Department of Dermatology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan; Biochemistry Department, National Defense Medical Center, Taipei, Taiwan.
Br J Dermatol. 2014 Aug;171(2):356-62. doi: 10.1111/bjd.13051. Epub 2014 Jul 26.
Loss of the DNA-binding activity of a transcription factor is detrimental to its function in responsive gene regulation. We diagnosed a Taiwanese family with nail-patella syndrome (NPS) whose members inherited the mutated LMX1b transcription factor with no DNA-binding homeodomain. The loss-of-function variants cause haploinsufficiency of LMX1b, leading to the clinical manifestation of NPS. The underlying molecular mechanism is unclear.
To test whether the recurrent pathogenic truncated LMX1b-R198X reported in our patients might be a functional protein. Its biochemical properties were explored.
The luciferase reporter driven by the human interleukin (IL)-6 gene promoter was assayed to measure the transcriptional activity of LMX1b. The nuclear localization of different enhanced green fluorescent protein-tagged LMX1b proteins was observed using fluorescence microscopy. Western blotting was employed to evaluate the expression of various transfected LMX1b constructs.
LMX1b-R198X enhanced the IL-6 promoter activity activated by the wild-type LMX1b and diminished the promoter activity induced by phorbol 12-myristate 13-acetate. LMX1b-R198X carried out its effect differentially in the expression of various human genes. The nuclear localization of the wild-type LMX1b was disrupted by the C-terminus truncation. The protein stability exhibited by LMX1b-R198X appears to be much higher than that of the wild-type protein.
We demonstrated that loss of function might not be the only way for mutated LMX1b to cause haploinsufficiency as the main pathogenic mechanism for NPS. LMX1b-R198X has less nuclear localization and higher stability than the wild-type protein; consequently, it might function as a competitor to sequester other effectors by protein-protein interaction to interfere with downstream transcriptional events.
转录因子的 DNA 结合活性丧失对其在响应基因调控中的功能是有害的。我们诊断了一个台湾的指甲髌骨综合征（NPS）家族，其成员遗传了突变的 LMX1b 转录因子，没有 DNA 结合同源结构域。功能丧失变异导致 LMX1b 的单倍不足，导致 NPS 的临床表现。其潜在的分子机制尚不清楚。
测试我们患者中报告的复发性致病性截断 LMX1b-R198X 是否可能是一种功能性蛋白质。探讨其生化特性。
通过人白细胞介素（IL）-6 基因启动子驱动的荧光素酶报告基因检测来测量 LMX1b 的转录活性。使用荧光显微镜观察不同增强型绿色荧光蛋白标记的 LMX1b 蛋白的核定位。采用 Western blot 评估各种转染的 LMX1b 构建体的表达。
LMX1b-R198X 增强了野生型 LMX1b 激活的 IL-6 启动子活性，并减弱了佛波醇 12-肉豆蔻酸 13-乙酸诱导的启动子活性。LMX1b-R198X 对不同的人基因的表达产生不同的作用。野生型 LMX1b 的核定位被 C 端截断破坏。LMX1b-R198X 的蛋白稳定性似乎明显高于野生型蛋白。
我们证明了功能丧失可能不是突变 LMX1b 导致单倍不足的唯一方式，因为 NPS 的主要发病机制。与野生型蛋白相比，LMX1b-R198X 的核定位较少，稳定性更高；因此，它可能通过蛋白-蛋白相互作用作为一种竞争性物质来隔离其他效应物，从而干扰下游转录事件。
