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)

基本信息

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
项目类别：
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

项目摘要

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) 家族性和散发性 ALS 中脊髓神经元细胞质中富含 TDP-43 的内含物是该疾病的两个近期酵母全基因组缺失的标志。功能毒性抑制剂筛选显示，TDP-43介导的毒性的最强抑制剂是编码金属磷酸酯酶（MPE）的基因的消除Dbr1 是唯一一种已知能水解内含子在剪接体从前 mRNA 切除过程中形成的 2',5'-磷酸二酯键的酶。Dbr1 活性的降低会导致 RNA 套索的积累，从而隔离致病性 TDP-43。，防止其干扰正常的 RNA 代谢，抑制酵母（一种人类神经元细胞系）和原代大鼠神经元中的脱支活性可以保护它们。所有真核物种中 Dbr1 蛋白催化结构域的高度序列同一性支持了以下观察结果：从酵母到人类，Dbr1 对 TDP-43 活性的影响是相似的。单独的RNA套索脱支酶和与含有与真核生物内含子RNA Dbr1中发现的分支点相同的真正分支点的合成RNA的复合物。溶组织内阿米巴 (Eh) 最容易结晶，揭示了 Dbr1 酶相对于其他 MPE 家族成员的几个意想不到的特征。所有 Dbr1 酶都是单核的，在所有先前表征的天冬氨酸位置上具有不变的活性位点半胱氨酸残基。 MPE 超家族成员，所有这些成员均具有双核酶活性。此外，所有 Dbr1 蛋白都含有高度保守的插入环，这在我们称为“MPE”的其他 MPE 中没有发现。 “套索识别环”（LRL），来自体内互补测定，使用以反式表达的多个 Dbr1 变体 ¿ dbr1 酵母支持所提出的单核酶机制，以及在晶体结构中观察到的各种独特结构元件的作用，这些结构还揭示了 Dbr1 如何区分 22,52-磷酸二酯键和更丰富的 32 的分子基础。 ,52-磷酸二酯键有了这些结果，我们现在拥有了测试 Dbr1 抑制代表一种新的治疗途径这一假设所需的工具。 TDP-43 介导的 ALS。1) 来自多个物种（包括人类）的大量纯化 Dbr1 蛋白；2) 经过测试的、强大的体外 RNA 脱支测定，适用于小分子文库的高通量筛选 (HTS)。 3) 与 HTS 抑制剂搜索并行，通过引入糖修饰和接头来合成分支 RNA 类似物的能力，以模拟我们观察到的与酶结合的分支 RNA 的构象晶体学上，这与溶液中这些物质的构象不同；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) 快速观察抑制剂原子细节的能力Dbr1 复合物能够合理设计具有更高亲和力和特异性的化合物；6）能够使用培养的人类神经元对候选 Dbr1 抑制剂化合物进行初步毒性筛选；7）能够完成人类 Dbr1 的结构。在开始在 TDP-43 介导的 ALS 毒性的细胞模型和小鼠模型中测试抑制剂之前，需要先完成本提案中概述的 Dbr1 酶工作。