Biochemical and genetic analysis of mesd function

描述（由申请人提供）：低密度脂蛋白受体发挥多种细胞作用，从内吞作用和货物运输到亚细胞运输和细胞信号传导。尽管在功能上存在这种多样性，脂蛋白相关受体（lrp）拥有一个由富含半胱氨酸的常见重复簇、补体样重复序列（配体结合域）、EGF重复序列和YWTD组成的细胞外结构域，这些结构域形成β -螺旋桨结构。这些基序的数量和排列因受体而异。LRP家族成员的复杂结构和大量的二硫键对细胞的翻译后加工提出了巨大的挑战。lrp的正确转运和不正确折叠受体的处理是由分泌途径中的许多蛋白质和酶辅助的。虽然大多数这些蛋白参与一般的质量控制，但细胞培养研究提供了新的MESD蛋白靶向LRP6螺旋桨的证据。如果MESD对相关lrp的螺旋桨结构域的折叠和运输至关重要，那么MESD的突变可能不仅对发育而且对成人生理有广泛的影响。与这一预测一致的是，缺少网状的胚胎不能形成原始条纹并分化中胚层。结合胚胎学、遗传学和生物化学，为理解这种新蛋白质的功能提供了有力的方法。为了开始解决mesd的功能，我们将：(I)通过比较mesd缺陷和LRP5-/-来确定mesd表型与LRP信号传导的关系；LRP6-/-表型和利用嵌合体分析鉴定需要mesd功能的组织；（II）确定MESD是否促进细胞培养中其他lrp的折叠和定位；（III）通过检测遗传剂量与LRP突变的相互作用和MESD的组织特异性敲除，确定MESD是否对体内LRP折叠至关重要；最后，（IV）确定MESD中对ES细胞分化和转基因小鼠功能重要的区域。结合胚胎学、遗传学和生物化学，为理解这种新蛋白质的功能提供了有力的方法。

DESCRIPTION (provided by applicant): The low-density lipoprotein receptors perform diverse cellular roles ranging from endocytosis and cargo transport to subcellular trafficking and cell signaling. Despite this diversity in function, lipoprotein-related receptors (LRPs) possess an extracellular domain comprised of common repeating clusters of cysteine-rich, complement-like repeats (ligand binding domains), EGF repeats, and YWTD containing domains that form beta-propeller structures. The number and arrangement of these motifs varies amongst receptors. The complex structure and high number of disulfide bonds in LRP family members pose a formidable posttranslational processing challenge to cells. The correct trafficking of LRPs and disposal of improperly folded receptors is assisted by many proteins and enzymes in the secretory pathway. While the majority of these proteins participate in general quality control, cell culture studies provide evidence that the novel MESD protein targets the LRP6 propeller. If MESD is essential for folding and trafficking the propeller domain of related LRPs, mutations in mesd could have wide ranging effects not only on development but also for adult physiology. Consistent with this prediction, embryos lacking mesd fail to form a primitive streak and differentiate mesoderm. Combined, embryology, genetics and biochemistry provide a powerful approach towards understanding the function of this novel protein. In order to begin to address mesd function, we will: (I) Determine how the mesd phenotype relates to LRP signaling by comparison of the mesd deficient and LRP5-/-; LRP6-/- phenotypes and utilize chimera analysis to identify tissues requiring mesd function; (II) Determine if MESD facilitates folding and localization of other LRPs in cell culture; (III) Determine if MESD is essential for LRP folding in vivo by examining genetic dosage interaction with LRP mutations and tissue specific knockout of mesd; and finally, (IV) Identify regions of MESD important for function in ES cell differentiation and transgenic mice. Combined, embryology, genetics and biochemistry provide a powerful approach towards understanding the function of this novel protein.