MOLECULAR BASIS OF PROPIONIC ACIDEMIA

丙酰-C0A羧基酶(PCC)缺陷会导致生命- 威胁人类的丙酸血症和智力低下。 该酶由两个基因(PCCA和PCCB)分别编码。 染色体，并在杂合子中表现出复杂的互补模式 和复合杂合子。PCCB中有12个独立的突变 已确认身份。其中四个发生在单个外显子内 与转羧酶的12个S亚基有相当大的同源性。没有一个 PCCA的突变还没有确定。人们对此知之甚少 任何一种基因的调控。此外，无论是结合位点还是 该酶的三级结构已被阐明。这些 实验旨在促进我们对人类先天PCC的理解 从生化和细胞水平到分子水平的错误。 这些研究旨在1)确定PCCA和PCCA的组织 和PCCB基因，定义它们的外显子/内含子边界，5‘和3’侧翼 区域；2)克隆并在细菌中联合表达α和βcDNA 为了证实突变对酶组装的抑制作用和 催化活性；3)制备足量的重组 用于生化和生物物理研究的正常和突变酶； 将这些蛋白质结晶，以进行后续的X射线分析；4)确定 酶结构中假定的CoA结合基序；5)适应 目前的方法，如SSCP或双脱氧DNA指纹分析，以筛选 阿尔法和贝塔基因突变。采用的具体技术包括： 在细菌中克隆和表达该酶的两个亚基；蛋白质 常规技术和亲和技术相结合的纯化方法； 免疫沉淀法；患者RNA和基因组DNA的制备 细胞；Southern、Northern和Western印迹；聚合酶链式反应 扩增，克隆，双链和单链DNA分析 测序。这些研究将阐明个体突变的作用。 这一先天代谢错误的发病机制。初级序列 α-和β-PCC是已知的；我们现在需要推进我们的理解 对酶的二级和三级结构以及酶的作用 禁用PCC的突变。这些研究将确立 导致改善治疗原理的基因/表型相关性， 最终包括基因治疗。

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.