Identification of neuroglobin-interacting proteins using yeast two-hybrid screening

Abstract

Neuroglobin (Ngb) is a globin protein that is highly and specifically expressed in brain neurons. A large volume of evidence has proven that Ngb is a neuroprotective molecule against hypoxic/ischemic brain injury and other related neurological disorder; however, the underlying mechanisms remain poorly understood. Aiming to provide more clues in understanding the molecular mechanisms of Ngb’s neuroprotection, we performed yeast two-hybrid screening to search for proteins that interact with Ngb. From a mouse brain cDNA library, we found totally 36 proteins that potentially interact with Ngb, and 10 of them were each identified in multiple positive clones. The shared sequences within these multiple clones are more likely to be Ngb-interacting domains. In primary cultured mouse cortical neurons, immuno-precipitation was performed to confirm the interactions of selected proteins with Ngb. The discovered Ngb-interacting proteins in this study include those involved in energy metabolism, mitochondria function and signaling pathways for cell survival and proliferation. Our findings provide molecular targets for investigating protein interaction-based biological functions and neuroprotective mechanisms of Ngb.

Mitochondrial distribution of neuroglobin and its response to oxygen–glucose deprivation in primary-cultured mouse cortical neurons

“…The detectable Ngb in mitochondria of cortical neurons under normal resting condition indicates a basal level of Ngb translocation into mitochondria because Ngb gene is encoded by nuclear genomic DNA, but not mitochondrial DNA (Burmester et al, 2000). Indeed, we have recently identified a group of Ngb-interacting proteins using yeast two-hybrid screening and confirmed their interaction by Co-IP (Yu et al, 2012), some of which are mitochondrial proteins such as VDAC, cytochrome c1 (Cyc1) - a subunit of mitochondria complex III (Zhu et al, 2011), and electron-transfer flavoprotein (Etfa). These findings provided additional evidence in support of the mitochondrial distribution of Ngb, although the detailed actions of Ngb in mitochondria warrant further investigations.…”

“…Our lab has demonstrated that Ngb over-expression preserved mitochondrial function in primary cortical neurons after hypoxia (Liu et al, 2009) and in an experimental model of glaucoma in mice (Wei et al, 2011). Our recent study using yeast two-hybrid screening further identified a group of Ngb-interacting proteins, some of which are mitochondrial proteins including cytochrome c1 (Cyc1), which is a subunit of mitochondria complex III; and VDAC, a component of mitochondria permeability transition pore (Yu et al, 2012). However, no direct evidence of Ngb’s physical interaction with mitochondria has been obtained.…”

Neuroglobin upregulation induced by 17β-estradiol sequesters cytocrome c in the mitochondria preventing H2O2-induced apoptosis of neuroblastoma cells

“…Similar NGB subcellular localization 28, 29 and the interaction of NGB with intra-mitochondrial proteins has been recently reported in other cell lines. 30, 31 …”

Neuroglobin, a Factor Playing for Nerve Cell Survival

“…Due to the high energy demand of the nervous tissue, it appears strongly correlated with mitochondrial dysfunction [12]. In this context, of particular interest is the evidence that NGB, an evolutionary highly conserved protein localized in nerve cells, is both physically and functionally related to mitochondrial functions [18,28,33]. In addition, it confers protection to nerve cells both in vitro and in vivo against a wide range of pathological conditions.…”

“…They appear to correspond to metabolically active, oxygen consuming cell populations [23,24], as exemplified by retinal cells [25]. NGB was mainly (~90%) localized in the cytosol [26], but accumulating evidence revealed that it is also associated with mitochondria, as demonstrated by immunohistochemistry [27], yeast two hybrid assays [28] and biochemical studies [29]. …”