DELTA ALA SYNTHASE--ISOZYMES AND SIDEROBLASTIC ANEMIA

Delta Ala 合酶——同工酶和铁粒幼细胞贫血

基本信息

批准号：
2444019
负责人：
DAVID Franklin BISHOP
金额：
$ 25.29万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-01-01 至 2000-06-30
项目状态：
已结题

项目摘要

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.

拟议研究的总体目标是调查人类δ组织特异性同工酶的生物化学和生物学氨基乙酰丙酸合酶 (ALA-S)，第一种酶和限速酶血红素生物合成的步骤。这些关于看家同工酶的研究 (ALAS1) 和红细胞特异性同工酶 (ALAS2) 旨在提供基本信息并理解 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 同工酶在运输和掺入过程中线粒体，3）潜在的遗传和表型异质性先天性血液病、X连锁侧幼细胞性贫血（XLSA）、 4) ALAS1 产生的生化和细胞生物学表型缺陷和 5) 血红素对 ALAS1 的转录调控 HepG2 细胞。我们对全长人类 cDNA 进行分离和测序以及编码 ALAS1 和 ALAS2 的基因组序列，两者的表达原核和真核系统中的同工酶，并发现 ALAS2 突变导致 XLSA 提供独特且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、HePG2 和 K562 以确定酶切位点线粒体导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 (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 ALAS1 ALAS2将用于抗体生产和结晶和 X 射线衍射以确定其三维结构。定点化学修饰和定点诱变将用于鉴定和评估参与活性位点的残基两种同工酶，以确定 PLP 辅因子结合位点，表征与血红素结合有关的基序，以确定参与蛋白水解加工和降解的残留物并检测参与亚基关联的位点。非细胞模型红细胞血红素缺乏症将通过引入特定的小鼠胚胎干细胞和人 HepG2 细胞中的 ALAS1 突变通过同源重组。这种血红素合成缺陷的细胞可以为研究各种血红素提供有价值的模型系统依赖性系统，例如普遍的细胞色素缺陷和由此产生的生化表型可能表明类似的人类遗传失调。将努力确定分子事件介导血红素对 ALAS1 转录的抑制。一个敏感且将使用准确的 RT-PCR 方法来定量 ALAS1 mRNA 评估 HepG2 中血红素介导的 ALAS1 转录控制细胞。 ALAS1 基因的转录活性区域是通过 DNase I 作图、足迹和转录鉴定与这些因素相互作用以响应血红素的因素将是特点。

项目成果

期刊论文数量（16）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Familial-skewed X-chromosome inactivation as a predisposing factor for late-onset X-linked sideroblastic anemia in carrier females.

DOI：
10.1182/blood.v96.13.4363
发表时间：
2000-12
期刊：
Blood
影响因子：
20.3
作者：
M. Cazzola;A. May;G. Bergamaschi;P. Cerani;V. Rosti;D. Bishop
通讯作者：
M. Cazzola;A. May;G. Bergamaschi;P. Cerani;V. Rosti;D. Bishop

The molecular biology and pyridoxine responsiveness of X-linked sideroblastic anaemia.

DOI：
发表时间：
1998
期刊：
Haematologica
影响因子：
10.1
作者：
A. May;D. Bishop
通讯作者：
A. May;D. Bishop

Human ABC7 transporter: gene structure and mutation causing X-linked sideroblastic anemia with ataxia with disruption of cytosolic iron-sulfur protein maturation.

DOI：
10.1182/blood.v96.9.3256.h8003256_3256_3264
发表时间：
2000
期刊：
Blood
影响因子：
20.3
作者：
Soumeya Bekri;G. Kispál;Heike Lange;Edward Fitzsimons;John Tolmie;Roland Lill;David F. Bishop
通讯作者：
Soumeya Bekri;G. Kispál;Heike Lange;Edward Fitzsimons;John Tolmie;Roland Lill;David F. Bishop

Pyridoxine-refractory congenital sideroblastic anaemia with evidence for autosomal inheritance: exclusion of linkage to ALAS2 at Xp11.21 by polymorphism analysis.

吡哆醇难治性先天性铁粒幼细胞贫血，有常染色体遗传的证据：通过多态性分析排除 Xp11.21 与 ALAS2 的连锁。