Proteasomes, PODs and Polyglutamine Diseases

蛋白酶体、POD 和多聚谷氨酰胺疾病

• 批准号: 6898835
• 负责人: MARTIN C RECHSTEINER
• 金额: $ 34.57万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 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: https://reporter.nih.gov/project-details/6898835
• 关键词: cell line; cellular pathology; cerebellum; chimeric proteins; cytotoxicity; disease /disorder etiology; fluorescence microscopy; genetically modified animals; glutamine; homopeptide; laboratory mouse; molecular pathology; mutant; neural degeneration; neurons; peptidases; proteasome; protein degradation; protein structure function; single cell analysis; transfection; transfection /expression vector; ubiquitin

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.

多发性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。伴随着神经元功能障碍的是多聚Q扩展蛋白的积聚，通常在核内包涵体中。我们对这一假设提出了几个测试，即PolyQ蛋白之所以积累是因为它们被泛素-蛋白酶体系统(UPS)降解得很差，实际上可能会抑制UPS。我们已经在不同的表达载体中将ATAXIN-7的正常和谷氨酰胺扩展区放置在泛素(Ub)和二氢叶酸还原酶(DHFR)或绿色荧光蛋白(GFP)之间。我们现在将确定多聚Q长度是否影响所产生的蛋白质泛素化的速度和/或程度， 它对异肽酶的敏感性以及它被26S蛋白酶体降解的速度。我们还将确定含有扩展多聚Q链的蛋白质的降解是否会导致UPS的抑制。这一问题将被解决：通过体外生化实验，通过大规模转染神经细胞系后的生化分析，以及通过对原代小脑神经元的显微分析。REGGamma是一种在大脑中高度表达的核蛋白酶体成分，它能抑制谷氨酰胺后蛋白酶体的切割。在与Gillian Bates的合作中，我们将REGGamma基因敲除小鼠与表达谷氨酰胺扩展的亨廷顿蛋白外显子1的R6/2小鼠进行了杂交。我们将确定REGGamma的缺失是否延缓或改善了多Q病理，并将在结果小鼠的不同脑区提取液中检测蛋白酶体活性。虽然野生型REGGamma抑制多谷氨酰胺的降解，但最近分离的REGGamma变异体在体外显著加快了蛋白酶体对多聚Q的破坏。这一发现为治疗聚谷氨酰胺疾病提供了一种可能的治疗方法。使用上述Ub-SCA7-DHFR/GFP载体，我们 将确定多聚Q毒性是否被抑制，泛素-蛋白酶体系统是否在培养细胞或表达突变蛋白酶体激活剂的原代神经元中幸免。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。