Studies on the Regulatory Mechanism of Neuronal Death: Isolation of Novel Factors and Title of Preparation of Model of Neurodegenerative Disease.

蛋白质二硫化物异构酶(PDI)存在于内质网(ER)中，协助未折叠分泌蛋白的成熟和运输。PDI有两个结构域，它们作为独立的活性位点发挥作用，与小的氧化还原活性蛋白硫氧还蛋白同源。我们证明了PDI是S亚硝化的，这是一个将一氧化氮(NO)基团转移到关键的半胱氨酸硫醇以影响蛋白质功能的反应。NO诱导的PDI的S亚硝化抑制了其酶活性，导致多泛素化蛋白的积累，并激活了未折叠的蛋白反应。此外，我们在表现为散发性帕金森氏症或阿尔茨海默病的大脑中显示，PDI是S亚硝化的。因此，PDI可以防止与内质网应激和蛋白质错误折叠相关的神经毒性，但没有通过PDI的S亚硝化来阻止这种保护作用。在泛素蛋白连接酶Parkin缺陷小鼠中，Parkin相关的内皮素受体样受体(PAEL-R)上调导致多巴胺能神经元死亡。这些动物的细胞死亡分别在ER伴侣ORP150(150 kDa氧气调节蛋白)上调或缺乏的小鼠中得到保护或增加。我们杂交Pael-R上调和Parkin基因敲除小鼠。在小鼠中，我们观察到大脑中的内质网应激，黑质和蓝斑中儿茶酚胺能神经元的选择性死亡，以及线粒体复合体I的缺乏。这些数据表明，在与帕金森病相关的环境中，内质网和多巴胺相关的应激是导致多巴胺能神经元活力下降的主要因素。

Protein-disulphide isomerase (PDI), which exists in endoplasmic reticulum (ER), and assists in the maturation and transport of unfolded secretory proteins. PDI has two domains that function as independent active sites with homology to the small, redox-active protein thioredoxin. We demonstrated that PDI was S-nitrosylated, a reaction transferring a nitric oxide (NO) group to a critical cysteine thiol to affect protein function. NO-induced S-nitrosylation of PDI inhibited its enzymatic activity, leads to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. Furthermore, we showed in brains manifesting sporadic Parkinson's or Alzheimer's disease, that PDI is S-nitrosylated. Thus, PDI prevents neurotoxicity associated with ER stress and protein misfolding, but NO blocks this protective effect in neurodegenerative disorders through the S-nitrosylation of PDI.Upregulation of Parkin associated endothelin-receptor like receptor (Pael-R) in the ubiquitin-protein ligase Parkin deficient mice leads to death of dopaminergic neurons. The cell death in these animals was protected or aggaravated in upregulated or deficient mice of the ER chaperone ORP150 (150 kDa oxygen-regulated protein), respectively. We interbreed Pael-R-upregulated and Parkin knockout mice. In the mice, we observed ER-stress in the brain, selective cell death of catecholaminergic neurons in the substantia nigra and the locus ceruleus, and deficiency of mitochondria complex I. These data suggest a model in which ER-and dopamine-related stress are major contributors to decreased viability of dopaminergic neurons in a setting relevant to Parkinson's disease.