The essential role of cyclin-dependent kinase CRK9 in trypanosome pre-mRNA processing

细胞周期蛋白依赖性激酶 CRK9 在锥虫前 mRNA 加工中的重要作用

• 批准号: 10570982
• 负责人: ARTHUR GUNZL
• 金额: $ 41万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-02 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10570982
• 关键词: Ablation; Affect; African Trypanosomiasis; Alanine; Animals; Architecture; Binding; Blood Circulation; Cell Cycle; Cell Cycle Completion; Cell Death; Cell Line; Cells; Chagas Disease; Chemicals; Classification; Clinical; Clinical Trials; Code; Complex; Cultured Cells; Cutaneous; Cyclin-Dependent Kinases; Cyclins; Cytokinesis; Data; Defect; Developing Countries; Development; Disease; Docking; Drug Targeting; Drug resistance; Enzyme Inhibition; Enzymes; Future; Gene Expression; Gene Order; Genes; Genetic Transcription; Germ; Human; Immunoprecipitation; In Vitro; Individual; Infection; Intervention; Learning; Leishmania; Leishmaniasis; Libraries; Link; Messenger RNA; Mitosis; Multienzyme Complexes; Mus; Mutate; Mutation; Nuclear; Parasite resistance; Parasites; Persons; Pharmaceutical Preparations; Phase II/III Trial; Phosphorylation; Phosphotransferases; Poly A; Polyadenylation; Pre-mRNA Polyadenylation Factor; Process; Proteins; RNA; RNA Processing; RNA Splicing; Reaction; Recombinants; Research; Role; Signal Pathway; Site; Spliced Leader RNA; Spliced Leader Sequences; Spliceosome Assembly Pathway; Spliceosomes; Structure; System; Technology; Testing; Therapeutic; Therapeutic Intervention; Trans-Splicing; Translations; Trypanocidal Agents; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Trypanosomiasis; Visceral; Wheat; analog; deviant; genome-wide; high throughput screening; human disease; inhibitor; mRNA Precursor; mimetics; neglected tropical diseases; overexpression; protein complex; small molecule inhibitor; tool; transcriptome sequencing

Summary The kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. affect millions of people worldwide, causing Human African Trypanosomiasis (HAT), Chagas’ disease, and various forms of Leishmaniasis, respectively. Since drugs for these neglected tropical diseases are limited, often toxic and difficult to administer, and parasite resistance to existing drugs is on the rise, it is important to develop new chemo- therapeutic strategies. Our research is focused on trypanosome gene expression because the underlying mechanisms deviate substantially from those in the human host. For example, polycistronic transcription of protein coding genes and processing of pre-mRNA by spliced leader trans splicing are parasite-specific steps in mRNA synthesis and maturation. We discovered in T. brucei that the activity of the cyclin-dependent kinase (CDK) CRK9 is required for trans splicing. By generating a cell line that expresses analog-sensitive CRK9 and no wild-type enzyme, we could chemically inhibit the enzyme in specific manner in cultured cells. Surprisingly, we observed an instant splicing block after applying the inhibitor, suggesting that CRK9 carries out essential reversible phosphorylation on the RNA processing machinery. Our preliminary data indicate that one of CRK9’s substrate is the SR protein and known splicing factor TSR1, and that blocking CRK9 activity affects the assembly of the spliceosome, the large and dynamic RNA-protein complex that carries out the splicing reaction. Consequently, we will determine the mechanism of how CRK9 aids or controls the splicing process. Furthermore, CDKs represent a highly druggable enzyme class, and CRK9 forms an unusual trimeric enzyme complex with a deviant L-type cyclin and a kinetoplastid-specific protein, suggesting that CRK9 is a promising target for chemotherapeutic intervention. Therefore, we propose to characterize the enzyme complex and determine a minimal complex that is active and can be expressed recombinantly as prerequisite for future high throughput inhibitor screens. Finally, based on preliminary data, we will test the hypothesis that CRK9 is the target of the compound SCYX-7158 which is currently in clinical trials against HAT.

概括 动质体寄生虫布氏锥虫、克氏锥虫和利什曼原虫。影响数百万人 全世界范围内的人群，导致人类非洲锥虫病 (HAT)、恰加斯病和各种形式的锥虫病 分别是利什曼病。由于治疗这些被忽视的热带疾病的药物有限，而且往往有毒且困难 由于寄生虫对现有药物的耐药性正在上升，因此开发新的化疗药物非常重要 治疗策略。我们的研究重点是锥虫基因表达，因为潜在的 机制与人类宿主的机制有很大不同。例如，多顺反子转录 蛋白质编码基因和通过剪接前导反式剪接对前 mRNA 的加工是寄生虫特异性步骤 mRNA 合成和成熟。我们在 T. brucei 中发现细胞周期蛋白依赖性激酶的活性 (CDK) 反式拼接需要 CRK9。通过生成表达模拟敏感 CRK9 的细胞系和 由于没有野生型酶，我们可以在培养细胞中以特定方式化学抑制该酶。出奇， 我们观察到应用抑制剂后立即出现剪接阻断，这表明 CRK9 执行了必要的 RNA 加工机器上的可逆磷酸化。我们的初步数据表明 CRK9 之一 底物是 SR 蛋白和已知的剪接因子 TSR1，阻断 CRK9 活性会影响组装 剪接体是进行剪接反应的大型动态 RNA-蛋白质复合物。 因此，我们将确定CRK9如何辅助或控制剪接过程的机制。此外， CDK 代表一种高度可成药的酶类，CRK9 形成一种不寻常的三聚酶复合物，具有 异常的 L 型细胞周期蛋白和动质体特异性蛋白，表明 CRK9 是一个有前途的靶点 化疗干预。因此，我们建议表征酶复合物并确定 具有活性且可重组表达的最小复合物，作为未来高通量的先决条件 抑制剂筛选。最后，根据初步数据，我们将检验CRK9是该研究的目标的假设。 化合物 SCYX-7158 目前正在进行针对 HAT 的临床试验。