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首页> 外文期刊>The Open Neuroendocrinology Journal >Misfolding of Mutated Vasopressin Causes ER-Retention and Activation of ER-Stress Markers in Neuro-2a Cells
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Misfolding of Mutated Vasopressin Causes ER-Retention and Activation of ER-Stress Markers in Neuro-2a Cells

机译：加压素突变的错误折叠会导致ER保留并激活Neuro-2a细胞中的ER应力标记。

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Arginine-vasopressin (AVP) is a peptide hormone normally secreted from neuroendocrine cells via theregulated secretory pathway. In Familial Neurohypophyseal Diabetes Insipidus (FNDI), an autosomal dominant form ofcentral diabetes insipidus, mutations of pro-vasopressin appear to accumulate in the endoplasmic reticulum (ER) causing alack of biologically active AVP in the blood. To investigate the effect of pro-vasopressin mutations regarding intracellularfunctions of protein targeting and secretion, we created two FNDI-associated amino acid substitution mutants, e.g., G14R,and G17V in frame with green fluorescent protein (GFP) and pro-vasopressin (VP) in frame with red fluorescent protein(VP-RFP). Fluorescence microscopy of Neuro-2a cells expressing these constructs revealed co-localization of VP-GFPand VP-RFP to punctate granules along the length and accumulating at the tips of neurites, characteristic of regulatedsecretory granules. In contrast, the two FNDI-associated amino acid substitution mutants, e.g., G14R-GFP, and G17VGFP,were localized to a perinuclear region of the Neuro-2a cells characteristic of the endoplasmic reticulum. Coexpressionof these mutants with VP-RFP showed VP-RFP was retained in the ER, co-localized with the mutantssuggesting the formation of heterodimers as found in FNDI. Stimulated secretion experiments indicated that VP-GFP wassecreted in an inducible manner whereas, G14R-GFP and G17V-GFP were retained to nearly 100% within the cells.Analysis by western blotting and semi-quantitative RT-PCR indicated an increased protein and mRNA expression for anER resident molecular chaperone, BiP. Further analysis of ER-storage disease-associated proteins such as caspase 12 andCHOP showed an increase in these as well. The results suggest that G14R-GFP and G17V-GFP are retained in the ER ofNeuro-2a cells, resulting in up-regulation of the molecular chaperone BiP, and activation of the ER-storage diseaseassociatedcaspase cascade system.
机译：精氨酸加压素（AVP）是一种通常通过调节分泌途径从神经内分泌细胞分泌的肽激素。在家族性中枢性尿崩症的常染色体显性形式的家族性神经下垂体尿崩症（FNDI）中，前加压素的突变似乎在内质网（ER）中积累，导致血液中缺乏生物活性AVP。为了研究促血管加压素突变对蛋白质靶向和分泌的细胞内功能的影响，我们在框架中创建了两个与FNDI相关的氨基酸替代突变体，例如G14R和G17V，并带有绿色荧光蛋白（GFP）和促血管加压素（VP）与红色荧光蛋白（VP-RFP）一致。表达这些构建体的Neuro-2a细胞的荧光显微镜显示VP-GFP和VP-RFP共同定位在点状颗粒上，并沿神经突的尖端积累，这是分泌型调节性颗粒的特征。相反，两个与FNDI相关的氨基酸取代突变体，例如G14R-GFP和G17VGFP，定位于内质网的Neuro-2a细胞的核周区域。这些突变体与VP-RFP的共表达表明VP-RFP被保留在ER中，与突变体共定位，这表明在FNDI中发现了异二聚体的形成。刺激的分泌实验表明VP-GFP以可诱导的方式分泌，而G14R-GFP和G17V-GFP保留在细胞内接近100％.Western印迹和半定量RT-PCR分析表明蛋白质和mRNA表达增加用于常驻分子伴侣蛋白BiP。对与ER储存疾病相关的蛋白（例如caspase 12和CHOP）的进一步分析表明，这些蛋白也有所增加。结果表明，G14R-GFP和G17V-GFP保留在Neuro-2a细胞的ER中，导致分子伴侣BiP上调，并激活了ER储存疾病相关的半胱天冬酶级联系统。

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