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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：
10227392
负责人：
Bryan Seguinot
金额：
$ 3.96万
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/10227392
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/antagonist 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complex protein degradation reconstitution small molecule sperm cell translational scientist ubiquitin ligase unpublished works vesicle transport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