DELTA ALA SYNTHASE--ISOZYMES AND SIDEROBLASTIC ANEMIMIA

拟议研究的总体目标是调查 人类δ-半乳糖苷酶的组织特异性同工酶的生物化学和生物学研究 氨基乙酰丙酸合成酶（ALA-S），第一种酶和限速酶 血红素生物合成步骤。 管家同工酶的这些研究 红系特异性同工酶（ALAS 1）和红系特异性同工酶（ALAS 2）被设计成 提供基本信息和理解1） 组织特异性人ALA-S的物理动力学特征 同工酶，2）正常加工和成熟的性质， ALA-s同工酶在它们的运输和掺入到 线粒体，3）遗传和表型异质性的基础， 先天性血液病，X连锁侧胚细胞性贫血（XLSA）， 4)ALAS 1导致的生物化学和细胞生物学表型 缺乏和5）ALAS 1的转录调控血红素在 hepg 2细胞 我们对全长人cDNA的分离和测序 以及编码ALAS 1和ALAS 2的基因组序列， 原核和真核系统中的同工酶， ALAS 2突变导致XLSA提供了独特和必要的资源， 来实现这些目标。 细胞加工和线粒体 将在多种细胞中研究这些同工酶的靶向作用 包括COS-1 CHO、HePG 2和K562，以确定 线粒体输入前导序列和任何进一步的性质 线粒体蛋白水解加工。 成熟的线粒体形式 两种同工酶的特征包括最适pH、K、K吡哆醛 5 '-磷酸盐（PLP）结合、pI、稳定性、分子量和形状， 以及金属、离子和核苷酸的影响。 纯化的重组ALAS 1 和ALAS 2将用于抗体生产和结晶 和X射线衍射来确定它们的三维结构。 定点化学修饰和定点诱变将 用于识别和评估活性位点中涉及的残基 两种同工酶，以确定PLP辅因子结合位点， 表征涉及血红素结合的基序，以确定 参与蛋白水解加工和降解的残基，并检测 参与亚基缔合的位点。 一个非- 红系血红素缺乏症将通过引入特定的 ALAS 1突变到小鼠胚胎干细胞和人HepG 2细胞中 通过同源重组。 这种血红素合成缺陷的细胞可以 为研究各种血红素提供了有价值的模型系统， 依赖系统，如广义细胞色素缺乏症和 由此产生的生化表型可能表明类似的人类遗传 紊乱 将努力确定分子事件 介导血红素对ALAS 1转录的抑制。 一个敏感而 将使用准确的RT-PCR方法定量ALAS 1 mRNA 评估血红素介导的ALAS 1在HepG 2中的转录控制 细胞 ALAS 1基因的转录活性区将被 通过DNA酶I作图和足迹法以及转录鉴定 与这些因子相互作用以响应血红素的因子将是 表征了

The overall objective of the proposed research is to investigate the biochemistry and biology of the tissue-specific isozymes of human delta- aminolevulinate synthase (ALA-S), the first enzyme and the rate-limiting step of heme biosynthesis. These studies of the housekeeping isozyme (ALAS1) and the erythroid-specific isozyme (ALAS2) are designed to provide fundamental information and understanding of 1) the physicokinetic characteristics of the tissue-specific human ALA-S isozymes, 2) the nature of the normal processing and maturation of the ALA-s isozymes during their transport and incorporation into mitochondria, 3) the genetic and phenotypic heterogeneity underlying the congenital hematological disorder, X-linked sideorblastic anemia (XLSA), 4) the biochemical and cell biologic phenotype resulting from ALAS1 deficiency and 5) the transcriptional regulation of ALAS1 by heme in HepG2 cells. Our isolation and sequencing of the full-length human cDNAs and genomic sequences encoding ALAS1 and ALAS2, expression of both isozymes in prokaryotic and eukaryotic systems, and discovery that mutations in ALAS2 cause XLSA provide the unique and necessary resources to accomplish these goals. The cellular processing and mitochondrial targeting of these isozymes will be studied in a variety of cells including COS-1. CHO, HePG2, and K562 to determine the cleavage site of the mitochondrial import leader sequence and the nature of any further mitochondrial proteolytic processing. The mature mitochondrial forms of both isozymes will be characterized including pH optima, K, K pyridoxal 5'-phosphate (PLP) binding, pI, stability, molecular weight and shape, and effects of metals, ions and nucleotide. Purified recombinant ALAS1 and ALAS2 will be used for antibody production and for crystallization and X-ray diffraction to determine their three-dimensional structures. Site-directed chemical modification and site-directed mutagenesis will be used to identify and evaluate residues involved in the active site of both isozymes, to identify the PLP co-factor binding site, to characterize the motifs implicated in heme binding, to determine the residues involved in proteolytic processing and degradation and to detect sites involved in subunit association. A cellular model for non- erythroid heme deficiency will be produced by introduction of specific ALAS1 mutations into murine embryonic stem cells and human HepG2 cells by homologous recombination. Such heme synthesis-deficient cells could provide valuable model systems for investigations of various heme- dependent systems such as generalized cytochrome deficiencies and the resulting biochemical phenotypes may suggest analogous human genetic disorders. Efforts will be directed to determine the molecular events mediating the repression of ALAS1 transcription by heme. A sensitive and accurate RT-PCR method for the quantitation of ALAS1 mRNA will be used to evaluate the heme-mediated control of ALAS1 transcription in HepG2 cells. The transcriptionally active regions of the ALAS1 gene will be identified by DNase I mapping and footprinting and the transcription factor(s) interacting with these in response to heme will be characterized.