In vivo and crude extract analysis of polyQ aggregation intermediates

描述(由申请人提供)：本项目的总体目标是了解驱动谷氨酰胺重复序列(PolyQ)蛋白质聚集的分子和细胞机制。在包括亨廷顿病和马查多-约瑟夫病在内的至少12种人类遗传病中发现了这样的多Q序列。体外实验证实，在将多聚Q蛋白组装成纤维的过程中，可能存在稳定的中间体或低聚体。这些低聚物已被证明在细胞中具有细胞毒性和致病特性。我们认为突变蛋白质中多聚Q延伸两侧的天然序列将极大地影响细胞环境中聚集物种的分布， 减轻蛋白质聚集的分子机制的活动可能会受到这种侧翼序列的影响。最近的一项研究利用小鼠神经母细胞瘤细胞系(2)的粗提物，利用新开发的具有荧光检测能力的分析性超速离心机鉴定了寡聚体的存在。这项技术将与FRAP(光漂白后荧光恢复，使用共聚焦显微镜进行)结合使用，以探索含有多Q的蛋白质在体内和线虫粗提物中的聚集状态。将探索蛋白质侧翼序列对寡聚体相对分布的影响，以及它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，D。 被用来发现两个研究得很好的伴侣(Hsp104和Hsp70)如何使用同一套荧光技术来减轻中间聚集态的影响。当首席研究员在宾夕法尼亚大学HHMI研究员南希·博尼尼博士的实验室休假时，关键试剂和飞行路线正在开发中。博尼尼实验室在研究几种含有多聚Q的蛋白质在果蝇发育和衰老中的作用方面拥有专业知识(3-8)。有关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克里斯托弗·林克博士的实验室进行。他在使用这种动物模型研究蛋白质聚集性疾病方面拥有专业知识(9-14)。这项关键工作的成果将被带回哈弗福德，并将提供重要的 本科生在他们的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员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，这将允许共享资源，并在现有研究计划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，该奖项还将对未来几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种人类疾病涉及多Q驱动的聚集，已证明隔离成包涵体或核包涵体的途径很复杂，涉及有毒寡聚体的积累。最近开发了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(使用共焦显微镜，并使用分析超速离心对分子种群进行表征)来探测多Q聚集，使用黑腹葡萄球菌和线虫模型系统。将使用伴侣来探索缓解潜在有毒中间种群的能力，伴侣是一种蛋白质，其功能是防止或减少蛋白质聚集。

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.