight the active participation of NGB in several cellular processes that can be upregulated in response to NGB overexpression, playing a role in the adaptive response to stress in neuroblastoma cells.
Highlights
We preliminarily investigated the effects of NGB gene overexpression, stably transfected in a cellular model of human neuroblastoma cells (SH-SY5Y), in the form of a FLAGtagged protein
NGB was uniquely identified in the NGB-FLAG samples and not in the CTRL, as expected, considering the low basal levels of endogenous NGB in SH-SY5Y cells, confirming the data obtained by immunoblotting detection
One protein of the differential dataset belonging to the cluster of the mitochondrial protein import, the voltage-dependent anion-selective channel protein 1 (VDAC1), a subunit of mitochondrial complex III and cytochrome c1, was chosen and tested as target of technical validation of the proteomic label-free quantitative (LFQ) experiment (Figure 2E)
Summary
NGB reversibly binds oxygen with an affinity higher than that of hemoglobin, stores oxygen to supply cells and plays an important neuroprotective role [2,3]. NGB is sensitive to changes in O2 availability and may play a role in ATP production [5]. This protein intervenes by inhibiting the intrinsic pathway of apoptosis, interfering with the release of cytochrome c (Cytc) from the mitochondria [6]. Regardless of the type of damage, the protective role of NGB appears to depend on two specific conditions: its overexpression and mitochondrial localization [16]. The redox state of the cell is strongly influenced by reactive oxygen species (ROS) which are by-products of aerobic metabolism [17]
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