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）：识别微管腔中的蛋白质微管是细胞骨架中普遍存在的动态细丝，几乎参与任何涉及运动的细胞活动。与细胞中的这些结构相关的是多种蛋白质，它们调节微管功能或使用微管作为在整个细胞内运输物质的轨道。这些管完全由一种蛋白质组成：a,b-微管蛋白。所有转运蛋白和几乎所有调节蛋白都与微管蛋白聚合物的外部或末端结合。迄今为止，微管腔中发生的调节过程只有一个例子：α-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，许多细胞微管并不是空的——它们实际上充满了颗粒。仅用相对较小的酶进行一次翻译后修饰无法解释如此大量的材料。微管腔内必须有额外的蛋白质来解释颗粒。这些颗粒的身份、目的和功能尚不清楚。了解腔蛋白的性质对于微管的基础生物学非常重要。它还可能提供对微管相关疾病的分子病理学的深入了解，并为化疗带来重要的新靶标。了解微管中特定物质的用途和功能的第一步是识别该物质。因此，该项目的目标是鉴定与细胞微管管腔颗粒相关的蛋白质。我们的方法是利用药物紫杉醇独特的微管结合特性，它以高亲和力结合组装微管蛋白上的单个管腔位点，用于将光亲和标签放置在活细胞微管的内部。将合成紫杉醇衍生物，其中光标记官能团将通过长聚醚链与紫杉醇连接。探针的设计使得光亲和标记将投射到微管腔深处。活细胞中微管结合探针的光解将使紫杉醇与附近的蛋白质交联，通过检测紫杉醇标签可以在细胞蛋白质的复杂混合物中识别出这些蛋白质。光标记的蛋白质将通过质谱法进行分析。我们假设所鉴定的一些蛋白质将是全新的发现。公共健康相关性：Susan Bane，首席研究员标题：识别微管腔的蛋白质微管是癌症化疗的一个非常重要的目标。此外，微管-蛋白质相互作用的缺陷与阿尔茨海默病等疾病有关。识别管内的蛋白质将在细胞中产生一个新的位置，该位置可以是针对癌症和其他疾病的化疗药物的靶标。