mitoNEET is a novel target of type II diabetes drug pioglitazone, and localizes on mitochondrial outer membrane via the N-terminal transmembrane a-helix. Genetics studies have shown that mitoNEET has a crucial regulatory role for energy metabolism in mitochondria, and has been associated with not only type II diabetes, but also human breast cancer proliferation and neurodegenerative diseases. The soluble C-terminal mitoNEET contains a [2Fe-2S] cluster with an unusual ligand arrangement of three cysteine and one histidine residues. Nevertheless, specific function of mitoNEET remains largely elusive. Preliminary studies indicated that the mitoNEET [2Fe-2S] clusters are fully reduced when expressed in Escherichia coli cells, and that the reduced mitoNEET [2Fe-2S] clusters are highly sensitive to nitric oxide, a physiological free radical that has been associated with a number of human diseases including diabetes. Upon exposure to nitric oxide, the mitoNEET [2Fe-2S] clusters are readily modified forming the mitoNEET-bound dinitrosyl iron complex (DNIC). The goal of this application is to test a hypothesis that the mitoNEET [2Fe-2S] cluster may act as a novel sensor of nitric oxide to modulate energy metabolism in mitochondria. There are two specific aims:
Aim 1 is to determine the specific reactivity of the mitoNEET [2Fe-2S] clusters with nitric oxide. The proposed research will determine the kinetics of the reaction between the reduced mitoNEET [2Fe-2S] clusters and nitric oxide and investigate modification of the mitoNEET [2Fe-2S] clusters in cultured cells by nitric oxide. Potential regulation of the nitric oxide-mediated modification of the mitoNEET [2Fe-2S] clusters by the type II diabetes drug pioglitazone will also be explored.
Aim 2 is to investigate the repair mechanism for the nitric oxide-modified [2Fe-2S] clusters in mitoNEET. To repair the nitric oxide-modified [2Fe-2S] clusters in mitoNEET, the modified lesion dinitrosyl iron complex (DNIC) in the protein must be removed before a new [2Fe-2S] cluster may be assembled. The proposed experiments will focus on how DNIC in mitoNEET may be removed by biological thiols via thiol ligand exchange. Biochemical analyses will be combined with the in vivo siRNA approaches to investigate the specific activity and physiological relevance of biological thiols in mediating removal of DNIC from mitoNEET. Success of the proposed research is expected to illustrate a potentially novel signal pathway of NO in mitochondria via the mitoNEET [2Fe-2S] clusters, to establish a link between NO signal and type II diabetes via the modification of the mitoNEET [2Fe-2S] clusters, and to provide information for developing new therapeutic treatments for type II diabetes. The results will also lay groundwork to unravel the potential regulatory roles of two mitoNEET-related mitochondrial proteins, the Wolfram Syndrome 2-related Miner1 and the function unknown protein Miner2, in human cells.
Human mitochondrial outer membrane protein mitoNEET is a new target of type II diabetes drug pioglitazone, and contains an unusual [2Fe-2S] cluster. The focus of this application is to test a hypothesis that the mitoNEET [2Fe-2S] cluster may act as a sensor of nitric oxide to regulate energy metabolism in mitochondria. The proposed research is expected to reveal a potentially novel link between nitric oxide and type II diabetics and to provide fundamental information for developing new therapeutic approaches for type II diabetes.