MOLECULAR BASIS OF PROPIONIC ACIDEMIA

缺乏丙炔- c0a羧化酶（PCC）会导致寿命降低

Deficiencies in propionyl-C0A carboxylase (PCC) precipitate life- threatening propionic acidemia in humans together with mental retardation. This enzyme is encoded by two genes (PCCA and PCCB) on separate chromosomes, and exhibits complex complementation patterns in heterozygotes and compound heterozygotes. Twelve separate mutations in PCCB have been identified. Four of these occur within a single exon that exhibits considerable homology with the 12 S subunit of transcarboxylase. None of the mutations have yet been characterized for PCCA. Little is known about the regulation of either gene. Additionally, neither the binding sites nor the tertiary structure of the enzyme have been elucidated. These experiments are designed to advance our understanding of human inborn PCC errors from the biochemical and cellular level to athe molecular level. These studies are aimed at 1) determining the organization of both the PCCA and PCCB genes, defining their exon/intron boundaries, 5'- and 3'-flanking regions; 2) cloning and jointly expressing alpha and beta cDNAs in bacteria to confirm the inhibitory effects of mutation on enzyme assembly and catalytic activity; 3) preparing sufficient quantities of recombinant normal and mutant enzyme for biochemical and biophysical studies; to crystallize these proteins for subsequent X-ray analysis; 4) ascertaining the presumptive CoA binding motifs in the enzyme structure; and 5) adapting current methods such as SSCP or dideoxy DNA fingerprinting, to screen for alpha and beta PCC mutations. Specific techniques employed will include: cloning and expressing both subunits of the enzyme in bacteria; protein purification by both conventional and affinity techniques; immunoprecipitation; preparation of RNA and genomic DNA from patients cells; Southern, northern and Western blots; polymerase chain reaction amplification, cloning, double- and single-stranded DNA analysis, DNA sequencing. These studies will clarify the role of individual mutations int he pathogenesis of this inborn metabolic error. The primary sequences of alpha- and betaPCC are known; we now need to advance our understanding to the secondary and tertiary structures of the enzyme and the role of mutations in disabling PCC. These studies will establish the genotype/phenotype correlations leading to improved therapeutic rationales, ultimately including gene therapy.