Huntingtin interacting proteins are genetic modifiers of neurodegeneration.
Huntingtin interacting proteins are genetic modifiers of neurodegeneration.
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DOI：
10.1371/journal.pgen.0030082
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发表时间：
2007-05-11
期刊：
影响因子：
4.5
通讯作者：
Hughes RE
中科院分区：
文献类型：
--
作者：
Kaltenbach LS;Romero E;Becklin RR;Chettier R;Bell R;Phansalkar A;Strand A;Torcassi C;Savage J;Hurlburt A;Cha GH;Ukani L;Chepanoske CL;Zhen Y;Sahasrabudhe S;Olson J;Kurschner C;Ellerby LM;Peltier JM;Botas J;Hughes RE
亨廷顿氏病（HD）是由亨廷顿蛋白（Htt）中的多聚谷氨酰胺束扩张引起的致命性神经退行性疾病。HD中的神经元毒性被认为至少部分是涉及突变型Htt的蛋白质相互作用的结果。因此，我们假设HD神经变性的遗传修饰剂应该在Htt蛋白相互作用物中富集。为了测试这个想法，我们确定了一个全面的一套Htt相互作用使用两种互补的方法：高通量酵母双杂交筛选和亲和下拉，然后通过质谱。这项工作导致了234个高置信度的Htt相关蛋白的鉴定，其中104个是用酵母方法发现的，130个是用下拉唐斯发现的。然后，我们测试了一组任意的60个基因编码相互作用的蛋白质的能力，表现为遗传修饰剂的神经退行性疾病的果蝇模型的HD。这种高含量的验证试验表明，测试的60个直系同源物中有27个是高置信度的遗传修饰剂，因为观察到多于一个等位基因的修饰。在相互作用者中看到的遗传修饰剂的45%命中率比通常在无偏遗传筛选中观察到的1%-4%高一个数量级。使用酵母双杂交和下拉/质谱方法发现的蛋白质中类似地代表了遗传修饰剂，支持这些互补技术在鉴定生物相关蛋白质中同样有用的观点。被证实为神经变性表型修饰剂的相互作用蛋白代表了多种生物学功能，包括突触传递、细胞骨架组织、信号转导和转录。在这些修饰剂中，有17种神经变性的功能丧失抑制剂，它们可以被认为是治疗干预的潜在靶点。最后，我们发现，7个相互作用的蛋白质，从11个测试能够与全长Htt从小鼠脑共免疫沉淀。这些研究表明，高通量筛选蛋白质相互作用结合在模式生物中的遗传验证是一种强有力的方法，用于确定新的候选修饰剂的聚谷氨酰胺毒性。亨廷顿氏病（HD）是一种致命的遗传性神经退行性疾病，其典型地开始于中年并且进展为严重不受控制的运动和认知功能障碍的症状。HD是一致致命的，死亡发生在症状发作后10至15年。目前尚无有效治疗HD的方法。亨廷顿舞蹈症的基因突变导致一种叫做亨廷顿蛋白（Htt）的蛋白质含有一段异常长的谷氨酰胺。这种延长的谷氨酰胺跨度改变了Htt蛋白的形状，这可能导致它以异常的方式与其他细胞蛋白相互作用。在这项研究中，我们已经确定了大量的新的蛋白质，结合正常和突变形式的Htt蛋白。为了确定这些相互作用的蛋白质在HD中的潜在作用，我们表明改变其中许多蛋白质的表达可以调节突变型Htt对苍蝇神经元的病理影响，这些神经元在表达突变型Htt时会恶化。鉴定与Htt结合并调节其病理活性的细胞蛋白可能有助于发现HD的有效治疗方法。
Huntington's disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%–4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to co-immunoprecipitate with full-length Htt from mouse brain. These studies demonstrate that high-throughput screening for protein interactions combined with genetic validation in a model organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity. Huntington's Disease (HD) is a fatal inherited neurodegenerative disease, which typically begins in middle age and progresses with symptoms of severe uncontrolled movements and cognitive dysfunction. HD is uniformly fatal with death occurring ten to 15 years after onset of symptoms. There is currently no effective treatment for HD. The genetic mutation underlying HD causes a protein called huntingtin (Htt) to contain an abnormally long tract of the amino acid glutamine. This extended span of glutamines changes the shape of the Htt protein, which can cause it to interact in abnormal ways with other cellular proteins. In this study, we have identified a large number of new proteins that bind to normal and mutant forms of the Htt protein. To establish a potential role for these interacting proteins in HD, we show that changing the expression of many of these proteins can modulate the pathological effects of mutant Htt on fly neurons that deteriorate when they express mutant Htt. Identifying cellular proteins that bind to Htt and modulate its pathological activity may facilitate the discovery of an effective treatment for HD.
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