Footz Tim, Idrees Faisal, Acharya Moulinath, Kozlowski Kathy, Walter Michael A
Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada.
Invest Ophthalmol Vis Sci. 2009 Jun;50(6):2599-606. doi: 10.1167/iovs.08-3251. Epub 2009 Feb 14.
To assess the effects of previously uncharacterized PITX2 missense mutations found in patients with Axenfeld-Rieger syndrome and to determine the functional roles of the C-terminal region of PITX2.
Recombinant PITX2 proteins were analyzed with the use of cellular immunofluorescence, electrophoretic mobility shift, reporter transactivation, and protein half-life assays in human trabecular meshwork cells.
Two homeobox mutations, R43W and R90C, resulted in severely reduced DNA-binding and transcriptional activation despite normal nuclear localization. L105V, located C-terminal to the homeodomain, resulted in normal localization, reporter gene transactivation, and protein half-life, but with an altered mobility shift pattern of protein-DNA complexes. N108T, also located C-terminal to the homeodomain, resulted in an altered mobility shift pattern and with slightly increased reporter transactivation and shortened protein half-life. The PITX2 C-terminal region contains at least three domains, each with distinct modulating effects on reporter transactivation.
PITX2 homeobox mutations predictably resulted in decreased function of the protein. However, the two C-terminal mutations exhibited only subtle defects on PITX2 transactivation and protein-DNA binding, suggesting that ocular development is sensitive to even slight alterations of PITX2 function. The C-terminal mutations L105V and N108T lie in a domain that inhibits PITX2 transcriptional activation. These two mutations produce electrophoretic mobility shift assay patterns representing altered protein-DNA interactions that may be important for accurate target gene selection. Additionally, N108T resulted in a less stable PITX2 mutant protein with elevated activity that may result in stochastic dysregulation during critical stages of development. Together, the results clearly indicate that stringent control of PITX2 is required for normal ocular development and function.
评估在Axenfeld-Rieger综合征患者中发现的此前未被描述的PITX2错义突变的影响，并确定PITX2 C末端区域的功能作用。
在人小梁网细胞中，利用细胞免疫荧光、电泳迁移率变动分析、报告基因反式激活及蛋白质半衰期测定法对重组PITX2蛋白进行分析。
两个同源异型框突变R43W和R90C，尽管核定位正常，但导致DNA结合和转录激活严重降低。位于同源异型结构域C末端的L105V，导致定位正常、报告基因反式激活及蛋白质半衰期正常，但蛋白质-DNA复合物的迁移率变动模式改变。同样位于同源异型结构域C末端的N108T，导致迁移率变动模式改变，报告基因反式激活略有增加，蛋白质半衰期缩短。PITX2 C末端区域至少包含三个结构域，每个结构域对报告基因反式激活具有不同的调节作用。
PITX2同源异型框突变可预见地导致该蛋白功能降低。然而，两个C末端突变仅对PITX2反式激活和蛋白质-DNA结合表现出细微缺陷，表明眼部发育对PITX2功能的即使是轻微改变也很敏感。C末端突变L105V和N108T位于一个抑制PITX2转录激活的结构域中。这两个突变产生的电泳迁移率变动分析模式代表了改变的蛋白质-DNA相互作用，这可能对准确选择靶基因很重要。此外，N108T导致PITX2突变蛋白稳定性降低且活性升高，这可能在发育关键阶段导致随机失调。总之，结果清楚地表明，正常眼部发育和功能需要对PITX2进行严格调控。
