ORM Protein Regulation of Sphingolipid Biosynthesis in Yeast and Mammals

描述（由申请人提供）：哮喘是困扰美国儿童的最常见的慢性疾病之一，发病率目前为10%，自1980年以来几乎翻了一番。虽然这种增加的原因尚未确定，但几个基因与这种疾病的发病机制有关。其中包括ORMDL 3，它是内质网（ER）蛋白ORM家族的一员，最近成为鞘脂生物合成的关键调节因子。鞘脂是一个具有重要细胞功能的脂质家族，因此其合成和降解的异常调节与许多疾病相关并不奇怪。尽管其重要性，负责鞘脂稳态的机制知之甚少。因此，最近发现ORM蛋白存在于与丝氨酸棕榈酰转移酶（SPT）（鞘脂生物合成的关键酶）的复合物（“SPOTS”）中是非常重要的。在酵母中的研究表明，通过ORM调节SPT是由其N-末端结构域的磷酸化和ER内SPOTS复合物的再分布控制的。重新分布与观察结果一致，即虽然可以容易地测量ORM对体内SPT活性的调节，但体外SPT活性不受ORM的影响。哺乳动物ORMDL也调节体内SPT活性。然而，它们缺乏在酵母中发现的N-末端延伸，这表明了不同的调节机制。使用我们实验室开发的一套高度创新的试剂和方法来研究酵母和人SPT的结构组织，我们将解决有关ORM调节SPT机制的关键未决问题。首先，我们将确定SPT活性是否受酵母和哺乳动物细胞中ER内SPOTS复合物的寡聚状态和/或再分布的调节。其次，我们将调查的机制，负责监管哺乳动物SPT的ORMDLs，其中缺乏的N-末端调控结构域中发现的酵母。从这些探索性研究中获得的数据将提供有关鞘脂生物合成调控的重要新信息，并为阐明ORMDL 3在哮喘病理生理学中的作用的动物和人类研究奠定基础。

DESCRIPTION (provided by applicant): Asthma is one of the most common chronic diseases afflicting American children and the rate of incidence, currently 10%, has nearly doubled since 1980. Although the reason for this increase has not been determined, several genes have been implicated in the pathogenesis of this disorder. Among these is ORMDL3, a member of the ORM family of endoplasmic reticulum (ER) proteins that have recently emerged as key regulators of sphingolipid biosynthesis. The sphingolipids are a diverse family of lipids with crucialcellular functions and it is therefore not surprising that aberrant regulation of their synthesis and degradation has been associated with numerous disorders. Despite their importance, the mechanisms responsible for sphingolipid homeostasis are poorly understood. Therefore, the recent discovery that the ORM proteins exist in a complex ("SPOTS") with serine palmitoyltransferase (SPT), the committed enzyme of sphingolipid biosynthesis, is very significant. Studies in yeast suggest that regulation of SPT by the ORMs is controlled by phosphorylation of their N-terminal domains and redistribution of the SPOTS complex within the ER. The redistribution is consistent with the observation that while ORM regulation of in vivo SPT activity can be easily measured, in vitro SPT activity is unaffected by the ORMs. Mammalian ORMDLs also regulate in vivo SPT activity. However, they lack the N-terminal extensions found in their yeast counterparts, suggesting a different mechanism of regulation. Using a set of highly innovative reagents and methods developed in our laboratory to study the structural organization of both yeast and human SPT, we will resolve key outstanding questions regarding the mechanism of regulation of SPT by the ORMs. First, we will determine whether SPT activity is regulated by the oligomeric state and/or redistribution of the SPOTS complex within the ER in yeast and mammalian cells. Second, we will investigate the mechanism responsible for regulation of mammalian SPT by the ORMDLs, which lack the N-terminal regulatory domain found in their yeast counterparts. The data obtained from these exploratory studies will provide important new information about the regulation of sphingolipid biosynthesis in general, and lay the groundwork for studies in animals and humans that will elucidate the role of ORMDL3 in the pathophysiology of asthma.