MOLECULAR BASIS OF PROPIONIC ACIDEMIA

丙酸血症的分子基础

• 批准号: 6581868
• 负责人: JAN P. KRAUS
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6581868
• 关键词: RNA splicing; X ray crystallography; active sites; chemical stability; enzyme deficiency; enzyme structure; gene expression; genetic mapping; human genetic material tag; introns; ketotic hyperglycinemia; molecular cloning; molecular pathology; mutant; northern blottings; nucleic acid sequence; propionyl coA carboxylase; protein purification; protein structure function; southern blotting; tissue /cell culture; western blottings

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.

丙酰辅酶A羧基酶(PCC)的缺乏会导致危及人类生命的丙酸血症和智力低下。PCC由位于不同染色体上的PCCA和PCCB两个基因编码，在复合杂合子中表现出复杂的互补模式。已确定PCCA和PCCB基因有58个不同的突变。在50个受影响的等位基因中，有21个在第12外显子的保守区和相同的14个核苷酸内发生了4个β突变。到目前为止，只有两个BetaPCC突变在大肠杆菌中表达并进行了鉴定。人们对这两种基因的区域一无所知。此外，该酶的配体结合部位和三级结构都还没有确定。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/内含子边界以及5‘和3’侧翼区；2)在我们的PCC基因组克隆中分离和鉴定人的PCCA和PCCB启动子；3)在大肠杆菌和人细胞中表达含有人α和βPCC突变的cDNA结构，以表征重组蛋白在线粒体输入、组装和催化特性上的突变；4)确定酶结构中的ATP和丙酰辅酶A结合基序；5)优化适合X-射线衍射法解析酶结构的PCC晶体的表达、纯化和结晶条件。采用的具体技术包括：在细菌和人类细胞中克隆和表达该酶的两个亚基；蛋白质纯化技术；RNA和基因组DNA的制备；Southern、Northern和Western blotting；DNA测序和结晶学。这个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使PCC失活中的作用。这些研究将改善基因型/表型的相关性，从而改善治疗原理。