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Characterization of the TBCEL-dependent Tubulin Degradation Mechanism

TBCEL 依赖性微管蛋白降解机制的表征

基本信息

批准号：
10480738
负责人：
Bryan Seguinot
金额：
$ 4.03万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10480738
关键词：
Actins Address Affect Architecture Binding Biochemical Biological Assay Brain Bypass Cell Culture Techniques Cell physiology Cells Cellular biology Complex Couples Cytoskeleton Data Defect Degradation Pathway Dependence Development Erythrocytes Event Excision Fertility Gel Goals Histologic Homeostasis Homologous Gene Image In Vitro Infertility Interphase Kidney Laboratories Lead Leucine-Rich Repeat Ligase Liver Malignant Neoplasms Mammals Maps Mass Spectrum Analysis Mediating Microtubules Mitotic Mitotic spindle Modeling Molecular Chaperones Morphology Mus Mutation Nature Neurodegenerative Disorders Organelles Pathway interactions Pattern Play Polymers Process Protein Biochemistry Proteins Proteome Proteomics Research Personnel Rest Reticulocytes Role Specific qualifier value Spermatogenesis Structure System Testing Testis Tissues Tubulin Tubulin Interaction Ubiquitin Ubiquitin-Activating Enzymes Ubiquitination Work base clinically relevant design developmental disease dimer fly inhibitor interest male mouse model multicatalytic endopeptidase complex protein degradation reconstitution small molecule sperm cell translational scientist ubiquitin ligase unpublished works vesicle transport

项目摘要

Characterization of the TBCEL-dependent Tubulin Degradation Mechanism Tubulin, polymerized into microtubules, plays a central role in the spatial organization of the cell. In interphase, microtubules are critical for organelle organization and cellular vesicle transport, while in mitotic cells microtubules form the mitotic spindle. Tubulin dyshomeostasis has been implicated in cancer, developmental disorders, and neurodegenerative diseases. Although tubulin is regulated at multiple levels, no dedicated endogenous tubulin elimination mechanism has been identified. TBCEL is a protein similar to the tubulin assembly chaperone TBCE, but it lacks tubulin chaperone activity. Instead, TBCEL promotes proteasome- dependent tubulin degradation. Work from the sponsoring laboratory revealed that TBCEL is highly upregulated during late erythrocyte development and is critical for the specific elimination of tubulin during erythrocyte differentiation. I propose to characterize the TBCEL-dependent tubulin degradation mechanism. Proteasome- mediated protein degradation is usually ubiquitin-dependent. However, ubiquitination can be bypassed if a substrate-specific proteasome-targeting mechanism exists. Because TBCEL has a ubiquitin-like domain, a type of domain that often directly interacts with the proteasome, I hypothesize that TBCEL promotes tubulin degradation by directly interacting with the proteasome, thus bypassing ubiquitination. Thus, I will investigate how TBCEL couples to the UPS using pull-down mass spectrometry proteomics to identify TBCEL interactors, biochemical reconstitutions, and cell culture assays to test this “ubiquitin-bypass” model. If this model is not confirmed, a likely alternative would be that TBCEL promotes tubulin ubiquitination by delivering a ligase to tubulin. TBCEL also interacts with tubulin and destabilizes tubulin dimers, an activity that I have mapped to the leucine-rich repeat (LRR) domain of TBCEL. The TBCEL-tubulin binding interface and how TBCEL destabilizes tubulin is not known, but I hypothesize that tubulin destabilization is required for tubulin to be degraded. For this, I will characterize the LRR domain-tubulin interaction by protein biochemistry and through structural studies. Since tubulin is key for many cellular functions, it is important to study the effect of TBCEL’s activity on cells. In fact, a mutation of the fly TBCEL homolog leads to spermatogenesis defects and infertility. TBCEL’s function in mammalian testes (spermatogenesis) is not known, but it is expressed at high levels in this tissue. To characterize TBCEL’s function in this and other nonerythroid tissues, I will use a Tbcel-/- mouse model to perform histological analysis on tissues of interest. The completion of this project will provide substantial information about the TBCEL-dependent tubulin degradation pathway and the significance of this pathway for cell biology. Due to tubulin’s high clinical relevance, researchers are trying to design small-molecule degraders for tubulin. Thus, our work could inspire translational researchers to develop new ways to target tubulin for degradation.

TBCEL 依赖性微管蛋白降解机制的表征微管蛋白聚合成微管，在细胞的空间组织中起着核心作用。在间期，微管对于细胞器组织和细胞囊泡运输至关重要，而在有丝分裂细胞中微管形成有丝分裂纺锤体。微管蛋白稳态失调与癌症、发育疾病和神经退行性疾病。尽管微管蛋白受到多个层面的调控，但没有专门的内源性微管蛋白消除机制已被确定。 TBCEL是一种类似于微管蛋白的蛋白质组装伴侣TBCE，但缺乏微管蛋白伴侣活性。相反，TBCEL 促进蛋白酶体- 依赖性微管蛋白降解。赞助实验室的工作表明 TBCEL 高度上调在红细胞发育后期，对于红细胞发育过程中微管蛋白的特异性消除至关重要差异化。我建议描述 TBCEL 依赖性微管蛋白降解机制。蛋白酶体- 介导的蛋白质降解通常依赖于泛素。然而，如果存在底物特异性蛋白酶体靶向机制。因为 TBCEL 具有类似泛素的结构域，所以经常与蛋白酶体直接相互作用的结构域，我假设 TBCEL 促进微管蛋白通过直接与蛋白酶体相互作用而降解，从而绕过泛素化。因此，我将调查 TBCEL 如何使用下拉质谱蛋白质组学与 UPS 耦合来识别 TBCEL 相互作用子，生化重建和细胞培养测定来测试这种“泛素旁路”模型。如果这个型号不是经证实，一种可能的替代方案是 TBCEL 通过向微管蛋白传递连接酶来促进微管蛋白泛素化。微管蛋白。 TBCEL 还与微管蛋白相互作用并破坏微管蛋白二聚体的稳定性，我已将这一活动映射到 TBCEL 的富含亮氨酸重复 (LRR) 结构域。 TBCEL-微管蛋白结合界面以及 TBCEL 如何不稳定微管蛋白尚不清楚，但我假设微管蛋白不稳定是微管蛋白降解所必需的。为了这，我将通过蛋白质生物化学和结构研究来表征 LRR 结构域-微管蛋白相互作用。由于微管蛋白对于许多细胞功能至关重要，因此研究 TBCEL 活性对细胞的影响非常重要。在事实上，果蝇 TBCEL 同源物的突变会导致精子发生缺陷和不育。 TBCEL 的功能哺乳动物睾丸（精子发生）尚不清楚，但它在该组织中高水平表达。到为了表征 TBCEL 在该组织和其他非红细胞组织中的功能，我将使用 Tbcel-/- 小鼠模型来执行对感兴趣的组织进行组织学分析。该项目的完成将提供大量信息关于 TBCEL 依赖性微管蛋白降解途径以及该途径对细胞生物学的意义。由于微管蛋白的高度临床相关性，研究人员正在尝试设计微管蛋白的小分子降解剂。因此，我们的工作可以激励转化研究人员开发靶向微管蛋白降解的新方法。