The Intron Debranching Enzyme (Dbr1) in Amyotrophic Lateral Sclerosis

肌萎缩侧索硬化症中的内含子脱支酶 (Dbr1)

• 批准号: 8733978
• 负责人: Peter JOHN HART
• 金额: --
• 依托单位: SOUTH TEXAS VETERANS HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-10-01 至 2018-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8733978
• 关键词: Ablation; Active Sites; Affect; Affinity; Amyotrophic Lateral Sclerosis; Aspartic Acid; Avidity; Binding; Biological Assay; Brain Stem; C-terminal; Catalytic Domain; Cell Line; Cells; Complex; Crystallization; Cysteine; Cytoplasm; Data; Databases; Disease; Disease Progression; Effectiveness; Elements; Entamoeba histolytica; Enzymes; Excision; Family member; Generations; Genes; Genetic; Gulf War; Hand; Health; Hour; Human; In Vitro; Introns; Kinetics; Length; Mediating; Metabolism; Modeling; Modification; Molecular; Molecular Conformation; Mononuclear; Motor Neurons; Mus; Mutation; Nature; Neuroblastoma; Neurodegenerative Disorders; Neurons; Organism; Paper; Patients; Persian Gulf; Pharmaceutical Chemistry; Pharmaceutical Preparations; Positioning Attribute; Progressive Disease; Property; Proteins; Publishing; RNA; RNA-Binding Proteins; Rattus; Relative (related person); Reporting; Resolution; Rilutek; Risk; Role; Signal Transduction; Solutions; Specificity; Spinal Cord; Spliceosomes; Structure; Testing; Toxic effect; Toxicity Tests; Variant; Veterans; Work; Yeasts; analog; arm; base; design; effective therapy; electron density; fluorophore; high throughput screening; in vivo; inhibitor/antagonist; lariat debranching enzyme; loss of function; mRNA Precursor; man; member; neuroblastoma cell; novel therapeutics; phosphodiester; prevent; protein TDP-43; research study; small molecule; small molecule libraries; sugar; synchrotron radiation; therapeutic development; tool; yeast genome

DESCRIPTION (provided by applicant): Mutations in the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS). Inclusions enriched in TDP-43 in the cytoplasm of spinal cord neurons in both familial and sporadic ALS are hallmarks of the disease. Two recent yeast genome-wide loss-of-function toxicity suppressor screens revealed the strongest suppressor of TDP-43-mediated toxicity is the ablation of the gene encoding the metallophosphoesterase (MPE) Dbr1, the only enzyme known to hydrolyze the 2',5'-phosphodiester bonds formed within introns during their excision from pre-mRNA by the spliceosome. Decreasing Dbr1 activity results in the accumulation of RNA lariats that are proposed to sequester pathogenic TDP-43, preventing it from interfering with normal RNA metabolism. Supporting this hypothesis, knockdown of debranching activity in yeast, a human neuronal cell line, and in primary rat neurons protects them from TDP-43-mediated toxicity. The high degree of sequence identity in the catalytic domains of Dbr1 proteins across all eukaryotic species supports the observation that Dbr1's influence on TDP-43 activity is similar from yeast to man. We recently determined the first crystal structures of an RNA lariat debranching enzyme alone and in complex with a synthetic RNA containing a bona fide branchpoint identical to those found in intron RNA lariats. Dbr1 from the eukaryotic organism Entamoeba histolytica (Eh) crystallized most readily, revealing several unexpected features of Dbr1 enzymes relative to other MPE family members. All Dbr1 enzymes are mononuclear, possessing an invariant active site cysteine residue in the position of the aspartic acid observed in all previously characterized MPE superfamily members, all of which are active as dinuclear enzymes. In addition, all Dbr1 proteins contain a highly conserved insertion loop not found in other MPEs we term the "lariat recognition loop" (LRL). Functional data coming from in vivo complementation assays using multiple Dbr1 variants expressed in trans in ¿dbr1 yeast support the proposed mononuclear enzymatic mechanism, as well as the roles assigned to the various unique structural elements observed in the crystal structures. The structures also reveal the molecular basis for how Dbr1 distinguishes 22,52-phosphodiester linkages from the far more abundant 32,52-phosphodiester linkages. With these results in-hand, we are now in possession of the tools needed to test the hypothesis that inhibition of Dbr1 represents a novel therapeutic avenue to treat TDP-43-mediated ALS. These tools include: 1) large quantities of purified Dbr1 proteins from multiple species, including human; 2) a tested, robust in vitro RNA debranching assay amenable to high throughput screening (HTS) of small molecule libraries for inhibitors; 3) the ability to synthesize, in parallel with the HTS inhibitor search, branched RNA analogs through the introduction of sugar modifications and linkers in order to mimic the conformation of the branched RNA we observe bound to the enzyme crystallographically, which differs from the conformations of these species in solution; 4) an in vivo complementation assay to test the effectiveness of potential inhibitors coming from both of the pipelines mentioned above; 5) the ability to rapidly observe the atomic details of inhibitor¿Dbr1 complexes to enable the rational design of compounds with greater affinity and specificity; 6) the ability to perform initial toxiciy screens of candidate Dbr1 inhibitor compounds using cultured human neurons; and 7) the ability to pursue the structure of human Dbr1. The completion of the work outlined in this proposal on the Dbr1 enzyme is required before testing inhibitors in cell-based and murine models of TDP-43 mediated toxicity in ALS can begin.

