Validation of Novel Pathogenic Htt Post-Translational Modifications (PTMs)

新型致病性 Htt 翻译后修饰 (PTM) 的验证

• 批准号: 9008084
• 负责人: Christopher A Ross
• 金额: $ 62.65万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9008084
• 关键词: Affinity Chromatography; Amino Acids; Autopsy; Behavior; Biochemical; Biological Assay; Body Weight; Brain; Cataloging; Catalogs; Cell Culture Techniques; Cell model; Cells; Circular Dichroism; Collaborations; Corpus striatum structure; Disease; Event; Future; Gel; Health; Human; Huntington gene; Immunoprecipitation; In Vitro; Injection of therapeutic agent; Knock-in Mouse; Length; Mammalian Cell; Maps; Mass Spectrum Analysis; Measures; Modification; Monitor; Mus; Neurons; Nuclear; Pathogenesis; Peptide antibodies; Performance; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Physical condensation; Post-Translational Modification Site; Post-Translational Protein Processing; Prions; Protein Conformation; Proteins; Proteolysis; Reaction; Role; Site; Staging; Techniques; Testing; Time; Tissues; Toxic effect; Transgenic Mice; Validation; Video Microscopy; Viral; Viral Vector; Wild Type Mouse; Yang; analytical ultracentrifugation; base; expression vector; in vivo; induced pluripotent stem cell; mouse model; mutant; neuropathology; novel; polyglutamine; programs; promoter; research study; small molecule; stoichiometry; targeted treatment; therapeutic development; therapeutic target

DESCRIPTION (provided by applicant): The best validated therapeutic target in HD remains Htt itself. Previously identified PTMs of expanded Htt (e.g. S13/16 and S421) are important modulators of HD pathogenesis. We previously studied proteolytic cleavage of Htt (Ratovitski et al., 2007, 2009, 2011), and more recently have been studying covalent PTMs of Htt, especially phosphorylation. Htt is very likely to have many other sites of PTM besides the currently known ones (described in the Significance section). We plan to characterize Htt PTMs systematically and quantitatively. Furthermore, our experiments include the use of human HD iPS cells for our continuing discovery studies, and a staged program beginning with mass spectrometry for discovery and progressing through in vitro and then in vivo confirmation and functional validation. Phosphorylation which enhances toxicity will be especially promising as a therapeutic target, if relevant kinases can be identified and inhibited. In Aim 1, we will define Htt PTMs usin Htt-N586-82Q mice, HD "knock-in" mice and human HD iPS cells, and will determine whether the polyQ expansion in Htt leads to changes in PTMs. In Aim 2, we will conduct in vitro functional studies of the effects of Htt PTMs on mutant Htt conformation and cellular toxicity. In Aim 3, we will test the effects of PTMs on mutant Htt toxicity in vivo, using our N-586-82Q transgenic mouse model or stereotactic injection of viral expression vectors encoding Htt with altered PTMs into the striatum of wild-type mice. These studies taken together will identify novel sites of PTM in mutant Htt, and functionally validate their role in pathogenesis in vitro and in vivo. The sites will then be candidate targets for therapeutic development.

描述（由申请人提供）：HD 中经过验证的最佳治疗靶点仍然是 Htt 本身。先前鉴定的扩展 Htt 的 PTM（例如 S13/16 和 S421）是 HD 发病机制的重要调节剂。我们之前研究了 Htt 的蛋白水解裂解 (Ratovitski et al., 2007, 2009, 2011)，最近一直在研究 Htt 的共价 PTM，特别是磷酸化。除了目前已知的位点（在“意义”部分中描述）之外，Htt 很可能还有许多其他 PTM 位点。我们计划系统地、定量地表征 Htt PTM。此外，我们的实验包括使用人类 HD iPS 细胞进行持续的发现研究，以及一个分阶段的计划，从质谱分析开始进行发现，并通过体外、然后体内确认和功能验证进行进展。如果能够识别和抑制相关激酶，增强毒性的磷酸化将特别有希望作为治疗靶点。在目标 1 中，我们将使用 Htt-N586-82Q 小鼠、HD“敲入”小鼠和人类 HD iPS 细胞定义 Htt PTM，并将确定 Htt 中的 PolyQ 扩展是否会导致 PTM 的变化。在目标 2 中，我们将进行 Htt PTM 对突变 Htt 构象和细胞毒性影响的体外功能研究。在目标 3 中，我们将使用我们的 N-586-82Q 转基因小鼠模型或将编码具有改变 PTM 的 Htt 的病毒表达载体立体定向注射到野生型小鼠的纹状体中，测试 PTM 对突变 Htt 毒性的体内毒性作用。这些研究结合在一起将鉴定突变体 Htt 中 PTM 的新位点，并在功能上验证它们在体外和体内发病机制中的作用。这些位点将成为治疗开发的候选目标。