REGULATION OF SKELETAL MUSCLE DEVELOPMENT AND MAINTENANCE BY PROTEIN O-GLUCOSYLTRANSFERASE 1 (POGLUT1)

摘要 碳水化合物添加（糖基化）是最常见的翻译后修饰之一 分泌蛋白和细胞表面蛋白。糖基化对于正常动物发育至关重要 生理学和糖基化相关基因突变会导致 100 多种人类疾病 具有不同的表型。然而，聚糖结构很复杂，每种形式的糖基化 可以在数十到数千种目标蛋白中找到。因此，了解分子 糖基化障碍的潜在机制构成了一项重大挑战。关键角色之一 蛋白质糖基化的作用是调节细胞间的通讯机制，称为 Notch信号通路。 Notch信号调节动物发育过程中的许多过程 成人保养。例如，对小鼠和细胞培养的研究表明，肌肉 哺乳动物损伤后的发育和肌肉修复依赖于 Notch 信号传导。然而， Notch 通路成分或调节剂的突变尚未在人类患者中报告 患有肌肉萎缩症。我们最近报道了一个近亲家庭，其中有几个 兄弟姐妹患有 LGMD-2Z，这是一种新型肢带型肌营养不良症。 LGMD-2Z是 由 POGLUT1 基因的隐性突变纯合性引起。 POGLUT1 是 糖基转移酶，将 O-连接的葡萄糖添加到多个跨膜上并分泌 蛋白质，包括 Notch 信号通路的多个成分。我们之前已经展示过 POGLUT1 调节果蝇和小鼠的 Notch 信号传导。肌肉组织分析 从上述患者中分离出的成肌细胞提供了证据表明受损的 Notch 信号传导在这种肌营养不良症的病理生理学中发挥着重要作用。 然而，Notch 通路和其他通路中 POGLUT1 的生物学相关靶点 肌肉不知道。在本提案中，我们将使用生化和细胞培养测定， 蛋白质组分析、小鼠遗传实验和 iPS 细胞实验以确定分子 POGLUT1 调节肌肉发育和维持的潜在机制 确定其相关目标。我们将使用来自患者的 iPS 细胞以及 CRISPR/Cas9 介导的 它们的修正版本，用于体外疾病建模和体内移植实验。这些 研究有可能为肌营养不良症的病理生理学提供新的见解 由异常糖基化引起，可能为未来的治疗建立新的框架 肌肉疾病的治疗方法。

SUMMARY Carbohydrate addition (glycosylation) is one of the most common posttranslational modifications of secreted and cell surface proteins. Glycosylation is critical for normal animal development and physiology, and mutations in genes involved in glycosylation cause more than a 100 human diseases with diverse phenotypes. However, glycan structures are complex, and each form of glycosylation can be found in tens to thousands of target proteins. Accordingly, understanding the molecular mechanisms underlying glycosylation disorders constitute a major challenge. One of the critical roles played by protein glycosylation is the regulation of a cell-to-cell communication mechanism called the Notch signaling pathway. Notch signaling regulates many processes during animal development and adult maintenance. For example, studies in mice and cell culture have shown that muscle development and muscle repair after injury depend on Notch signaling in mammals. However, mutations in Notch pathway components or modulators have not been reported in human patients with muscular dystrophy. We have recently reported a consanguineous family in which several siblings suffer from LGMD-2Z, which is a new form of limb-girdle muscular dystrophy. LGMD-2Z is caused by homozygosity for a recessive mutation in a gene called POGLUT1. POGLUT1 is a glycosyltransferase which adds O-linked glucose to a number of transmembrane and secreted proteins, including multiple components of the Notch signaling pathway. We have previously shown that POGLUT1 regulates Notch signaling in fruit flies and mice. Analysis of the muscle tissues and myoblasts isolated from the above-mentioned patients provided evidence suggesting that impaired Notch signaling plays an important role in the pathophysiology of this form of muscular dystrophy. However, the biologically-relevant targets of POGLUT1 in the Notch pathway and other pathways in the muscle are not known. In this proposal, we will use biochemical and cell culture assays, proteomic profiling, mouse genetic experiments and iPS cell experiments to determine the molecular mechanisms underlying the regulation of muscle development and maintenance by POGLUT1 and to identify its relevant targets. We will use iPS cells from patients, along with a CRISPR/Cas9-mediated corrected version of them, for in vitro disease modeling and in vivo engraftment experiments. These studies have the potential to provide novel insight into the pathophysiology of a muscular dystrophy caused by abnormal glycosylation and might establish a new framework for future therapeutic approaches for muscle diseases.