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，是许多人中必不可少的辅因子或传感器元素蛋白质。血红素的生产必须得到严格控制以充分提供这些功能，但要避免生产过多，因为游离血红素和血红素前体的积累是有毒的。血红素中的第一个投入步骤生物合成是甘氨酸和琥珀酰辅酶A的凝结，可产生5-氨基乙酸（ALA）。这个反应用ALA合酶（Alas）催化，该合酶使用吡ido醇5ʹ-磷酸（PLP，维生素B6的活性形式）作为必不可少的辅助因子。在动物中，有两个不同表达的Alas同工型。 Alas1存在于大多数细胞，而Alas2是一种红细胞特异性酶，在红色细胞中大大更新发展。在人类中，ALAS2中的突变引起两种疾病：（1）X连接的Sideroblastic贫血（XLSA）当酶活性太低而无法支持血红素产生和红细胞生成的健康水平时，（2）红细胞X连锁原畸形（XLPP），来自功能获得的Alas2突变，过量产生Ala，引起有毒血红素生物合成中间体的积累。 Alas的生命周期在步骤中受到严格调节包括线粒体进口和蛋白质更新。这两个步骤都是通过血红素结合控制的反馈。酶活性（和/或稳定性）也受到调节，这些过程受到与其他相互作用的影响酶，包括LON蛋白酶，琥珀酰-COA合成酶（SCS），以及可能最后两个也是血红素合成中的关键酶。重要的是，我们最近发现a a也是通过线粒体CLPX（MTCLPX）动态刺激，这是AAA+蛋白展开酶家族的成员。 MTCLPX能量依赖性的进展将PLP的基础设施加速到ALA和CLPX部署引起脊椎动物的贫血。我们还解决了无PLP Alas（来自酵母）和活动的结构 PLP结合的酶，它阐明了会议变化与PLP行业相结合的酶了解PLP的MTCLPX促进负载的重要信息。这些结构还提供了对酶的真核特异性调节C末端结构域的首次观察。这个域结构提出可检验的机制来解释XLPP突变并包含SC的结合位点，我们将进一步研究。继续研究MTCLPX如何与Alas进行物理相互作用，并测试模型 PLP加载的机制有望发现MTCLPX-ALAS2相互作用与一些XLSA等位基因类。在最近的另一个激动人心的突破中，我们的合作者发现了一个显性人Clpx突变似乎过度激活了，导致MTCLPX连接的红细胞生成原化畸形（EPP）。该疾病的机械基础将在分子，结构和细胞水平。通过探测控制Alas酶的复杂机制，我们将阐明新的分子调节手段。我们认为，这项工作反过来会激发新颖的治疗策略打击因不良调控而导致的使人衰弱的疾病。