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不同的细胞作用，从内吞和货物运输到亚细胞运输和细胞信号传递。尽管功能不同，脂蛋白相关受体(LRP)仍具有一个胞外结构域，由富含半胱氨酸、补体样重复序列(配基结合域)、EGF重复序列和YWTD结构域组成，这些结构域形成了β-螺旋桨结构。这些基序的数量和排列因受体而异。LRP家族成员复杂的结构和大量的二硫键给细胞的翻译后加工带来了巨大的挑战。LRPs的正确运输和不正确折叠的受体的处理在分泌途径中的许多蛋白质和酶的帮助下进行。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的证据表明，新型MESD蛋白质以LRP6推进器为靶标。如果MESD对于折叠和运输相关LRP的螺旋体结构域是必不可少的，那么MESD的突变不仅会对发育产生广泛的影响，还会对成年生理产生广泛的影响。与这一预测一致的是，缺乏MESD的胚胎无法形成原始条纹并分化出中胚层。胚胎学、遗传学和生物化学相结合，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的功能，我们将：(1)通过比较MESD缺陷的LRP5-/-和LRP5-/-确定MESD的表型与LRP信号的关系；(3)通过嵌合体分析确定需要MESD功能的组织；(2)确定MESD是否促进了其他LRP在细胞培养中的折叠和定位；(3)通过检测MESD与LRP突变的遗传剂量的相互作用和组织特异性MESD的敲除，确定MESD是否对体内的LRP折叠是必不可少的；以及(4)确定MESD在ES细胞分化和转基因小鼠中对于功能的重要区域。胚胎学、遗传学和生物化学相结合，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.