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

新型致病性 Htt 翻译后修饰 (PTM) 的验证

基本信息

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
项目类别：
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

项目摘要

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）：高清中最佳验证的治疗靶标仍然是HTT本身。先前鉴定出的扩展HTT的PTM（例如S13/16和S421）是HD发病机理的重要调节剂。我们先前研究了HTT的蛋白水解裂解（Ratovitski等，2007，2009，2011），最近正在研究HTT的共价PTM，尤其是磷酸化。 HTT除了当前已知的其他地点（在“意义”部分中描述）外，HTT很可能还有许多其他PTM站点。我们计划在系统和定量上对HTT PTM进行表征。此外，我们的实验包括使用人类HD IPS细胞进行我们的持续发现研究，以及从质谱开始的阶级程序，以发现和通过体外进行发现和进展，然后在体内确认和功能验证。如果可以鉴定和抑制相关的激酶，那么增强毒性的磷酸化将特别有望作为治疗靶点。在AIM 1中，我们将定义HTT PTMS USIN HTT-N586-82Q小鼠，HD“敲入”小鼠和人类HD IPS细胞，并确定HTT中的PolyQ扩展是否会导致PTMS的变化。在AIM 2中，我们将对HTT PTM对突变体HTT构象和细胞毒性的影响进行体外功能研究。在AIM 3中，我们将使用我们的N-586-82Q转基因小鼠模型或立体定向注射病毒表达向量编码HTT，用改变PTM的变化ptms tos ptms测试PTM对体内突变HTT毒性的影响。这些研究一起将识别PTM在突变体HTT中的新部位，并在功能上验证其在体外和体内发病机理中的作用。然后，这些站点将成为治疗开发的候选目标。