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

总结 碳水化合物添加（糖基化）是最常见的翻译后修饰之一。 分泌和细胞表面蛋白。糖基化对正常动物发育至关重要， 在生理学上，与糖基化有关的基因突变会导致100多种人类疾病 不同的表型。然而，聚糖结构是复杂的，每种形式的糖基化 可以在成千上万的靶蛋白中找到。因此，了解分子 糖基化障碍的潜在机制构成了主要挑战。其中一个关键的角色 蛋白质糖基化所起的作用是调节细胞与细胞之间的通讯机制， Notch信号通路。Notch信号转导调节动物发育过程中的许多过程， 成人保养例如，在小鼠和细胞培养中的研究表明， 在哺乳动物中，损伤后的发育和肌肉修复依赖于Notch信号传导。然而，在这方面， Notch途径组分或调节剂中的突变尚未在人类患者中报道 肌肉萎缩症我们最近报道了一个近亲家庭，其中几个 兄弟姐妹患有LGMD-2Z，这是一种新形式的肢带型肌营养不良症。LGMD-2Z是 是由POGLUT 1基因的隐性突变纯合性引起的。POGLUT1是一个 糖基转移酶，其将O-连接的葡萄糖添加到许多跨膜和分泌的 蛋白质，包括Notch信号通路的多种组分。我们先前已经表明 POGLUT1调节果蝇和小鼠的Notch信号。肌肉组织分析和 从上述患者中分离的成肌细胞提供的证据表明， Notch信号传导在这种形式的肌营养不良的病理生理学中起重要作用。 然而，POGLUT 1在Notch途径和其他途径中的生物学相关靶点在肿瘤中的表达并不明显。 肌肉是未知的。在这个建议中，我们将使用生化和细胞培养分析， 蛋白质组学分析、小鼠遗传实验和iPS细胞实验，以确定 POGLUT 1调节肌肉发育和维持的潜在机制， 确定相关目标。我们将使用来自患者的iPS细胞，沿着CRISPR/Cas9介导的 它们的校正版本，用于体外疾病建模和体内移植实验。这些 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 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.