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，但脂蛋白相关受体（LRP）具有由富含半胱氨酸的常见重复簇、补体样重复（配体结合结构域）、EGF重复和形成β-螺旋桨结构的含YWTD的结构域组成的胞外结构域。 这些基序的数量和排列在受体之间变化。 LRP家族成员复杂的结构和大量的二硫键对细胞的翻译后加工构成了巨大的挑战。 LRP的正确运输和不正确折叠的受体的处置由分泌途径中的许多蛋白质和酶辅助。 虽然这些蛋白质中的大多数参与一般质量控制，但细胞培养研究提供了新MESD蛋白靶向LRP 6推进器的证据。 如果MESD对于折叠和运输相关LRP的螺旋桨结构域是必不可少的，那么mesd中的突变不仅对发育而且对成人生理学具有广泛的影响。 与这一预测一致，缺乏mesd的胚胎不能形成原始条纹和分化中胚层。胚胎学、遗传学和生物化学的结合为理解这种新蛋白质的功能提供了一种强有力的方法。 为了开始研究mesd功能，我们将：（I）通过比较mesd缺陷型和LRP 5-/-; LRP 6-/-表型来确定mesd表型如何与LRP信号传导相关，并利用嵌合体分析来鉴定需要mesd功能的组织;（II）确定MESD是否促进细胞培养物中其它LRP的折叠和定位;（III）通过检查与LRP突变的遗传剂量相互作用和mesd的组织特异性敲除来确定MESD是否是体内LRP折叠所必需的;最后，（IV）鉴定MESD的对ES细胞分化和转基因小鼠中的功能重要的区域。 胚胎学、遗传学和生物化学的结合为理解这种新蛋白质的功能提供了一种强有力的方法。

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.