Macromolecular interactions controlling the ALA synthases, keystone enzymes that initiate heme biosynthesis

控制 ALA 合成酶（启动血红素生物合成的关键酶）的大分子相互作用

基本信息

批准号：
9752583
负责人：
TANIA A BAKER
金额：
$ 41.94万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-15 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9752583
关键词：
ATP Hydrolysis Active Sites Adopted Affect Alleles Aminolevulinic Acid Anemia Animals Binding Binding Proteins Binding Sites Biochemical Biological Assay Biophysics C-terminal Cell Culture Techniques Cells ClpX protein Complex Coupled Defect Deuterium Development Disease Elements Enzymes Erythrocytes Erythroid Erythroid Cells Erythropoiesis Erythropoietic Protoporphyria Eukaryota Family Feedback Foundations Genes Glycine Heme Hemoglobin Hereditary Sideroblastic Anemia Human Hydrogen In Vitro Inherited Life Cycle Stages Ligand Binding Link MEL Gene Mass Spectrum Analysis Mediating Mitochondria Modeling Molecular Molecular Chaperones Molecular Conformation Molecular Structure Motor Mus Mutagenesis Mutation N-terminal Organism Oxygen Peptide Hydrolases Peptides Phenotype Physical condensation Porphyrias Process Production Protein Family Protein Isoforms Proteins Pyridoxal Pyridoxal Phosphate Reaction Regulation Role Signal Transduction Site-Directed Mutagenesis Structure Succinate-CoA Ligases Surface Testing Vertebrates Vitamin B6 Work Yeasts cofactor combat differential expression dimer endopeptidase La enzyme activity experimental study ferrochelatase gain of function heme a heme biosynthesis inorganic phosphate insight knock-down member monomer mutant novel novel therapeutic intervention novel therapeutics protein degradation protoporphyrin IX sensor small molecule succinyl-coenzyme A targeted treatment unfoldase

项目摘要

Heme is the oxygen-binding ligand of hemoglobin and is an essential cofactor or sensor element in many proteins. Heme production must be tightly controlled to adequately supply these functions but to avoid overproduction, as accumulation of free heme and heme precursors is toxic. The first committed step in heme biosynthesis is the condensation of glycine and succinyl-CoA to yield 5-aminolevulinic acid (ALA). This reaction is catalyzed by ALA synthase (ALAS), which uses pyridoxal 5ʹ-phosphate (PLP, the active form of vitamin B6) as an essential cofactor. In animals, there are two differentially expressed ALAS isoforms. ALAS1 is present in most cells, whereas ALAS2 is an erythroid-specific enzyme that is dramatically upregulated during red cell development. In humans, mutations in ALAS2 cause two diseases: (1) X-linked sideroblastic anemia (XLSA) when enzyme activity is too low to support healthy levels of heme production and erythropoiesis and (2) Erythroid X-linked protoporphyria (XLPP), from gain-of-function ALAS2 mutations that overproduce ALA, causing build up of toxic heme biosynthetic intermediates. The life cycle of ALAS is tightly regulated at steps including mitochondrial import and protein turnover. Both these steps are feedback controlled by heme-binding. Enzyme activity (and/or stability) is also regulated and these processes are affected by interaction with other enzymes, including Lon protease, succinyl-CoA synthetase (SCS), and perhaps ferrochelatase (FECH), the final two also critical enzymes in heme synthesis. Importantly, we recently discovered that ALAS activity is also dramatically stimulated by mitochondrial ClpX (mtClpX), a member of the AAA+ family of protein unfoldases. The mtClpX energy-dependent unfoldase accelerates incorporation of PLP into ALAS and CLPX depletion causes anemia in vertebrates. We also solved structures of both PLP-free ALAS (from yeast) and the active PLP-bound enzyme, which illuminates the conformational changes coupled to PLP incorporation and provides important information for understanding mtClpX-promoted loading of PLP. These structures also provide the first observation of the eukaryotic-specific regulatory C-terminal domain of the enzyme. This domain structure suggests testable mechanisms to explain the XLPP mutations and contains the binding site for SCS, which we will further study. Continuing to investigate how mtClpX physically interacts with ALAS and to test models for the mechanism of PLP-loading holds promise for uncovering a link between mtClpX-ALAS2 interactions and some classes of XLSA alleles. In another recent, exciting breakthrough, our collaborators discovered a dominant human CLPX mutation that appears to hyperactivate ALAS, leading to mtClpX-linked erythropoietic protoporphyria (EPP). The mechanistic basis of this disease will be scrutinized at the molecular, structural and cellular level. Thus, by probing the complex mechanisms that control ALAS enzymes we will elucidate new molecular means of regulation. We believe that this work, in turn, will inspire novel therapeutic strategies for combating the debilitating illnesses caused by misregulated ALAS.

血红素是血红蛋白的氧结合配体，是许多疾病中重要的辅助因子或传感器元件。必须严格控制血红素的产生，以充分提供这些功能，但要避免。生产过剩，因为游离血红素和血红素前体的积累是有毒的。生物合成是甘氨酸和琥珀酰辅酶A缩合生成5-氨基乙酰丙酸(ALA)。由 ALA 合酶 (ALAS) 催化，该酶使用 5ʹ-磷酸吡哆醛（PLP，维生素 B6 的活性形式）作为一种重要的辅助因子，ALAS1 存在于两种差异表达的亚型中。大多数细胞，而 ALAS2 是一种红细胞特异性酶，在红细胞在人类中，ALAS2 突变会导致两种疾病：(1) X 连锁铁粒幼细胞贫血 (XLSA)。当酶活性太低而无法支持血红素生成和红细胞生成的健康水平时以及 (2) 红系 X 连锁原卟啉症 (XLPP)，源自过度产生 ALA 的功能获得性 ALAS2 突变，导致有毒血红素生物合成中间体的积累 ALAS 的生命周期受到严格控制。包括线粒体输入和蛋白质周转，这两个步骤都是由血红素结合控制的反馈。酶活性（和/或稳定性）也受到调节，并且这些过程受到与其他物质相互作用的影响酶，包括 Lon 蛋白酶、琥珀酰辅酶 A 合成酶 (SCS)，或许还有亚铁螯合酶 (FECH) 最后两种也是血红素合成的关键酶，重要的是，我们最近发现 ALAS 活性也很重要。线粒体 ClpX (mtClpX)（蛋白质解折叠酶 AAA+ 家族的成员）可显着刺激。 mtClpX 能量依赖性解折叠酶加速 PLP 掺入 ALAS 和 CLPX 消耗我们还解析了不含 PLP 的 ALAS（来自酵母）和活性物质的结构。 PLP 结合酶，阐明了与 PLP 掺入相关的构象变化，并提供这些结构还提供了了解 mtClpX 促进的 PLP 加载的重要信息。首次观察到该酶的真核特异性调节 C 末端结构域。提出了可测试的机制来解释 XLPP 突变，并包含 SCS 的结合位点，我们将继续研究 mtClpX 如何与 ALAS 物理相互作用并测试模型。 PLP 加载机制有望揭示 mtClpX-ALAS2 相互作用与在最近的另一个令人兴奋的突破中，我们的合作者发现了一些 XLSA 等位基因。人类 CLPX 突变显性似乎过度激活 ALAS，导致 mtClpX 相关的红细胞生成原卟啉症（EPP）将从分子、结构和机制方面进行详细研究。因此，通过探索控制 ALAS 酶的复杂机制，我们将阐明新的机制。我们相信这项工作反过来将激发新的治疗策略。对抗因 ALAS 失调引起的衰弱性疾病。