Phe28B10 Induces Channel-Forming Cytotoxic ... - ACS Publications

Nov 15, 2016 - Phe28B10 Induces Channel-Forming Cytotoxic Amyloid Fibrillation in Human Neuroglobin, the Brain-Specific Hemoglobin. Sheetal Uppal† ...
0 downloads 0 Views 3MB Size
Article pubs.acs.org/biochemistry

Phe28B10 Induces Channel-Forming Cytotoxic Amyloid Fibrillation in Human Neuroglobin, the Brain-Specific Hemoglobin Sheetal Uppal,† Amit Kumar Singh,† Richa Arya,† Debanjan Tewari,‡ Neha Jaiswal,† Abhijeet Kapoor,†,§ Amal Kanti Bera,‡ Alo Nag,† and Suman Kundu*,† †

Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, India Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India



S Supporting Information *

ABSTRACT: 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 in vitro 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 Phe28B10 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 in vivo, 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.

O

It is thus evident that the large number of investigations of Ngb emphasized only one aspect of its physiological relevance, neuroprotection. Considering the fact that a large number of neuroprotectants, as outlined below, also exhibit key roles in neurotoxicity or neurodegeneration through amyloid formation, it is also imperative that the significance of Ngb in such processes be investigated. An increased incidence in the amyloid-induced neurodegenerative diseases suggests that amyloid formation not only is associated with the usual suspects that have been widely studied, viz., α-synuclein, amyloid precursor protein (Aβ42), prion, and tau,13 but also might be caused by the formation of amyloid fibrils from novel proteins that are yet to be explored. It is well established that the abnormal aggregation of misfolded neuronal proteins plays a central role in the pathogenesis of neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD).14,15 The normal physiological roles of many of these neuronal proteins

ver the past two decades, rapid accumulation of sequenced genomes allowed the identification of new globins in a wide variety of organisms.1,2 It is now believed that hemoglobins (Hbs) are ubiquitous to life forms, suggesting their diverse and essential biological functions.3 Of special significance has been the discovery of a unique globin in human and other vertebrate brains, aptly named neuroglobin (Ngb).4 Ngb is a “neuronal” heme protein that shares little sequence similarity (