Isolation and characterization of a novel NADPH-dependent flavin reductase in C. elegans and humans

NADPH-cytochrome P-450 reductase (CPR) is a flavin-containing enzyme, associated with the endoplasmic reticulum membrane, found in bacteria, plants, yeast, and animals. Its main function is the transfer of electrons from NADPH via FAD and FMN cofactors to cytochrome P-450 isoenzymes as well as to other heme-containing enzymes such as heme oxygenase and cytochrome b5. It also plays a critical role in the bioactivation and detoxification of one-electron acceptors such as the therapeutically important anticancer agents mitomicin c and tirapazepine. I identified a novel NADPH-dependent flavin reductase in C. elegans (FRE-1) with high similarity to cytochrome P450 reductase and the nitric oxide synthases (NOS). FRE-1 is transcribed from the same promoter as a two-gene operon with a novel protein, which we called HIT-1. It is a new member of the histidine triad (HIT) superfamily of proteins, which function as nucleotidyl transferases and hydrolases. This HIT family of proteins is highly conserved in nature, but the physiological role has not been identified for its members. I isolated a human ortholog of FRE-1 from a variety of tissues, including kidney, placenta and brain. This was the same protein as NR1, recently isolated from cancer cells. I also obtained a human sequence sharing significant similarity with C. elegans HIT-1, which has been named HHT1 (human histidine triad protein !). The presence of FRE-1 and HIT-1 in an operon in C. elegans suggests their common function in the cell. Using C. elegans and human embryonic kidney cells (HEK 7X) as models I show that the novel flavin reductase and a novel histidine triad protein are stress induced proteins and that they perform cooperative functions in stress conditions such as exposure to drugs. In this thesis I show that NR1 is induced by stress such as exposure to various concentrations of menadione and that it can reduce cytochrome c, a heme containing protein. Here, I use a model quinone menadione (MD) as an electron acceptor, which can undergo one-electron reduction by NR1. The results show that treatment of HEK expressing NR1 with menadione leads to increased cytotoxicity as compared to HEK cells expressing endogenous levels of NR1. I also show that HHT1 can significantly reduce the cytotoxicity induced by menadione in NR1 expressing and that HHT1 physically associates with and negatively regulates activity of NR1. This could represent a novel mechanism to regulate xenobiotic response, drug metabolism and activation of environmental chemicals.Medicine, Faculty ofGraduat

Human chromosome 7:DNA sequence and biology

DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate genes for developmental diseases including autism