In vivo and crude extract analysis of polyQ aggregation intermediates

PolyQ 聚集中间体的体内和粗提物分析

• 批准号: 8432253
• 负责人: Robert Fairman
• 金额: $ 35.06万
• 依托单位: HAVERFORD COLLEGE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-15 至 2016-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8432253
• 关键词: Aging; Animal Model; Award; Back; Behavioral; Biological; Biological Models; Biology; Caenorhabditis elegans; Cell Line; Cells; Cellular Structures; Colorado; Complex; Crude Extracts; Detection; Development; Disease; Drosophila genus; Exons; Faculty; Fluorescence; Fluorescence Recovery After Photobleaching; Future Generations; Glutamine; Goals; Hereditary Disease; Human Genetics; Huntington Disease; Imaging Techniques; In Vitro; Inclusion Bodies; Kinetics; Laboratories; Larva; Lead; Learning; Left; Length; Life; Link; Machado-Joseph Disease; Measures; Methods; Microscope; Microscopy; Molecular; Molecular Chaperones; Mus; Nuclear Inclusion; Pathogenicity; Pennsylvania; Play; Population; Principal Investigator; Process; Property; Proteins; Protocols documentation; Reagent; Relative (related person); Research; Research Infrastructure; Research Personnel; Resource Sharing; Role; Scientist; Staging; Stretching; System; Techniques; Toxic effect; Training and Education; Transgenic Organisms; Universities; Work; aggregation pathway; analytical ultracentrifugation; base; biomedical scientist; college; cytotoxic; cytotoxicity; disease phenotype; fly; human Huntingtin protein; human disease; in vivo; insight; monomer; mutant; neuroblastoma cell; polyglutamine; prevent; programs; protein aggregation; research study; sedimentation velocity; tool; undergraduate student

DESCRIPTION (provided by applicant): The over-arching goal of this project is to understand the molecular and cellular mechanisms that drive glutamine-repeat (polyQ) protein aggregation. Such polyQ sequences are found in at least twelve human genetic diseases including Huntington's disease and Machado-Joseph disease. It is well established from in vitro experiments that stable intermediates, or oligomers, may be present during the assembly of polyQ proteins into fibrils. Such oligomers have been shown to have cytotoxic and pathogenic properties in cells. We propose that native sequences flanking the polyQ stretches in mutant proteins will greatly influence the distribution of aggregation species in the context of the cell, and that the activities of molecular machinery that mitigate protein aggregation may be influenced by such flanking sequences. A recent study, using crude extracts from a mouse neuroblastoma cell line (2), has identified the presence of oligomers using the newly developed analytical ultracentrifuge with fluorescence detection capability. This technique will be used, combined with FRAP (fluorescence recovery after photobleaching, carried out using a confocal microscope), to probe the aggregation states of polyQ-containing proteins both in vivo and in crude extracts in the worm, C. elegans. The effect of protein flanking sequences will be explored on the relative distribution of oligomers, and its correlation to cytotoxicity. To further assess biological significance for the role of various aggregation states in disease, D. melanogaster will be used to discover how two well-studied chaperones (Hsp104 and Hsp70) might mitigate the effects of intermediate aggregation states, using the same set of fluorescence techniques. Key reagents and fly lines are being developed while the principal investigator is on sabbatical leave in the laboratory of Dr. Nancy Bonini, an HHMI investigator at the University of Pennsylvania. The Bonini laboratory has expertise in the study of the role of several polyQ- containing proteins on fly development and aging (3-8). Preliminary work with C. elegans will be carried out in the laboratory of Dr. Christopher Link, at the University of Colorado, Boulder in the summer of 2012. He has expertise in the use of this animal model to study protein aggregative diseases (9-14). The products of this critical work will be brought back to Haverford, and will provide an important set of tools for undergraduate students to use in their senior research work. Two faculty in the Biology Department at Haverford College already use these animal models for their research, so there is adequate infrastructure support in place, which will allow for a sharing of resources, and provide new synergies in the existing research programs. By involving undergraduate researchers in all aspects of the project, this award will also contribute significantly to the education and training of future generations of biomedical scientists. PUBLIC HEALTH RELEVANCE: Twelve human diseases involve polyQ-driven aggregation, and the pathway to sequestration into inclusion bodies or nuclear inclusions has been shown to be complex, involving the accumulation of toxic oligomers. New biophysical and imaging techniques have been recently developed that allow scientists to probe aggregation both in vivo and in crude extracts, and the PI plans to use fluorescence techniques (using a confocal microscope, and characterization of molecular populations using an analytical ultracentrifuge) to probe polyQ aggregation using D. melanogaster and C. elegans model systems. The ability to mitigate potentially toxic intermediate populations will be explored using chaperones, proteins whose function is to prevent or reduce protein aggregation.

