An orderly approach to toxic mechanism by disorderly peptides

无序肽毒性机制的有序方法

• 批准号: 8365310
• 负责人: ANDREW D. MIRANKER
• 金额: $ 34.97万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365310
• 关键词: AIDS/HIV problem; Address; Affinity; Alzheimer' s Disease; Amyloid; Amyloid fibers; Amyloidosis; Attention; Bacterial Toxins; Beta Cell; Binding; Biochemical; Biological; Biological Assay; Cause of Death; Cell Death; Cell Line; Cell membrane; Cells; Cellular Membrane; Cereals; Consensus; Deposition; Diabetes Mellitus; Disease; Disease Progression; Ensure; Environment; Extravasation; Fiber; Filament; Fluorescence; Fluorescence Resonance Energy Transfer; Functional disorder; HIV Infections; Homeostasis; Human; In Vitro; Joints; Kinetics; Lateral; Life; Lipid Bilayers; Malignant Neoplasms; Measures; Mediating; Membrane; Modeling; Molecular; Monitor; Neurons; Non-Insulin-Dependent Diabetes Mellitus; Organelles; Pancreas; Parkinson Disease; Pathology; Pathway interactions; Penetration; Peptides; Physiological; Play; Process; Property; Protein Conformation; Proteins; Publishing; Research; Role; Severity of illness; Site-Directed Mutagenesis; Societies; Solutions; Structure; System; Testing; Toxic effect; Toxin; Vesicle; Work; antimicrobial; base; conformational conversion; cytotoxic; cytotoxicity; design; design and construction; dimer; gain of function; in vivo; insight; particle; peptide A; protein aggregate; protein folding; protein misfolding; research study; self assembly; simulation; single molecule; stem; therapeutic target

DESCRIPTION (provided by applicant): Protein misfolding and fibrillar aggregation play a critical role in the pathology of some of the most serious diseases facing society today. Diseases long known to have such a relationship include Alzheimer's and Parkinson's. More recently, diabetes, cancer and HIV/AIDS have also been shown to have proteins for which self association of misfolded states into ?-sheet rich fibers is associated with disease progression. It is also long known, however, that disease severity does not correlate with the amount of aggregated protein deposited as plaques (termed amyloid). Numerous in vitro and in vivo studies in recent years have drawn attention instead to the role of non-fibrillar, pre-amyloid species as the relevant agents of pathology. Specifically, cellular dysfunction appears to be initiated by interactions between specific oligomeric species and cellular membranes, resulting in disruption of cellular homeostasis. These membrane-associated oligomers need not have amyloid- like structure and indeed in several systems are characterized by ?-helical structure. The challenge we propose to address here stems from observations that toxic gains-of-function emanate from subsets of a dynamic and heterogeneous oligomeric ensemble. By investigating the molecular mechanisms by which these ensembles form, we will determine the molecular basis of toxicity and illuminate a pathway towards relevant therapeutic targets. The aims of this proposal are designed to elucidate the mechanism by which amyloid-associated oligomers form, undergo conformational change, and disrupt and translocate across lipid bilayers. We focus on two, ~40 residue peptides, A? and IAPP, implicated in Alzheimer's and Type II diabetes respectively. These peptides share many biophysical and biochemical properties. Key to this proposal is that they both undergo disorder to ?-helical transitions upon binding to membranes and that each forms heterogeneous arrays of membrane- associated oligomers that are capable of disrupting lipid bilayers in vitro and cause cell death when added to cultured cell lines. For both systems, targeted nonspecific disruption of their ?-helical states has been demonstrated to rescue cellular toxicity. By studying these peptides in tandem, we will elucidate a common molecular basis for cellular gains of toxic function, as well as pinpoint the basis for disease-specific differences. We propose to dissect mechanism through the use of complimentary ensemble and single molecule assays designed to elucidate structures, kinetics and energies associated with membrane bound conformational conversions. Molecular level insights will be achieved by using coarse grain simulations informed by experimentally determined constraints. Finally, we will ensure biological relevance in our findings by conducting parallel efforts on synthetic bilayers, purified sub-cellular organelles and whole cells. PUBLIC HEALTH RELEVANCE: Type II diabetes and Alzheimer's disease each have a small, disease-specific protein which shows almost no structure in solution, but can bind cell membranes. Once bound, a common structure emerges that is associated with pathology as they cause the death of insulin secreting cells and neurons respectively. The aim of this proposal is to elucidate the membrane bound molecular changes that these two systems have in common, as well as to determine what makes them distinct, in order to identify which structures are actually responsible for cell death.

描述(申请人提供)：蛋白质错误折叠和纤维聚集在当今社会面临的一些最严重疾病的病理中起着关键作用。人们早就知道这种关联的疾病包括阿尔茨海默氏症和帕金森氏症。最近，糖尿病、癌症和艾滋病毒/艾滋病也被证明含有蛋白质，这些蛋白质的错误折叠状态自我关联成丰富的纤维，与疾病的进展有关。它 然而，人们很早就知道，疾病的严重程度与聚集的蛋白质沉积成斑块(称为淀粉样蛋白)的数量无关。近年来，大量的体外和体内研究已经引起了人们的注意，而不是非纤维、前淀粉样蛋白作为相关的病理介质的作用。具体地说，细胞功能障碍似乎是由特定的寡聚体物种与细胞膜之间的相互作用启动的，导致细胞内稳态的破坏。这些膜相关低聚物不需要具有淀粉样结构，实际上在几个系统中都具有螺旋结构的特征。我们在这里提出的挑战来自于观察到有毒的功能收益来自动态和不同种类的寡聚体集合的子集。通过研究这些集合形成的分子机制，我们将确定毒性的分子基础，并阐明通往相关治疗靶点的途径。这项提议的目的是为了阐明淀粉样蛋白相关寡聚体的形成、构象变化以及破坏和跨脂双层转移的机制。我们专注于两个~40个残基的多肽，A？和IAPP，分别与阿尔茨海默氏症和II型糖尿病有关。这些多肽具有许多生物物理和生化特性。这一建议的关键是，它们都在与膜结合时都经历了无序到螺旋的转变，并且每个都形成了膜相关低聚物的异质阵列，能够在体外破坏脂质双层，并在加入培养的细胞系时导致细胞死亡。对于这两个系统，都已证明有针对性地非特异性破坏其？-螺旋状态可以挽救细胞毒性。通过对这些多肽的研究，我们将阐明细胞获得毒性功能的共同分子基础，以及疾病特异性差异的基础。我们建议通过使用互补的系综和单分子分析来剖析与膜结合的构象转换相关的结构、动力学和能量。分子水平的洞察将通过使用由实验确定的约束条件提供的粗粒度模拟来实现。最后，我们将通过对合成双层、纯化的亚细胞器和整个细胞进行平行努力，确保我们的发现具有生物学相关性。 公共卫生相关性：II型糖尿病和阿尔茨海默病都有一种小的、疾病特有的蛋白质，这种蛋白质在溶液中几乎没有结构，但可以结合细胞膜。一旦结合，就会出现一个与病理相关的共同结构，因为它们分别导致胰岛素分泌细胞和神经元的死亡。这一建议的目的是阐明这两个系统共同的膜结合分子变化，以及确定它们的不同之处，以便确定哪些结构实际上是导致细胞死亡的原因。