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

• 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
• 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

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，在许多生物中是必需的辅因子或感应元件。 蛋白质。必须严格控制血红素的生产，以充分供应这些功能，但要避免 生产过剩，因为积累游离的血红素和血红素前体是有毒的。亚铁血红素的第一个承诺步骤 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)。这种反应 是由丙氨酸合成酶(ALAS)催化的，它使用5-磷酸吡哆醛(Plp)，维生素B6的活性形式。 作为一个重要的辅因。在动物中，有两种差异表达的ALAS亚型。ALAS1出现在 大多数细胞，而ALAS2是一种红系特有的酶，在红细胞中显著上调 发展。在人类中，ALAS2的突变会导致两种疾病：(1)X连锁铁粒母细胞性贫血(XLSA) 当酶活性太低，不能支持健康的血红素产生和红细胞生成水平时，以及(2) 红系X连锁原红细胞增多症(XLPP)，由过度产生ALA的功能获得ALAS2突变所致， 导致有毒的血红素生物合成中间体的积聚。ALAS的生命周期受到严格的分步调控 包括线粒体进口和蛋白质周转。这两个步骤都是由血红素结合控制的反馈。 酶的活性(和/或稳定性)也是受调节的，这些过程受其他生物相互作用的影响。 酶，包括Lon蛋白酶，琥珀酰辅酶A合成酶(SCS)，可能还有铁络合酶(FECH)， 最后两种也是血红素合成中的关键酶。重要的是，我们最近发现，唉，活动也是 受到线粒体ClpX(MtClpX)的戏剧性刺激，线粒体ClpX是AAA+蛋白质解折叠酶家族的成员。 MtClpX能量依赖解折叠酶加速PLP掺入ALAS和CLPX耗竭 会导致脊椎动物贫血。我们还解决了无PLP的ALAS(来自酵母)和活性ALAS的结构 PLP结合酶，它照亮与PLP掺入有关的构象变化，并提供 对于理解mtClpX促进PLP负载的重要信息。这些结构还提供了 首次观察到该酶的真核特异性调控C-末端结构域。此域结构 建议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。继续调查mtClpX如何与ALAS进行物理交互，并测试 PLP负载的机制有望揭示mtClpX-ALAS2相互作用和 XLSA等位基因的某些类别。在最近另一项令人兴奋的突破中，我们的合作者发现了一种 显性人类CLPX突变似乎高激活ALAS，导致mtClpX连锁红细胞生成 原卟啉(EPP)。本病的发病机制将从分子、结构和分子水平进行详细研究。 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，这项工作反过来将启发新的治疗策略 与监管失当导致的衰弱疾病作斗争。