Peroxisome biogenesis disorders (PBDs)

NCATS的ADST实验室与我们的合作者合作，并在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，使用基于显微镜的高含量自动成像分析方法，进行了针对人类全基因组的20K基因siRNA文库的筛选。通过PMP70抗体的免疫荧光(IF)染色，进一步评估了其敲除促进了过氧化体组装的活性基因，从而确定了几个新的候选基因。基因本体论分析表明，一些活性基因与蛋白质去动力过程密切相关，蛋白质去动力过程在调节CRL活性方面起着关键作用。根据过氧化氢酶和PMP70共同染色的结果，敲除患者成纤维细胞中的这些基因大大增加了过氧化物体的数量，并改善了功能。此外，通过Western blotting分析，过氧体AGPS蛋白的加工率增加。有趣的是，PEX6，它与PEX1形成一个六角体，并促进过氧化体的导入，被证明在siRNA转染后上调；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.