描述（由申请人提供）： RNA结合蛋白TDP-43的突变导致肌萎缩侧索硬化症（ALS）。在家族性和散发性ALS中，脊髓神经元细胞质中富含TDP-43的包涵体是该疾病的标志。两个最近的酵母全基因组功能丧失毒性抑制剂筛选揭示了TDP-43介导的毒性的最强抑制剂是编码金属磷酸酯酶（MPE）Dbr 1的基因的消融，金属磷酸酯酶Dbr 1是已知水解内含子内形成的2 '，5'-磷酸二酯键的唯一酶，该内含子在通过剪接体从前mRNA切除期间形成。Dbr 1活性降低导致RNA变异体的积累，这些变异体被提议隔离致病性TDP-43，防止其干扰正常的RNA代谢。支持这一假设，敲低酵母，人类神经元细胞系，和原代大鼠神经元的脱支活性保护他们从TDP-43介导的毒性。在所有真核生物物种的Dbr 1蛋白的催化结构域的高度序列同一性支持观察Dbr 1的TDP-43活性的影响是类似的从酵母man. We最近确定的第一个晶体结构的RNA lactoblastrinedebranchingenzyme单独和复杂的合成RNA含有一个真正的branchpoint相同的内含子RNA lariats中发现的。来自真核生物溶组织内阿米巴（Eh）的Dbr 1最容易结晶，揭示了Dbr 1酶相对于其他MPE家族成员的几个意想不到的特征。所有Dbr 1酶是单核的，在所有先前表征的MPE超家族成员中观察到的天冬氨酸的位置中具有不变的活性位点半胱氨酸残基，所有这些酶都具有双核酶的活性。此外，所有的Dbr 1蛋白都含有一个在其他MPE中没有发现的高度保守的插入环，我们称之为“LRL识别环”（LRL）。来自体内互补试验的功能数据，使用多个Dbr 1变体在反式表达的dbr 1酵母支持提出的单核酶机制，以及分配给晶体结构中观察到的各种独特的结构元素的作用。这些结构还揭示了Dbr 1如何区分22，52-磷酸二酯键与更丰富的32，52-磷酸二酯键的分子基础。有了这些结果，我们现在拥有了测试这一假设所需的工具，即抑制Dbr 1代表了治疗TDP-43介导的ALS的一种新的治疗途径。这些工具包括：1）来自多个物种（包括人）的大量纯化的Dbr 1蛋白; 2）经过测试的、稳健的体外RNA去分支测定，其适合于抑制剂的小分子文库的高通量筛选（HTS）; 3）合成的能力，与HTS抑制剂搜索并行，通过引入糖修饰和接头来模拟我们观察到的与酶结合的分支RNA的构象，晶体学上，其不同于这些物质在溶液中的构象; 4）体内互补测定，以测试来自上述两个管道的潜在抑制剂的有效性; 5）快速观察抑制剂<$Dbr 1复合物的原子细节的能力，以使得能够合理设计具有更大亲和力和特异性的化合物; 6）使用培养的人神经元进行候选Dbr 1抑制剂化合物的初始毒性筛选的能力;和7）追踪人Dbr 1结构的能力。在开始在基于细胞的和小鼠模型中测试TDP-43介导的ALS毒性抑制剂之前，需要完成本提案中概述的Dbr 1酶工作。