Proteasomes, PODs and Polyglutamine Diseases
蛋白酶体、POD 和多聚谷氨酰胺疾病
基本信息
- 批准号:6898835
- 负责人:
- 金额:$ 34.57万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2004
- 资助国家:美国
- 起止时间:2004-06-01 至 2009-04-30
- 项目状态:已结题
- 来源:
- 关键词:cell linecellular pathologycerebellumchimeric proteinscytotoxicitydisease /disorder etiologyfluorescence microscopygenetically modified animalsglutaminehomopeptidelaboratory mousemolecular pathologymutantneural degenerationneuronspeptidasesproteasomeprotein degradationprotein structure functionsingle cell analysistransfectiontransfection /expression vectorubiquitin
项目摘要
Polyglutamine diseases result from neuronal expression of proteins containing expanded glutamine (Q) tracts. Neuron dysfunction is accompanied by the accumulation of the polyQ expanded protein often in intranuclear inclusions. We propose several tests of the hypothesis that the polyQ proteins accumulate because they are poorly degraded by the ubiquitin-proteasome system (UPS) and may in fact inhibit the UPS. We have placed normal and glutamine expanded regions from ataxin-7 between ubiquitin (Ub) and dihydrofolate reductase (DHFR) or green fluorescent protein (GFP) in various expression vectors. We will now determine whether polyQ length affects the rate and/or extent of ubiquitylation of the resulting protein,
its susceptibility to isopeptidases and the rate at which it is degraded by the 26S proteasome. We will also determine whether degradation of proteins containing expanded polyQ tracts leads to inhibition of the UPS. This issue will be addressed: by in vitro biochemical experiments, by biochemical analysis after large scale transfections of neuronal cell lines and by microscopic analysis of primary cerebellar neurons. REGgamma is a nuclear proteasome component highly expressed in brain that suppresses proteasomal cleavage after glutamine. In collaboration with Gillian Bates, we have crossed REGgamma knock-out mice to R6/2 mice that express glutamine-expanded exon 1 of huntingtin. We will determine whether the absence of REGgamma delays or ameliorates polyQ pathology and will assay for proteasome activity in extracts from various brain regions in the resulting mice. Whereas wild-type REGgamma suppresses polyglutamine degradation, a recently isolated REGgamma variant dramatically speeds polyQ destruction by the proteasome in vitro. This discovery suggests a possible therapy for polyglutamine diseases. Using the Ub-SCA7-DHFR/GFP vectors described above, we
will determine whether polyQ toxicity is suppressed and the ubiquitin-proteasome system spared in culture cells or primary neurons expressing the mutant proteasome activator. Obtaining such a result would justify the search for therapeutic agents able to convert wild-type proteasome activator to the mutant form.
多聚谷氨酰胺疾病是由含有扩展的谷氨酰胺 (Q) 束的蛋白质的神经元表达引起的。神经元功能障碍通常伴随着聚 Q 扩展蛋白在核内包涵体中的积累。我们对以下假设进行了多项测试:polyQ 蛋白之所以会积累,是因为它们很难被泛素蛋白酶体系统 (UPS) 降解,并且实际上可能会抑制 UPS。我们已将 ataxin-7 的正常和谷氨酰胺扩展区域放置在各种表达载体中的泛素 (Ub) 和二氢叶酸还原酶 (DHFR) 或绿色荧光蛋白 (GFP) 之间。我们现在将确定 PolyQ 长度是否影响所得蛋白质的泛素化速率和/或程度,
它对异肽酶的敏感性以及它被 26S 蛋白酶体降解的速率。我们还将确定含有扩展的 PolyQ 束的蛋白质的降解是否会导致 UPS 的抑制。该问题将通过体外生化实验、神经元细胞系大规模转染后的生化分析以及原代小脑神经元的显微镜分析来解决。 REGgamma 是一种在大脑中高表达的核蛋白酶体成分,可抑制谷氨酰胺后的蛋白酶体裂解。我们与 Gillian Bates 合作,将 REGgamma 敲除小鼠与表达亨廷顿蛋白谷氨酰胺扩展外显子 1 的 R6/2 小鼠杂交。我们将确定 REGgamma 的缺失是否会延迟或改善 PolyQ 病理学,并将测定所得小鼠各脑区提取物中的蛋白酶体活性。野生型 REGgamma 抑制多聚谷氨酰胺降解,而最近分离的 REGgamma 变体在体外可显着加速蛋白酶体对多聚 Q 的破坏。这一发现提出了一种治疗多聚谷氨酰胺疾病的可能方法。使用上述 Ub-SCA7-DHFR/GFP 载体,我们
将确定在表达突变蛋白酶体激活剂的培养细胞或原代神经元中,polyQ 毒性是否受到抑制,泛素蛋白酶体系统是否受到保护。获得这样的结果将证明寻找能够将野生型蛋白酶体激活剂转化为突变形式的治疗剂是合理的。
项目成果
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MARTIN C RECHSTEINER其他文献
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{{ truncateString('MARTIN C RECHSTEINER', 18)}}的其他基金
Proteasomes, PODs and Polyglutamine Diseases
蛋白酶体、POD 和多聚谷氨酰胺疾病
- 批准号:
6826106 - 财政年份:2004
- 资助金额:
$ 34.57万 - 项目类别:
Proteasomes, PODs and Polyglutamine Diseases
蛋白酶体、POD 和多聚谷氨酰胺疾病
- 批准号:
7056150 - 财政年份:2004
- 资助金额:
$ 34.57万 - 项目类别:
Proteasomes, PODs and Polyglutamine Diseases
蛋白酶体、POD 和多聚谷氨酰胺疾病
- 批准号:
7216180 - 财政年份:2004
- 资助金额:
$ 34.57万 - 项目类别:
Proteasomes, PODs and Polyglutamine Diseases
蛋白酶体、POD 和多聚谷氨酰胺疾病
- 批准号:
7391680 - 财政年份:2004
- 资助金额:
$ 34.57万 - 项目类别:
PROPERTIES OF AN ATP-UBIQUITIN-DEPENDENT PROTEASE
ATP 泛素依赖性蛋白酶的特性
- 批准号:
2178633 - 财政年份:1986
- 资助金额:
$ 34.57万 - 项目类别:
PROPERTIES OF AN ATP/UBIQUITIN-DEPENDENT PROTEASE
ATP/泛素依赖性蛋白酶的特性
- 批准号:
6329672 - 财政年份:1986
- 资助金额:
$ 34.57万 - 项目类别:
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