MOLECULAR BASIS OF PROPIONIC ACIDEMIA

丙酸血症的分子基础

• 批准号: 6271987
• 负责人: JAN P. KRAUS
• 金额: $ 18.78万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6271987
• 关键词: RNA splicing; X ray crystallography; active sites; chemical stability; complementary DNA; enzyme structure; gene expression; genotype; human genetic material tag; human subject; introns; ketotic hyperglycinemia; molecular cloning; molecular pathology; mutant; northern blottings; nucleic acid sequence; phenotype; polymerase chain reaction; propionyl coA carboxylase; protein structure function; single strand conformation polymorphism; southern blotting; western blottings

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.

丙酰-C 0A羧化酶（PCC）沉淀物寿命缺陷- 威胁人类的丙酸血症和智力迟钝。 这种酶由两个基因（PCCA和PCCB）编码，分别位于 染色体，并表现出复杂的互补模式，在杂合子 和复合杂合子。 PCCB中的12个独立突变已经被证实， 鉴定 其中四个发生在一个外显子中， 与转羧酶的12 S亚基具有相当大的同源性。 没有一 这些突变已经被表征为PCCA。 知之甚少 基因的调控。 此外，结合位点和 该酶的三级结构已被阐明。 这些 实验旨在提高我们对人类先天性PCC的理解 从生物化学和细胞水平到生物分子水平的错误。 这些研究旨在：1）确定预防和打击腐败局的组织结构， 和PCCB基因，定义它们的外显子/内含子边界，5 '-和3'-侧翼 2）在细菌中克隆和联合表达α和β cDNA 为了证实突变对酶组装的抑制作用， 催化活性; 3）制备足够量的重组 用于生物化学和生物物理研究的正常和突变酶; 使这些蛋白质结晶以用于随后的X射线分析; 4）确定 酶结构中假定的CoA结合基序;以及5）适应 目前的方法，如SSCP或双脱氧DNA指纹，以筛选 α和β PCC突变。 采用的具体技术将包括： 在细菌中克隆和表达酶的两个亚基;蛋白质 通过常规和亲和技术进行纯化; 免疫沉淀;从患者制备RNA和基因组DNA 细胞; Southern、北方和Western印迹;聚合酶链反应 扩增、克隆、双链和单链DNA分析、DNA 测序 这些研究将阐明个体突变的作用 这种先天性代谢缺陷的发病机制。 一级序列 α-和β-PCC是已知的;我们现在需要推进我们的理解 酶的二级和三级结构以及 使PCC失效的突变。 这些研究将建立 基因型/表型相关性导致改善的治疗原理， 最终包括基因治疗。