Peroxisome biogenesis disorders (PBDs)

在我们的合作者以及全球过氧化物酶体疾病基金会（GFPD）和Wynne Mateffy基金会的博士后支持下，NCATS的ADST实验室使用了一种高含量的基于自动显微镜的成像检测方法，对针对人类全基因组的20K基因siRNA文库进行了筛选。通过对过氧化物酶体膜蛋白PMP70抗体的免疫荧光（IF）染色，进一步评估敲低促进过氧化物酶体组装的活性基因，从而鉴定出几个新的候选基因。基因本体分析显示，多个活性基因与cullin-RING泛素连接酶（ubiquitin ligase， CRL）活性调控的蛋白去eddyation过程密切相关。通过过氧化氢酶和PMP70共染色测定，在患者成纤维细胞中敲除这些基因大大增加了功能改善的过氧化物酶体的数量。此外，从Western blotting分析判断，过氧化物酶体AGPS蛋白的加工百分比升高。有趣的是，与PEX1形成六聚体并促进过氧化物酶体输入的PEX6在siRNA转染后被上调；而其他PEX蛋白保持不变。

In conjunction with our collaborators and postdoctoral support by the Global Foundation for Peroxisome Disorders (GFPD) and the Wynne Mateffy Foundation the ADST laboratory of NCATS has used a high content automated microscopy-based imaging assay to conduct the screening of a 20K gene siRNA library targeting the human whole genome. Active genes whose knocking-down promoted peroxisome assembly were further evaluated in follow-up testing by immunofluorescence (IF) staining with antibodies to peroxisomal membrane protein PMP70 leading to the identification of several novel candidate genes. Gene ontology analysis revealed that several active genes were closely associated with the protein deneddylation process which plays critical roles in regulating cullin-RING ubiquitin ligase (CRL) activity. Knocking-down these genes in patient fibroblasts largely increased the number of peroxisomes with improved function as determined by catalase and PMP70 co-staining. In addition, the processing percentage of peroxisomal AGPS proteins was elevated as judged from Western blotting analysis. Intriguingly, PEX6, which forms a hexamer with PEX1 and facilitates peroxisomal import, was shown to be upregulated upon the siRNA transfection; whereas the other PEX proteins remain unchanged.