Identifying Proteins in the Microtubule Lumen

识别微管腔中的蛋白质

基本信息

批准号：
8367606
负责人：
SUSAN L BANE
金额：
$ 32.1万
依托单位：
STATE UNIVERSITY OF NY,BINGHAMTON
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8367606
关键词：
Accounting Acetylation Affinity Alzheimer&apos s Disease Antibodies Binding Binding Proteins Binding Sites Biology Carrier Proteins Cells Cellular Structures Cellular biology Chemotherapy-Oncologic Procedure Complex Complex Mixtures Cryoelectron Microscopy Cytoskeleton Defect Disease Environment Enzymes Equilibrium Filament Goals In Vitro Institution Kinetics Label Laboratory Research Lead Length Life Link Location Lysine Malignant Neoplasms Mass Spectrum Analysis Measures Microtubule Proteins Microtubules Motor Movement Nature Organic Chemistry Paclitaxel Parkinson Disease Pharmaceutical Preparations Photoaffinity Labels Photochemistry Polycystic Kidney Diseases Polymers Positioning Attribute Post-Translational Protein Processing Principal Investigator Process Property Protein Binding Proteins Proteomics Qualifying Radioactive Relative (related person)Research Research Project Grants Site Structure Structure-Activity Relationship Students Surface Taxane Compound Tube Tubulin Work cancer therapy chemotherapy crosslink design experience flexibility functional group genetic regulatory protein graduate student insight macromolecule molecular pathology particle photolysis protein transport research study taxane tomography tumor undergraduate student

项目摘要

DESCRIPTION (provided by applicant): Identifying Proteins in the Microtubule Lumen Microtubules are ubiquitous, dynamic filaments of the cytoskeleton that participate in practically any cellular activity that involves movement. Associated with these structures in cells are a diverse group of proteins that regulate microtubule function or use microtubules as tracks to transport materials throughout the cell. The tubes are composed entirely of a single protein:a,b- tubulin. All of the transporter proteins and virtually all of the regulatory proteins bind to the outside or to the ends of the tubulin polymers. To date there is only one example of a regulatory process that takes place in the microtubule lumen: acetylation of lysine-40 of a-tubulin. Cryoelectron tomography studies have demonstrated that many cellular microtubules are not empty - they are in fact filled with particles. Just one posttranslational modification with a relatively small enzyme cannot account for such a large amount of material. There must be additional proteins with the microtubule lumen to account for the particles. The identity, purpose and function of these particles are not known. Understanding the nature of luminal proteins is important in fundamental biology of microtubules. It may also provide insight into the molecular pathology of microtubule-associated disease and lead to important new targets for chemotherapy. The first step toward understanding the purpose and function of the particular matter in microtubules is to identify the material. Thus, the goal of this project is to identify proteins associated with the lumen particles of cellular microtubules. Our approach is to harness the unique microtubule binding properties of the drug paclitaxel, which binds with high affinity to a single, luminal site on assembled tubulin, to place photoaffinity labels in the interior of microtubules in living cells. Paclitaxel derivatives will be synthesized in which a photolabeling functional group will be linked to paclitaxel through a long polyether tether. The probes are designed such that the photoaffinity label will be projected deep within the microtubule lumen. Photolysis of the microtubule-bound probe in live cells will crosslink paclitaxel to nearby proteins, which will be identified in the complex mixtures of cellular proteins by detecting the paclitaxel tag. Photolabeled proteins will be analyzed by mass spectrometry. We hypothesize that some of the proteins identified will be entirely new discoveries. PUBLIC HEALTH RELEVANCE: Susan Bane, principal investigator Title: Identifying Proteins of the Microtubule Lumen Microtubules are a very important target for cancer chemotherapy. In addition, defects in microtubule-protein interactions are involved in diseases such as Alzheimer's. Identifying proteins inside of tube will yield a new place in the cell that can be the target for chemotherapeutic drugs against cancer and other diseases.

描述（由申请人提供）：识别微管管腔微管中的蛋白质是无处不在的动态细丝的细胞骨架的动态细丝，这些细丝实际上参与了任何涉及运动的细胞活动。与细胞中这些结构相关的是一组多样的蛋白质，这些蛋白质调节微管功能或使用微管作为在整个细胞中运输材料的轨道。管完全由单个蛋白质组成：a，b-微管蛋白。所有转运蛋白和几乎所有调节蛋白的转运蛋白与外部或小管蛋白聚合物的末端结合。迄今为止，仅在微管腔中发生的调节过程的一个例子：A-微管蛋白的赖氨酸-40的乙酰化。冷冻电子断层扫描研究表明，许多细胞微管不是空的 - 实际上它们充满了颗粒。只有一个相对较小的酶的翻译后修饰不能解释如此大量的材料。微管腔必须有其他蛋白质来解释颗粒。这些颗粒的身份，目的和功能尚不清楚。了解腔蛋白的性质在微管的基本生物学中很重要。它还可以洞悉微管相关疾病的分子病理学，并导致重要的化疗靶标。了解微管中特定物质的目的和功能的第一步是识别材料。因此，该项目的目的是鉴定与细胞微管的管腔颗粒相关的蛋白质。我们的方法是利用药物紫杉醇的独特微管结合特性，该特性具有高亲和力的结合组装小管蛋白上的一个单位位点，将光性标签放在活细胞中微管内部。将合成紫杉醇衍生物，其中光标记官能团将通过长聚醚系带连接到紫杉醇。探针的设计使得光亲和力标签将在微管腔内深处投影。活细胞中微管结合的探针的光解会通过检测紫杉醇标签在细胞蛋白的复杂混合物中交叉紫杉醇与附近的蛋白质交联。光标记蛋白将通过质谱法分析。我们假设确定的一些蛋白质将是全新的发现。公共卫生相关性：苏珊·贝恩（Susan Bane），首席研究员的职称：识别微管腔管道微管的蛋白质是癌症化学疗法的非常重要的靶标。另外，微管 - 蛋白质相互作用的缺陷涉及阿尔茨海默氏症等疾病。识别管子内部的蛋白质将在细胞中产生一个新位置，可以是针对癌症和其他疾病的化学治疗药物的靶标。