Biochemical and genetic analysis of mesd function

med功能的生化和遗传分析

• 批准号: 6921930
• 负责人: BERNADETTE C HOLDENER
• 金额: $ 33.86万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6921930
• 关键词: SDS polyacrylamide gel electrophoresis; affinity chromatography; biochemistry; biological signal transduction; cell differentiation; developmental genetics; embryology; gene mutation; genetically modified animals; immunocytochemistry; laboratory mouse; low density lipoprotein receptor; mesoderm; phenotype; protein folding; protein localization; protein structure function; tissue /cell culture

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.

描述（由申请人提供）：低密度脂蛋白受体执行多种细胞作用，从内吞作用和货物运输到亚细胞运输和细胞信号传导。 尽管存在这种功能多样性，脂蛋白相关受体 (LRP) 仍具有由富含半胱氨酸、补体样重复序列（配体结合结构域）、EGF 重复序列和包含形成 β-螺旋桨结构的 YWTD 结构域的常见重复簇组成的胞外结构域。 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带来了巨大的翻译后加工挑战。 分泌途径中的许多蛋白质和酶有助于 LRP 的正确运输和不正确折叠的受体的处理。 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证据表明新型 MESD 蛋白质以 LRP6 螺旋桨为目标。 如果 MESD 对于相关 LRP 的螺旋桨结构域的折叠和运输至关重要，那么 mesd 的突变不仅可能对发育而且对成人生理学产生广泛的影响。 与这一预测一致，缺乏 mesd 的胚胎无法形成原条并分化中胚层。胚胎学、遗传学和生物化学相结合，为理解这种新型蛋白质的功能提供了一种强有力的方法。 为了开始解决mesd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-/- 表型并利用嵌合体分析来识别需要 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) 通过检查基因剂量与 LRP 突变的相互作用以及 mesd 的组织特异性敲除，确定 MESD 是否对 LRP 体内折叠至关重要；最后，(IV) 确定对 ES 细胞分化和转基因小鼠功能重要的 MESD 区域。 胚胎学、遗传学和生物化学相结合，为理解这种新型蛋白质的功能提供了一种强有力的方法。