描述（由申请人提供）：该项目的首要目标是了解驱动谷氨酰胺重复 (polyQ) 蛋白质聚集的分子和细胞机制。这种polyQ序列存在于至少12种人类遗传疾病中，包括亨廷顿舞蹈病和马查多-约瑟夫病。体外实验明确表明，在将 PolyQ 蛋白组装成原纤维的过程中，可能存在稳定的中间体或寡聚物。此类寡聚物已被证明在细胞中具有细胞毒性和致病性。我们认为突变蛋白中 PolyQ 延伸侧翼的天然序列将极大地影响细胞环境中聚集种类的分布， 减轻蛋白质聚集的分子机制的活动可能会受到此类侧翼序列的影响。最近的一项研究使用小鼠神经母细胞瘤细胞系的粗提物 (2)，利用新开发的具有荧光检测功能的分析超速离心机鉴定了寡聚体的存在。该技术将与 FRAP（光漂白后的荧光恢复，使用共焦显微镜进行）结合使用，探测线虫体内和粗提物中含有 PolyQ 的蛋白质的聚集状态。将探讨蛋白质侧翼序列对寡聚体相对分布的影响及其与细胞毒性的相关性。为了进一步评估各种聚集状态在疾病中的作用的生物学意义，黑腹果蝇将 可用于发现两个经过充分研究的分子伴侣（Hsp104 和 Hsp70）如何使用同一组荧光技术减轻中间聚集状态的影响。关键试剂和蝇系正在开发中，而首席研究员正在宾夕法尼亚大学 HHMI 研究员 Nancy Bonini 博士的实验室休假。 Bonini 实验室在研究几种含 PolyQ 的蛋白质对果蝇发育和衰老的作用方面拥有专业知识 (3-8)。线虫的初步工作将于 2012 年夏天在科罗拉多大学博尔德分校 Christopher Link 博士的实验室进行。他拥有使用该动物模型研究蛋白质聚集疾病的专业知识 (9-14)。这项关键工作的产品将被带回哈弗福德，并将提供重要的 供本科生在高级研究工作中使用的一套工具。哈弗福德学院生物系的两名教师已经使用这些动物模型进行研究，因此有足够的基础设施支持，这将允许资源共享，并在现有研究项目中提供新的协同效应。通过让本科生研究人员参与该项目的各个方面，该奖项还将为下一代生物医学科学家的教育和培训做出重大贡献。 公共卫生相关性：12 种人类疾病涉及 PolyQ 驱动的聚集，并且隔离成包涵体或核包涵体的途径已被证明是复杂的，涉及有毒低聚物的积累。最近开发出新的生物物理和成像技术，使科学家能够探测体内和粗提物中的聚集，PI 计划使用荧光技术（使用共聚焦显微镜，并使用分析超速离心机表征分子群），使用黑腹果蝇和线虫模型系统来探测 PolyQ 聚集。将利用伴侣蛋白（其功能是防止或减少蛋白质聚集）来探索减轻潜在毒性中间群体的能力。