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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：
7216180
负责人：
MARTIN C RECHSTEINER
金额：
$ 32.78万
依托单位：
UNIVERSITY OF UTAH
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：
https://reporter.nih.gov/project-details/7216180
关键词：
26S proteasome Address Affect Biochemical Biological Biological Assay Brain Brain region Cell Line Cell Survival Cells Chimeric Proteins Collaborations Corpus striatum structure Cultured Cells Degradation Pathway Dihydrofolate Reductase Disease Embryo Exons Fluorescence Functional disorder Glutamine Goals Green Fluorescent Proteins Huntington Disease Hybrids In Vitro Ki antigen Knockout Mice Length Mammalian Cell Measurement Measures Mediating Methionine Microscopic Mus N-terminal Neurons Nuclear Nuclear Inclusion Oryctolagus cuniculus Pathology Pathway interactions Predisposition Proteins Proteolysis Rate Rattus Recombinant Proteins Recombinants Relative (related person)Reticulocytes SCA7 protein Source Speed Symptoms System Testing Therapeutic Agents Toxic effect Transfection Ubiquitin Variant expression vector human Huntingtin protein hybrid protein isopeptidase multicatalytic endopeptidase complex mutant polyglutamine protein degradation protein expression research study vector

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.

多聚谷氨酰胺疾病由含有扩展的谷氨酰胺（Q）束的蛋白质的神经元表达引起。神经元功能障碍伴随着polyQ扩展蛋白的积累，通常在核内包涵体中。我们提出了几个测试的假设，聚Q蛋白的积累，因为他们是很差的泛素-蛋白酶体系统（UPS）降解，实际上可能会抑制UPS。我们已经在各种表达载体中将来自共济失调蛋白-7的正常和谷氨酰胺扩展区域置于泛素（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和它被26 S蛋白酶体降解的速率。我们还将确定含有扩增的polyQ片段的蛋白质的降解是否导致UPS的抑制。这一问题将在以下方面得到解决：通过体外生物化学实验、通过神经元细胞系的大规模转染后的生物化学分析和通过原代小脑神经元的显微镜分析。REGgamma是在脑中高度表达的核蛋白酶体组分，其抑制谷氨酰胺后的蛋白酶体切割。与Gillian Bates合作，我们将REG γ基因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的缺失是否延迟或改善polyQ病理学，并将测定所得小鼠不同脑区提取物中的蛋白酶体活性。尽管野生型REGgamma抑制聚谷氨酰胺降解，但最近分离的REGgamma变体在体外显著加速蛋白酶体对polyQ的破坏。这一发现为多聚谷氨酰胺疾病提供了一种可能的治疗方法。使用上述Ub-SCA 7-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毒性是否被抑制以及泛素-蛋白酶体系统是否幸免。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体形式的治疗剂是合理的。