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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：
10214597
负责人：
TANIA A BAKER
金额：
$ 41.73万
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 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10214597
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 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，并且是许多免疫反应中的重要辅因子或传感元件。 proteins.血红素生产必须严格控制，以充分提供这些功能，但要避免过量生产，因为游离血红素和血红素前体的积累是有毒的。血红素合成的第一步生物合成是甘氨酸和琥珀酰辅酶A的缩合以产生5-氨基乙酰丙酸（ALA）。该反应由ALA合成酶（ALAS）催化，该合成酶使用吡哆醛5 β-磷酸（PLP，维生素B6的活性形式）作为一个重要的辅助因子。在动物中，存在两种差异表达的ALAS同种型。ALAS 1存在于大多数细胞，而ALAS 2是一种红细胞特异性酶，发展在人类中，ALAS 2突变导致两种疾病：（1）X连锁铁粒幼细胞性贫血（XLSA）当酶活性太低而不能支持血红素产生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），来自过度产生ALA的功能获得性ALAS 2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反馈控制的。酶的活性（和/或稳定性）也受到调节，并且这些过程受到与其它酶的相互作用的影响。酶，包括Lon蛋白酶，琥珀酰辅酶A合成酶（SCS），可能还有亚铁螯合酶（FECH），最后两个也是血红素合成的关键酶。重要的是，我们最近发现，ALAS活动也是线粒体ClpX（mtClpX）是AAA+蛋白质解折叠酶家族的成员。 mtClpX能量依赖性解折叠酶加速PLP掺入ALAS和CLPX耗尽导致脊椎动物贫血我们还解析了无PLP的ALAS（来自酵母）和活性肽的结构。 PLP结合酶，其阐明了与PLP掺入偶联的构象变化，并提供了这是理解mtClpX促进的PLP装载的重要信息。这些结构还提供了第一次观察到的真核生物特异性调控C-末端结构域的酶。这种域结构提出了可测试的机制来解释XLPP突变，并包含SCS的结合位点，我们将进一步研究。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-ALAS 2相互作用之间的联系，一些XLSA等位基因的类别。在最近另一个令人兴奋的突破中，我们的合作者发现了一种显性人类CLPX突变似乎过度激活ALAS，导致mtClpX连锁的红细胞生成原卟啉症（EPP）。这种疾病的机制基础将在分子，结构和细胞水平。因此，通过探索控制ALAS酶的复杂机制，我们将阐明新的分子调控手段。我们相信，这项工作，反过来，将激发新的治疗策略，对抗由ALAS失调引起的衰弱性疾病。