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complex protein degradation reconstitution small molecule sperm cell translational scientist ubiquitin ligase unpublished works vesicle transport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依赖性小管蛋白降解机制的表征聚合成微管中的微管蛋白在细胞的空间组织中起着核心作用。在相间，微管对于细胞器组织和细胞囊泡的转运至关重要，而在有丝分裂细胞中微管形成有丝分裂纺锤体。微管蛋白dyshomeostasis在癌症中隐含疾病和神经退行性疾病。尽管小管蛋白在多个层面上受到调节，但没有专用已经确定了内源性小管蛋白消除机制。 TBCEL是类似微管蛋白的蛋白质组装伴侣TBCE，但缺乏小管蛋白伴侣活性。相反，tbcel促进了蛋白酶体 - 依赖性小管蛋白降解。赞助实验室的工作表明，TBCEL已大大更新在红细胞晚期发育中，对于红细胞时的特异性消除微管蛋白至关重要分化。我建议表征依赖于TBCEL的小节蛋白降解机制。蛋白酶体 - 介导的蛋白质降解通常是泛素依赖性的。但是，如果一个存在底物特异性蛋白质组靶向机制。因为TBCEL具有类似泛素的域，一种类型我假设TBCEL促进微管蛋白通过直接与蛋白酶体相互作用来降解，从而绕过泛素化。那我会调查如何使用下拉的质谱蛋白质组学与UPS夫妇识别TBCEL Interactors，生化的重构和细胞培养测定法测试了这种“泛素 - 型”模型。如果此模型不是证实，可能是TBCEL通过将连接酶传递到微管蛋白。 TBCEL还与微管蛋白相互作用并破坏微管蛋白二聚体，这是我已映射到该二聚体 TBCEL的富含亮氨酸重复（LRR）结构域。 TBCEL-PUBULIN结合界面以及TBCEL如何破坏稳定微管蛋白尚不清楚，但我假设微管蛋白不稳定是降解微管蛋白。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。由于小管蛋白是许多细胞功能的关键，因此研究TBCEL活性对细胞的影响很重要。事实，苍蝇的突变导致精子发生缺陷和不育。 tbcel的功能哺乳动物测试（精子发生）尚不清楚，但在该组织中以高水平表达。到表征了TBCEL在此和其他非刺激性组织中的功能，我将使用tbcel - / - 鼠标模型对感兴趣的组织的组织学分析。该项目的完成将提供大量信息关于TBCEL依赖性小管蛋白降解途径以及该途径对细胞生物学的重要性。由于小管素的临床相关性很高，研究人员正在尝试为小动物设计小分子降解器。那就可以激发翻译研究人员开发靶向小管蛋白以降解的新方法。