Investigation of the disease mechanism causing Ankyloblepharon-ectodermal defects-cleft lip/palate syndrome and Ectrodactyly-ectodermal dysplasia-clefting syndrome

p53蛋白家族由三个成员组成：p53、p63和p73。敲除小鼠的研究表明，p63参与了许多细胞过程，如上皮组织发育过程中的细胞命运承诺、细胞增殖、分化和组织稳态。在上皮细胞和表皮细胞转化过程中，p63缺失的多能干细胞被阻断在简单上皮细胞和层状表皮细胞之间，不能转化为基底表皮角质形成细胞。对p63-/-小鼠的分析使得人类p63基因的突变能够识别出至少五种人类综合征和两种非综合征性人类疾病，其特征是不同程度的1)肢体变形，2)唇腭裂和/或3)外胚层发育不良。两种最具特征的综合征，EEC和AEC综合征，分别与DNA结合域和SAM结构域的突变有关。最近我们已经证明AEC综合征是基于功能机制的增益。SAM结构域的突变导致p63不可逆聚集。令人惊讶的是，突变体的变性温度仍然明显高于体温。因此，疾病机制不能用简单的热力学不稳定性来解释。我们建议进一步研究其发病机制。我们将使用质谱法鉴定共聚集蛋白，测量SAM结构域突变体的折叠动力学，并表征它们与伴侣蛋白的相互作用。此外，我们将描述目前已知的EEC突变体，并将其在细胞分析中的残余DNA结合亲和力和转录活性程度与患者表型的严重程度相关联。

The p53 protein family consists of three members: p53, p63 and p73. Knock out mouse studies have revealed that p63 is involved in many cellular processes such as cell fate commitment during development, cell proliferation, differentiation and tissue homeostasis in epithelial tissues. During epithelial and epidermal commitment, pluripotent stem cells depleted of p63 are blocked between the simple epithelial and stratified epidermal fates, and cannot commit to basal epidermal keratinocytes. Analysis of the p63-/- mouse has enabled the identification of mutations in the human p63 gene to be responsible for at least five human syndromes and two non-syndromic human disorders that are characterized by varying degrees of 1) limb deformation, 2) cleft lip/palate and/or 3) ectodermal dysplasia. The two best characterized syndromes, EEC and AEC syndrome, are linked to mutations in the DNA binding domain and the SAM domain, respectively. Recently we have shown that the AEC syndrome is based on a gain of function mechanism. Mutations in the SAM domain cause irreversible aggregation of p63. Surprisingly, the denaturation temperature of the mutants is still significantly higher than the body temperature. The disease mechanism can, therefore, not be explained with a simple thermodynamic destabilization. We now propose to further investigate the disease mechanism. We will identify co-aggregating proteins using mass spectrometry, measure the folding kinetics of the SAM domain mutants and characterize their interaction with chaperones. In addition we will characterize currently known EEC mutants and correlate their degree of residual DNA binding affinity and transcriptional activity in cellular assays with the severity of the patient phenotypes.