Liu Yuk-Chien, Singh Upinder
Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.
Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Int J Parasitol. 2014 Sep;44(10):729-35. doi: 10.1016/j.ijpara.2014.05.002. Epub 2014 Jun 11.
A plethora of information has been gained by sequencing the genome of the human parasite Entamoeba histolytica, however a lack of robust genetic tools hampers experimental elucidation of gene functions. We adapted the destabilization domain approach for modulation of protein levels in E. histolytica using the destabilization domains of FK506 binding protein (ddFKBP) and dihydrofolate reductase (ddDHFR), respectively. In our studies, the ddFKBP appears to be more tightly regulated than ddDHFR, with minimal detectable protein in trophozoites in the absence of the stabilizing compound. The on- and off-rate kinetics for ddFKBP were rapid, with stabilization and degradation within 3h of addition or removal of stabilizing compound, respectively. The kinetics for ddDHFR was different, with rapid stabilization (within 3h of stabilizing compound being added) but much slower degradation (protein not destabilized until 24h after compound removal). Furthermore, we demonstrated that for the ddFKBP, the standard stabilizing compound Shield-1 could be effectively replaced by two cheaper alternatives (rapamycin and FK506), indicating that the more cost-effective alternatives are viable options for use with E. histolytica. Thus, the destabilization domain approach represents a powerful method to study protein functions in E. histolytica and adds to the catalog of genetic tools that could be used to study this important human pathogen.
通过对人类寄生虫溶组织内阿米巴的基因组进行测序，我们已经获得了大量信息，然而，缺乏强大的遗传工具阻碍了对基因功能的实验性阐明。我们分别采用FK506结合蛋白（ddFKBP）和二氢叶酸还原酶（ddDHFR）的去稳定结构域，调整去稳定结构域方法以调节溶组织内阿米巴的蛋白质水平。在我们的研究中，ddFKBP似乎比ddDHFR受到更严格的调控，在没有稳定化合物的情况下，滋养体中可检测到的蛋白质极少。ddFKBP的结合和解离速率动力学很快，分别在添加或去除稳定化合物后3小时内实现稳定和降解。ddDHFR的动力学则不同，稳定速度很快（在添加稳定化合物后3小时内），但降解速度慢得多（直到去除化合物24小时后蛋白质才去稳定）。此外，我们证明，对于ddFKBP，标准稳定化合物Shield-1可以被两种更便宜的替代品（雷帕霉素和FK506）有效替代，这表明更具成本效益的替代品是用于溶组织内阿米巴的可行选择。因此，去稳定结构域方法是研究溶组织内阿米巴蛋白质功能的一种强大方法，并增加了可用于研究这种重要人类病原体的遗传工具目录。
