Proteasomes, PODs and Polyglutamine Diseases

多谷氨酰胺疾病是由于含有扩大的谷氨酰胺(Q)束的蛋白质的神经元表达。神经元功能障碍通常伴随着聚q扩增蛋白在核内包涵体中的积累。我们提出了几种测试假设，polyQ蛋白积累是因为它们被泛素-蛋白酶体系统（UPS）降解不良，实际上可能抑制UPS。我们将ataxin-7的正常区和谷氨酰胺扩增区置于泛素（Ub）和二氢叶酸还原酶（DHFR）或绿色荧光蛋白（GFP）之间。我们现在将确定多聚q长度是否会影响产生的蛋白质泛素化的速率和/或程度，

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.