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

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.