Neuroglobin regulates autophagy through mTORC1/RAPTOR/ULK-1 pathway in human neuroblastoma cells

Abstract

Abstract Neuroglobin (NGB) is a hexacoordinated hemeprotein mainly expressed in neurons. Following its upregulation and mitochondrial localization, NGB plays a pro-survival role against neuronal stress. Previously, we built a stable NGB-FLAG-overexpressing neuroblastoma cell line and showed that NGB promotes autophagy and localizes in autophagolysosomes. Here we studied the interactome of NGB-FLAG cells to identify novel autophagy-related NGB-binding partners and investigate how its upregulation could induce autophagy. LC3-II and p62 levels as well as mTORC1 activity were analyzed to evaluate autophagy in NGB-FLAG cells. NGB interactors were identified by affinity purification-mass spectrometry and protein-protein interaction network analysis and validated by immunoprecipitation. The increase of LC3-II and decrease of p62 in NGB-FLAG compared to control confirmed that NGB overexpression promotes autophagy. Interactome analysis identified the Regulatory associated protein of mTOR (RPTOR) as one of 134 putative NGB interactors, further validated by immunoprecipitation. NGB overexpression also determined a consistent increment of RPTOR phosphorylation at Ser792 which is required for mTORC1 inhibition, then confirmed by lower levels of phospho-mTOR and phospho-ULK1 in NGB-FLAG compared to control. Collectively, our data suggests that NGB is a positive regulator of autophagy. Through association with RPTOR, NGB may promote its activation and inhibit mTORC1 repressive activity on autophagy initiation.

Keywords

• Neuroglobin
• Raptor
• Autophagy
• Neuroblastoma cells