Characterization of the TBCEL-dependent Tubulin Degradation Mechanism

TBCEL依赖的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， 在有丝分裂细胞中，微管是细胞器组织和细胞泡运输的关键。 微管形成有丝分裂纺锤体。微管蛋白代谢紊乱与癌症、发育期 精神障碍和神经退行性疾病。尽管微管蛋白在多个层面上受到调控，但没有专门的 内源性微管蛋白的清除机制已经确定。TBCEL是一种类似于微管蛋白的蛋白质 装配伴侣TbCE，但缺乏微管蛋白伴侣活性。相反，TBCEL促进蛋白酶体- 依赖微管蛋白的降解。赞助实验室的工作表明，TBCEL的表达高度上调 在红细胞发育后期，对微管蛋白在红细胞中的特异性消除至关重要 差异化。我建议对依赖于TBCEL的微管蛋白降解机制进行表征。蛋白酶体- 介导的蛋白质降解通常是泛素依赖的。然而，泛素化可以绕过，如果 底物特异的蛋白酶体靶向机制存在。因为TBCEL有一个类似泛素的结构域，一个类型 在经常直接与蛋白酶体相互作用的结构域中，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如何与UPS偶联使用下拉质量谱蛋白质组学来识别TBCEL相互作用子， 生化重组和细胞培养分析来测试这种“泛素旁路”模式。如果此型号不是 证实，一种可能的替代方案是，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，破坏微管蛋白二聚体的稳定性，我已经将这一活动映射到 TBCEL的富亮氨酸重复序列(LRR)结构域。TBCEL-微管蛋白结合界面及TBCEL如何失稳 微管蛋白尚不清楚，但我推测微管蛋白失稳是微管蛋白降解所必需的。为了这个， 我将通过蛋白质生物化学和结构研究来表征LRR结构域与微管蛋白的相互作用。 由于微管蛋白是许多细胞功能的关键，因此研究TBCEL的活性对细胞的影响很重要。在……里面 事实上，苍蝇TBCEL同源物的突变会导致精子发生缺陷和不育。TBCEL在中的功能 哺乳动物的睾丸(精子发生)尚不清楚，但它在这个组织中表达水平很高。至 描述TBCEL在这个和其他非红系组织中的功能，我将使用Tbcel-/-小鼠模型来执行 感兴趣组织的组织学分析。该项目的完成将提供大量的信息 关于TBCEL依赖的微管蛋白降解途径以及该途径对细胞生物学的意义。 由于微管蛋白具有很高的临床相关性，研究人员正试图为微管蛋白设计小分子降解剂。 因此，我们的工作可以激励翻译研究人员开发新的方法来靶向微管蛋白的降解。

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.