This is a competitive renewal application for a Research Scientist Award (RSA). The overall objectives of this project are to further study the structure, function and the regulation of monoamine oxidase (MAO) A and B. MAO is an important enzyme in catecholamine metabolism. Abnormal levels of MAO activity have been shown in a number of mental disorders. These objectives will be accomplished using human MAO A, MAO B, cDNAs and human MAO A and B genomic clones isolated in this laboratory.
The specific aims of this five-year RSA application are described below: (I) To further investigate the structure and function of MAO A and B: The amino acids and domains responsible for MAO A and B substrate and inhibitor specificities will be identified by site-directed mutagenesis and chimeric enzymes constructed among MAO A, MAO B and trout MAO (trout MAO exhibits nonclassical inhibitor sensitivities). These mutants will be expressed in yeast. The kinetic parameters for substrates (Km) and inhibitors (Ki) will be determined. The regions responsible for mitochondrial targeting will be identified by using a series of C- terminal deletions and chimeric mutants. The kinetic parameter (Km, Ki) determination and Western blot will be performed on purified mitochondria to demonstrate if the MAO A or B protein is targeted to mitochondria. (II) To further investigate the mechanisms of regulation of MAO A and B gene expression: A new gene upstream of MAO A core promoter will be cloned and characterized. A novel repressor which down regulates MAO B promoter activity will be isolated by screening an expression library and by affinity column chromatography. Cell-specific elements (enhancers and silencers) and transcription factors for MAO A and B gene expression will be identified by transient expression assay, combined with mutations, deletions, gel retardation and DNaseI footprinting assays. The mechanisms of hormone stimulation of MAO A and B will be investigated. Further, the regulation of MAO A expression by the concentration and DNA binding efficiency of Sp1 in cells will be studied. Whether the increased MAO B catalytic activity in aged human brains and Alzheimer's patients is related to altered promoter sequences and/or the concentrations of transcription factors will be examined. These studies will provide most fundamental knowledge on the regulation of MAO A and B gene expression. The new knowledge may ultimately lead to a novel approach for designing specific MAO inhibitors which modulate MAO activity at the gene level, and may be used for patients with mental disorders.
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