10.1021/acs.biochem.6b00617.s001 Sheetal Uppal Sheetal Uppal Amit Kumar Singh Amit Kumar Singh Richa Arya Richa Arya Debanjan Tewari Debanjan Tewari Neha Jaiswal Neha Jaiswal Abhijeet Kapoor Abhijeet Kapoor Amal Kanti Bera Amal Kanti Bera Alo Nag Alo Nag Suman Kundu Suman Kundu Phe28<sup>B10</sup> Induces Channel-Forming Cytotoxic Amyloid Fibrillation in Human Neuroglobin, the Brain-Specific Hemoglobin American Chemical Society 2016 channel formation brain disorders Human Neuroglobin Ngb ligand binding kinetics apo states holo form fibril formation Recent reports blood hemoglobin ambient temperature Phe 28 B 10 Induces Channel-Forming Cytotoxic Amyloid Fibrillation Elevated temperature Ngb fibril Ngb amyloid fibril understanding amyloid-related brain disorders holo forms neuron-specific oxygen binding hemoglobin neuroblastoma cell lines ischemic stress-induced degeneration amyloid fibril-related amyloid formation brain stroke acid side chain Phe 28 B 10 cell membrane Brain-Specific Hemoglobin reference hemoglobins lipid bilayer membranes propidium iodide uptake assay role novel prospect neurodegenerative disorders 2016-11-15 00:00:00 Journal contribution https://acs.figshare.com/articles/journal_contribution/Phe28_sup_B10_sup_Induces_Channel-Forming_Cytotoxic_Amyloid_Fibrillation_in_Human_Neuroglobin_the_Brain-Specific_Hemoglobin/4269992 Since its discovery, neuroglobin (Ngb), a neuron-specific oxygen binding hemoglobin, distinct from the classical myoglobin and blood hemoglobin, has attracted attention as an endogenous neuroprotectant. Recent reports suggest that Ngb protects neurons from brain stroke, ischemic stress-induced degeneration, and other brain disorders. Proteins with a specific role in neuroprotection are often associated with neurodegeneration, as well, depending on the cellular environment or specific cellular triggers that tilt the balance one way or the other. This investigation explored the potential role of Ngb in amyloid fibril-related neuronal disorder. Ngb was capable of amyloid formation <i>in vitro</i> at neutral pH and ambient temperature, in both apo and holo forms, albeit at a slower rate in the holo form, unlike other hemoglobins that exhibit such behavior exclusively in the apo states. Elevated temperature enhanced the rate of fibril formation significantly. The B-helix, which is known to play a major role in Ngb ligand binding kinetics, was found to be amyloidogenic with the Phe28<sup>B10</sup> amino acid side chain as the key inducer of fibrillation. The Ngb amyloid fibril was also significantly cytotoxic to neuroblastoma cell lines, compared to those obtained from reference hemoglobins. The Ngb fibril probably promoted toxicity by inducing channel formation in the cell membrane, as investigated here using synthetic lipid bilayer membranes and the propidium iodide uptake assay. These findings imply that Ngb plays a role in neurodegenerative disorders <i>in vivo</i>, for which there seems to be indirect evidence by association. Ngb thus presents a novel prospect for understanding amyloid-related brain disorders beyond the limited set of proteins currently investigated for such diseases.