Deciphering the Mechanism of Transporters by Design and Experiments

通过设计和实验破译转运蛋白的机制

• 批准号: 8214098
• 负责人: Nathan Joh
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8214098
• 关键词: Abate; Address; Antineoplastic Agents; Apoptosis; Behavior; Binding; Biological; Biological Models; Biological Process; Biophysics; Carrier Proteins; Cell Death; Cell Proliferation; Cell Survival; Cells; Chemicals; Complex; Coupled; Deposition; Disease; Drug Delivery Systems; Drug Design; Drug Efflux; Engineering; Environment; Equilibrium; Etiology; Event; Evolution; Galactose; Gene Duplication; Generations; Genetic Screening; Heart Diseases; Integrins; Investigation; Kinetics; Link; Liposomes; Malignant Neoplasms; Malignant neoplasm of prostate; Maps; Mediating; Membrane; Membrane Proteins; Metals; Methods; Modeling; Molecular; Molecular Conformation; Motion; Movement; Nature; Normal Cell; Nutrient; Organelles; P-Glycoproteins; Pathway interactions; Peptides; Physiological; Physiology; Process; Protein Biochemistry; Protein Engineering; Proteins; Research; Role; Signal Transduction; Signaling Molecule; Structure; Testing; Thermodynamics; Training; Transmembrane Domain; Transportation; Work; Zinc; design; gaze; innovation; insight; meetings; neoplastic cell; novel; protein structure; protein structure function; public health relevance; research study; structural biology; sugar; theories; therapeutic development; zinc-binding protein

DESCRIPTION (provided by applicant): Transporters, a major class of membrane proteins, mediate substrate translocations that are implicated in a variety of disease-related physiology, including aberrant cellular deposition of nutrients, ab- normal cell death or proliferation, and efflux of drug. So, understanding the transport mechanism is theo- retically significant, as well as fundamental to addressing relevant questions, such as disease etiology at the molecular level and drug design. Despite the importance, efforts to gain structural understanding of transport mechanism have long been hampered by the experimental difficulty associated with the complex nature of the bilayer environment where these proteins reside. Very recently, an idea of common transport mechanism has been highlighted by the breakthroughs in structural investigations, which revealed that functionally diverse transporters share a surprisingly similar topology composed of dual structural subunits with inverted symmetry. Here, I plan to pursue basic biomedical quest on establishing a unified transport mechanism by investigating metal- and sugar-transportation as model systems, using methods of experimental, computational and struc- tural biophysics. In implementing the work, my expertise in membrane protein structure and thermodynamic folding will be broadened by the leading research of my sponsor, William DeGrado, in diverse fields of protein structure-function-dynamics relationship, molecular biophysics of membrane proteins and de novo protein design. Specific Aims: Following specific aims will be pursued: Aim 1. To test the importance of symmetric dual topo- logy in transport function, I will use de novo protein design approach to generate zinc transport function in a minimalist model transporter composed of a symmetrically designed helix bundle. Aim 2. To test the impor- tance of conformational dynamics in function, I will examine whether the activity by galactose transporter vSGLT, as well as de novo zinc transporter peptides, reflect the expected conformational change with respect to kinetic behaviors. Aim 3. To test the corollary of the importance of structural dynamics in transportation, I will use both protein design and genetic screening approaches to generate a transmembrane peptide that allosterically locks the vSGLT or model transporter in one conformational state by binding the target trans- porter, and test the transportation inhibition effect of the de novo allosteric binder peptide. Reemphasis of the proposal's innovation: Abated zinc translocation is related to the lack of zinc-induced apoptosis in prostate cancer, while specific sugar transporters are up regulated in other tumor cells to meet the increased demand for nutrient. So, understanding the metal- and sugar-transportation function using the minimalist zinc transporters and vSGLT as model systems is extensively relevant. Completion of the proposed project will not only provide an excellent training opportunity, but also present conceptual mechanistic theories for transport function, which is potentially important in therapeutics development. PUBLIC HEALTH RELEVANCE: Transporters are proteins that reside in the biological membrane and control the movement of variety of molecules in and out of cells, as well as cellular organelles. As maintaining the chemical balance is critical for cell survival, understanding how transporters function is central to exploring physiologically central questions, such as how transport function is generated at the molecular level, how impaired transport function is related to diseases, such as cancer or heart disease, and drug design. This proposal is aimed at providing the conceptual understanding for transporter mechanism, along with novel ways of selectively regulating the transport function, achieving of which will be theoretically significant, as well as potentially fundamental to therapeutics development.

描述（由申请人提供）：转运蛋白是一类主要的膜蛋白，介导底物易位，涉及多种疾病相关的生理，包括营养物质的异常细胞沉积，非正常细胞死亡或增殖，以及药物的外排。因此，了解转运机制具有重要的理论意义，同时也是解决相关问题的基础，例如分子水平的疾病病因学和药物设计。尽管具有重要意义，但长期以来，由于这些蛋白质所在的双层环境的复杂性，实验困难，对转运机制的结构理解受到阻碍。最近，结构研究的突破突出了共同转运机制的想法，揭示了功能不同的转运体具有惊人的相似拓扑结构，由具有倒对称的双结构亚基组成。在这里，我计划通过实验、计算和结构生物物理学的方法，研究金属和糖的运输作为模型系统，以建立统一的运输机制为基础的生物医学探索。在实施这项工作的过程中，我在膜蛋白结构和热力学折叠方面的专业知识将被我的赞助人William降格罗在蛋白质结构-功能-动力学关系、膜蛋白分子生物物理学和从头蛋白质设计等多个领域的领先研究所拓宽。具体目标：将追求以下具体目标：目标1。为了测试对称对偶拓扑在转运功能中的重要性，我将使用从头开始的蛋白质设计方法在由对称设计的螺旋束组成的极简模型转运体中生成锌转运功能。目标2。为了测试构象动力学在功能中的重要性，我将研究半乳糖转运蛋白vSGLT的活性，以及新生锌转运蛋白肽的活性，是否反映了与动力学行为相关的预期构象变化。目标3。为了测试结构动力学在运输中的重要性，我将使用蛋白质设计和遗传筛选方法来生成一种跨膜肽，通过结合靶转运蛋白来变构锁定vSGLT或模型转运蛋白在一种构象状态，并测试新生变构结合肽的运输抑制作用。再次强调该提案的创新之处：锌转运减少与前列腺癌中锌诱导的细胞凋亡缺乏有关，而其他肿瘤细胞中特异性糖转运蛋白上调以满足对营养物质增加的需求。因此，利用极简锌转运体和vSGLT作为模型系统来理解金属和糖的转运功能具有广泛的意义。拟议项目的完成不仅将提供一个很好的培训机会，而且还将提出运输功能的概念机制理论，这对治疗学的发展具有潜在的重要意义。