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.

聚谷氨酰胺疾病是由含有膨胀谷氨酰胺（Q）的蛋白质的神经元表达引起的。神经元功能障碍伴随着核内夹杂物中Polyq膨胀蛋白的积累。我们提出了几项关于Polyq蛋白积累的假设的测试，因为它们被泛素 - 蛋白酶体系统（UPS）降解，并且实际上可能会抑制UPS。我们已经在各种表达载体中放置了正常和谷氨酰胺扩展的区域，从泛素7（UB）和二氢叶酸还原酶（DHFR）或绿色荧光蛋白（GFP）之间的ataxin-7扩展。现在，我们将确定polyq长度是否影响所得蛋白质的泛素化的速率和/或程度。它对等肽酶的敏感性及其被26S蛋白酶体降解的速率。我们还将确定含有扩展的PolyQ块蛋白的降解是否导致UPS抑制。将解决这个问题：通过在大规模转染神经元细胞系和原发性小脑神经元的微观分析后，通过体外生化实验，通过生化分析。 Reggamma是一种在大脑中高度表达的核蛋白酶体成分，可抑制谷氨酰胺后蛋白酶体裂解。与吉利安·贝茨（Gillian Bates）合作，我们将雷格加马敲除小鼠越过了亨廷顿（Huntingtin）表达谷氨酰胺膨胀外显子1的R6/2小鼠。我们将确定是否没有雷格马延迟或改善PolyQ病理，并将测定所得小鼠各个大脑区域提取物的蛋白酶体活性。而野生型雷格马抑制了多谷氨酰胺降解，而最近分离的雷格玛变异体在体外会大大加速polyq的破坏。这一发现表明了多谷氨酰胺疾病的可能疗法。使用上述UB-SCA7-DHFR/GFP向量，我们将确定PolyQ毒性是否受到抑制，并在表达突变体蛋白酶体激活剂的培养细胞或原发性神经元中保留的泛素 - 蛋白酶体系统。获得这样的结果将证明对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的搜索是合理的。