MOLECULAR BASIS OF PROPIONIC ACIDEMIA

丙酰辅酶A羧化酶（PCC）的缺陷导致人类中危及生命的丙酸血症以及精神发育迟滞。PCC是由两个基因，PCCA和PCCB，在不同的染色体上编码，并表现出复杂的互补模式的复合杂合子。已经鉴定了PCCA和PCCB基因中的58个单独的突变。4个β突变发生在外显子12的保守区域内，并且在50个受影响等位基因中的21个中发生在相同的14个核苷酸内。到目前为止，只有两个betaPCC突变已在大肠杆菌中表达。大肠杆菌中表达，并进行了表征。我们对这两个基因的区域一无所知。此外，配体结合位点和酶的三级结构均未被鉴定。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。这些研究的目的是：1）确定PCCA和PCCB基因的结构，确定外显子/内含子边界和5 ′-和3 ′-侧翼区; 2）分离和表征我们PCC基因组克隆中的人PCCA和PCCB启动子;第三章在大肠杆菌和人细胞中表达含有人α和β PCC突变的cDNA构建体的cDNA构建体，以表征重组蛋白的线粒体输入、组装和催化性质的突变; 4）确定酶结构中的ATP和丙酰CoA结合基序; 5）包含以优化用于产生PCC晶体的技术的表达、纯化和结晶条件，所述PCC晶体适于通过X射线衍射解析酶的结构。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两个亚基;蛋白质纯化技术; RNA和基因组DNA的制备; Southern、北方和Western印迹; DNA测序和晶体学。该项目的主要目的是阐明酶的三级和四级结构以及突变在使PCC失效中的作用。这些研究将改善基因型/表型相关性，从而改善治疗原理。

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.