MOLECULAR BASIS OF PROPIONIC ACIDEMIA

丙酰辅酶a羧化酶（PCC）缺乏可导致危及生命的丙酸血症和智力迟钝。PCC由位于不同染色体上的两个基因PCCA和PCCB编码，在复合杂合子中表现出复杂的互补模式。PCCA和PCCB基因已鉴定出58个不同的突变。其中四个突变发生在12号外显子的一个保守区域内，50个受影响等位基因中有21个发生在相同的14个核苷酸内。迄今为止，只有两种betaPCC突变在大肠杆菌中表达并被鉴定。对这两个基因所在的区域都一无所知。此外，既没有确定配体结合位点，也没有确定酶的三级结构。这些实验旨在提高我们对PCC先天突变的生物化学、遗传学和分子细胞生物学的理解。这些研究旨在1)确定PCCA和PCCB基因的组织，确定外显子/内含子边界和5'-和3'-侧翼区域；2)在我们的PCC基因组克隆中分离和鉴定人类PCCA和pcccb启动子；3)在大肠杆菌和人细胞中表达含有人α和β PCC突变的cDNA构建体，表征重组蛋白在线粒体导入、组装和催化性能方面的突变；4)确定ATP和丙酰辅酶a在酶结构中的结合基序；5)含有优化表达、纯化和结晶条件的PCC晶体的生成技术适用于x射线衍射解酶结构。所采用的具体技术将包括：在细菌和人类细胞中克隆和表达酶的亚基；蛋白质纯化技术；RNA和基因组DNA的制备；南部、北部和西部斑点；DNA测序和晶体学。该项目的主要目的是阐明该酶的三级和四级结构以及突变在PCC致残中的作用。这些研究将改善基因型/表型相关性，从而改善治疗依据。

Deficiencies in propionyl-CoA carboxylase (PCC) precipitate life- threatening propionic acidemia in humans together with mental retardation. PCC is encoded by two genes, PCCA and PCCB, on separate chromosomes, and exhibits complex complementation patterns in compound heterozygotes. 58 separate mutations in PCCA and PCCB genes have been identified. Four of the beta mutations occur within a conserved region in exon 12 and within the same 14 nucleotides in 21 out of 50 affected alleles. To date only two betaPCC mutations have been expressed in E. coli and characterized. Nothing is known about the region of either gene. Additionally, neither the ligand binding sites nor the tertiary structure of the enzyme have been identified. These experiments are designed to improve our understanding of the biochemistry, genetics, and molecular cell biology of inborn mutations of PCC. These studies are aimed at 1) determining the organization of both the PCCA and PCCB genes, defining the exon/intron boundaries and 5'- and 3'- flanking regions; 2) isolating and characterizing the human PCCA and PCCB promoters in our PCC genomic clones; 3) expressing cDNA constructs containing cDNA constructs containing the human alpha and beta PCC mutations in Escherichia coli and in human cells to characterize the mutations on mitochondrial import, assembly, and catalytic properties of the recombinant protein; 4) ascertaining the ATP and propionyl CoA binding motifs in the enzyme structure; 5) containing to optimize the expression, purification, and the crystallization conditions for techniques for generating PCC crystals suitable for solving the structure of the enzyme by X-ray diffraction. Specific techniques employed will include: cloning and expressing both subunits of the enzyme in bacterial and human cells; protein purification techniques; preparation of RNA and genomic DNA; Southern, Northern and Western blots; DNA sequencing, and crystallography. The major aim of this project is to elucidate the tertiary and quaternary structure of the enzyme and the role of mutations in disabling PCC. These studies will improve the genotype/phenotype correlations leading to improved therapeutic rationales.