Dystrophic epidermolysis bullosa: quantitative proteomics approach to molecular mechanisms

营养不良大疱性表皮松解症（DEB）是一种单基因皮肤疾病，其特征是胶原蛋白VII功能的丧失或紊乱。患者表现出表皮-真皮粘连减弱，皮肤起泡和随后的疤痕。到目前为止，编码VII型胶原蛋白的COL7A1基因已经发现了800多种不同的突变。观察到广泛的临床表型，这部分是由于各自突变的性质，其范围从孤立的指甲营养不良，到四肢局部起泡，到全身性起泡和致残性畸形，粘膜累及，皮肤鳞状细胞癌风险增加，以及因过早死亡而无法生长。在最严重的疾病形式中，由于零突变，胶原蛋白VII蛋白和锚定原纤维都缺失。在较温和的形式中，基于错义突变，如三螺旋结构域内的甘氨酸取代，胶原VII存在，但稳定性改变，导致锚定原纤维的功能改变。尽管DEB的基因型-表型相关性具有一定的剂量依赖性，表达部分功能的胶原VII的个体比完全缺乏胶原VII的个体受到的影响更轻微，但很明显，这并不严格适用于许多个体。这一观察结果清楚地表明，DEB的疾病表现不仅受VII型胶原功能丧失的支配，而且重要的是由次要机制决定。我们的目标是通过鉴定迄今为止未知的与疾病发病机制和表型变异有关的基因和蛋白质，来产生关于DEB潜在分子机制的新知识。我们利用细胞培养和小鼠模型研究皮肤成纤维细胞-角化细胞串扰、受干扰的信号转导和炎症在疾病进展中的作用。结合小鼠模型、细胞生物学和蛋白质生化方法，以及基于质谱的蛋白质组学、免疫组织化学和荧光技术，将产生全面的数据集，从而更好地了解DEB的临床表型。临床表型将与分子途径联系起来，从而为设计新的分子疗法奠定了重要的先决条件。我们的目标是对DEB中蛋白质动力学的系统级理解，并设想我们的方法将成为其他人类遗传（皮肤）疾病研究的典范。

Dystrophic epidermolysis bullosa (DEB) is a monogenic skin disorder characterized by loss or perturbation of collagen VII functions. Patients exhibit diminished epidermal-dermal adhesion, skin blistering and subsequent scarring. Up to now, more than 800 different mutations have been disclosed in the gene COL7A1 which encodes collagen VII. A broad range of clinical phenotypes is observed, which is partially due to the nature of respective mutations and which range from isolated nail dystrophy, through localized blistering in the extremities, to generalized blistering and mutilating deformities, mucosal involvement, increased risk of skin squamous cell carcinoma and failure to thrive with premature demise. In the most severe form of the disease, due to null mutations, both collagen VII protein and anchoring fibrils are absent. In milder forms, based on missense mutations, such as glycine substitutions within the triple helical domain, collagen VII is present, but has altered stability and leads to altered functionality of anchoring fibrils. Although genotype-phenotype correlations in DEB have a certain dose-dependence, with individuals expressing partially functional collagen VII being more mildly affected than individuals completely devoid of collagen VII, it has become evident that this does not strictly apply for many individuals. This observation clearly indicates that disease manifestation in DEB is not only governed by loss of collagen VII function but importantly is also determined by secondary mechanisms. We aim to generate new knowledge of underlying molecular mechanisms of DEB by identifying hitherto unknown genes and proteins involved in disease pathogenesis and phenotypic variability. We study skin fibroblast-keratinocyte crosstalk, perturbed signal transduction and the role of inflammation in disease progression using cell culture as well as mouse models. Combination of a mouse model, cell biological and protein biochemical approaches, together with mass spectrometry-based proteomics and immunohistochemistry and -fluorescence will generate comprehensive datasets that will allow a better understanding of clinical phenotypes in DEB. Clinical phenotypes will be linked to molecular pathways, thus establishing an important prerequisite for the design of novel molecular therapies. We aim at a systems-level understanding of protein dynamics in DEB and envision that our approach will be exemplary for the investigation of other human genetic (skin) diseases.