The Nano-mechanics of Polycystin-1

Polycystin-1 的纳米力学

• 批准号: 7149071
• 负责人: ANDRES F. OBERHAUSER
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-11 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7149071
• 关键词: acidity /alkalinity; atomic force microscopy; biomechanics; biotechnology; chemical models; chemical stability; circular dichroism; computer simulation; elasticity; fluorescence spectrometry; gene expression; gene mutation; mechanoreceptors; membrane proteins; model design /development; nanotechnology; point mutation; polycystic kidney; protein engineering; protein folding; protein structure function; structural biology; thermodynamics; urea

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common life-threatening genetic diseases, and is a leading cause of renal failure. The majority of cases are caused by mutations in the PKD1 gene, which encodes for polycystin-1 (PC1). Recent evidence suggests that PC1 acts as a mechanosensor, receiving signals from the primary cilia, neighboring cells and extracellular matrix and transduces them into cellular responses that regulate proliferation, adhesion and differentiation that are essential for the control of renal tubules and kidney morphogenesis. PC1 is a large membrane protein that has an unusually long extracellular region (approximately 3000 aa) with a multi-modular structure. Proteins with a similar architecture have structural and mechanical roles. Our hypothesis is that PC1 is a mechano-transducer with a novel molecular architecture and elastic properties well-suited for sensing and transmitting distinct mechanical signals with a wide range of strengths. Consistent with this hypothesis we have found that most of the PC1 extracellular region is made of mechanically-elastic domains, providing direct support to the idea that the ectodomain may function as an effective force transmitter. Mutations may alter PC1's mechanical properties and so lead to the altered signal transduction and abnormal tissue development characteristic of ADPKD. To begin to understand the sensing mechanism of PC1, we will use using a combination of recombinant DNA and atomic force microscopy (AFM) techniques to assess the mechanical and biophysical properties of the extracellular region of normal and mutant forms of PC1. This technique offers the most direct way of studying the stability and elasticity of proteins that are exposed to mechanical forces and hence more closely approximate the conditions found in vivo. In Aim 1 we will use single molecule force spectroscopy to determine the mechanical properties of PC1-ectodomain. In Aim 2 we will determine whether missense mutations affect the mechanical and thermodynamic stability of PC1-ectodomain. In Aim 3 we will use protein engineering and computer simulations to develop a molecular model for PC1-ectodomain to examine the significance of its modular architecture and to enable a structural interpretation of the effects of the mutations. Our long term plan is to elucidate the structure and biophysical properties of the PC1 molecule and help us understand the effects of mutations on the PKD1 gene. These experiments should provide a solid basis to investigate the molecular mechanisms of the signaling events that link PC1 mutations with the ADPKD phenotype.

描述（由申请人提供）：常染色体显性多囊性肾脏疾病（ADPKD）是最常见的威胁生命的遗传疾病之一，并且是肾衰竭的主要原因。大多数病例是由PKD1基因中的突变引起的，该突变编码为Polycystin-1（PC1）。最近的证据表明，PC1充当机械传感器，从原发性纤毛，邻近细胞和细胞外基质接收信号，并将其转换为细胞反应，从而调节肾小管和肾脏小管和肾脏形态发生至关重要的增殖，粘附和分化。 PC1是一种大型膜蛋白，其具有多模块化结构的异常长的细胞外区域（约3000 AA）。具有相似结构的蛋白质具有结构和机械作用。我们的假设是，PC1是一种机械转换器，具有新颖的分子结构和弹性特性非常适合具有多种强度范围的不同机械信号。与这一假设一致，我们发现大多数PC1细胞外区域都是由机械弹性的域制成的，为eTodopomain可能充当有效的力发射器的想法提供了直接支持。突变可能会改变PC1的机械性能，从而导致ADPKD的信号转导和异常组织发育特征改变。 为了开始了解PC1的传感机制，我们将使用重组DNA和原子力显微镜（AFM）技术的组合来评估PC1正常和突变形式的细胞外区域的机械和生物物理特性。该技术提供了研究暴露于机械力的蛋白质稳定性和弹性的最直接方法，因此更接近体内发现的条件。在AIM 1中，我们将使用单分子力光谱来确定PC1-骨基域的机械性能。在AIM 2中，我们将确定错义突变是否影响PC1-骨基域的机械和热力学稳定性。在AIM 3中，我们将使用蛋白质工程和计算机模拟来开发PC1- eTodobain的分子模型，以检查其模块化结构的重要性，并能够对突变的影响进行结构性解释。我们的长期计划是阐明PC1分子的结构和生物物理特性，并帮助我们了解突变对PKD1基因的影响。这些实验应提供可靠的基础，以研究将PC1突变与ADPKD表型联系起来的信号传导事件的分子机制。