Factors modulating the interaction of huntingtin with lipid membranes: Implications for Huntington's Disease

描述（由申请人提供）：在理解亨廷顿（htt）蛋白突变形式与扩展的polyQ束形成的小聚集体如何获得导致亨廷顿病（HD）的毒性生物学特性方面存在根本性差距，更具体地说，这些蛋白如何与由脂质组成的细胞表面相互作用。这种差距的持续存在代表了一个重要的问题，因为这些相互作用可能代表了HTT诱导的细胞毒性的基本步骤，对这种现象的理解可以导致治疗干预的新靶点。我们研究的长期目标是了解导致毒性的生物大分子的纳米级病理性自组装的物理化学方面和分子机制。本申请的目的是 通过测定HTT聚集体对细胞和亚细胞膜表面完整性的影响，阐明脂质对HTT错误折叠、聚集和相关毒性的作用。我们的中心假设是，突变体htt和其聚集体形式的结合主要由脂质组成的亚细胞表面是与毒性相关的基本步骤，并通过膜的脂质组成和htt的翻译后修饰（PTM）来促进。该假设基于初步数据，表明突变亨廷顿蛋白（htt）片段和合成的polyQ肽积累在模型脂质双层上并使其不稳定，这取决于特定侧翼序列的可用性，如htt的第17个氨基酸或聚脯氨酸结构域。拟议的工作的理由是，详细的知识突变htt蛋白在脂质膜界面的聚集过程中，可以预期最终导致开发新的治疗和神经保护的目标HD。在我们的初步研究的指导下，将通过追求两个具体目标来测试该假设：1）鉴定在htt/o膜结合中起作用的脂质组分;和2）确定翻译后修饰如何调节htt和脂质膜之间的相互作用。在第一个目标下，扫描探针和各种基于囊泡的测定将被用来表征和测量htt和富含胆固醇、鞘磷脂或GM 1的膜之间发生的内源性相互作用。根据第二个目标，光谱，质谱和扫描探针技术的组合将被应用到生物PTM在调节HTT/LipD相互作用中发挥的作用的研究。拟议的研究是创新的，因为它关注的是htt（及其聚集形式）结合和插入脂质膜的物理和机械后果的影响，这可能最终导致细胞功能障碍和死亡。这一贡献是重要的，因为它预计将提供在脂质表面的htt相互作用和聚集的机制知识，这可能代表HD病理学的基本步骤。

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how small aggregates formed by mutant forms of the huntingtin (htt) protein with expanded polyQ tracts gain toxic biological properties causing Huntington's disease (HD) and, more specifically, how these proteins interact with cellular surfaces comprised of lipids. Continued existence of this gap represents an important problem because these interactions may represent a fundamental step in htt- induced cellular toxicity and understanding of this phenomenon can lead to new targets for therapeutic intervention. The long-term goal of our research is to understand the physicochemical aspects and molecular mechanisms of nanoscale, pathological self-assembly of biological macromolecules that lead to toxicity. The objective in this application is to elucidate the role of lipids on htt misfolding, aggregation, and related toxicity by determinng the impact htt aggregates have on the integrity of cellular and subcellular membranous surfaces. Our central hypothesis is that the binding of mutant htt and its aggregate forms to subcellular surfaces comprised predominately of lipids is a fundamental step associated with toxicity and is facilitated by lipid composition of the membrane and post-translational modifications (PTMs) of htt. This hypothesis is based on preliminary data demonstrating that mutant huntingtin (htt) fragments and synthetic polyQ peptides accumulate on and destabilize model lipid bilayers depending on the availability of specific flanking sequences, such as the firs 17 amino acids of htt or a polyproline domain. The rationale for the proposed work is that detailed knowledge of the aggregation process of mutant htt proteins at lipid membrane interfaces can be expected to ultimately lead to the development of novel therapeutic and neuroprotective targets for HD. Guided by our preliminary studies, this hypothesis will be tested by pursuing two specific aims: 1) Identify lipid components that play a role in the binding of htt o membranes; and 2) Determine how post-translational modification regulate the interaction between htt and lipid membranes. Under the first aim, scanning probe and a variety of vesicle-based assays will be employed to characterize and measure the endogenous interactions occurring between htt and membranes enriched with cholesterol, sphingomyelin, or GM1. Under the second aim, a combination of spectroscopic, mass spectrometry, and scanning probe techniques will be applied to the study of the role of biologically PTMs play in modulating htt/lipd interactions. The proposed research is innovative because it focuses on the impact of physical and mechanical consequences of htt (and its aggregate forms) binding and inserting into lipid membranes that may ultimately lead to cellular dysfunction and death. This contribution is significant because it is expected to provide mechanistic knowledge of htt interaction and aggregation at lipid surfaces, which potentially represents a fundamental step in HD pathology.