Serine Palmitoyl Transferase and Hereditary Neuropathy

描述（由申请人提供）： 丝氨酸棕榈酰转移酶（SPT）催化鞘脂合成中的关键和限速步骤。编码SPT的Sptlclp亚基的SPTLC 1基因突变导致遗传性感觉神经病1型（HSN 1），这是最常见的遗传性神经病。因此，异常鞘脂合成可能有助于这种疾病的病理生理。SPT含有至少一个额外的亚基Sptlc 2 p，其与Sptlclp形成异二聚体。HSN 1突变体Sptlclp蛋白凭借其形成无催化活性的稳定异源二聚体的能力而表现为SPT活性的显性负抑制剂。我们的长期目标是确定参与鞘脂合成的基因及其在人类疾病中的作用。使用一个模型系统的组合适合遗传和生化操作，我们将确定如何在SPT的突变有助于HSN 1的病理生理。具体而言，我们建议：1）分离和表征哺乳动物SPT复合物并鉴定鞘氨醇生物合成途径的下游组分; 2）绘制对催化、异源二聚化、调节Sptlc 2 p稳定性重要的结构域，并确定Sptlclp和Sptlc 2 p的拓扑结构，并确定SPT的重定位是否调节活性; 3）阐明HSN 1显性负突变降低SPT活性的机制;构建表达SPTLC 1-HSN 1显性负等位基因的转基因小鼠和SPTLC 1“敲除”小鼠。将对SPT复合物进行免疫纯化，并通过MALDI-TOF质谱法鉴定其组分。两个杂交筛选将用于鉴定Sptlclp和Sptlc 2 p相互作用蛋白。将使用酵母遗传学和哺乳动物细胞中正常和突变蛋白表达的组合进行结构域作图、拓扑结构测定、细胞内定位和显性失活突变体表征，其中siRNA用于消除内源性基因表达。将对野生型或突变型SPTLC 1转基因小鼠的行为、形态、生物化学和电生理变化进行表征。将在SPTLC 1条件性基因敲除小鼠中完成平行分析。总之，这些研究将阐明鞘脂的生物学，其调节和HSN 1的疾病过程的基本问题。

DESCRIPTION (provided by applicant): Serine palmitoyltransferase (SPT) catalyzes the committed and rate-limiting step in sphingolipid synthesis. Mutations in the SPTLC1 gene, encoding the Sptlclp subunit of SPT, result in hereditary sensory neuropathy, type 1 (HSN1), the most common inherited neuropathy. Thus, aberrant sphingolipid synthesis may contribute to the pathophysiology of this disease. SPT contains at least one additional subunit, Sptlc2p, which forms a heterodimer with Sptlclp. HSN1 mutant Sptlclp proteins behave as dominant negative inhibitors of SPT activity by virtue of their ability to form catalytically inactive stable heterodimers. Our long-range goal is to identify the genes involved in sphingolipid synthesis and their role in human disease. Using a combination of model systems amenable to genetic and biochemical manipulation, we will determine how the mutations in SPT contribute to the pathophysiology of HSN1. Specifically we propose to: 1) isolate and characterize the mammalian SPT complex and identify the downstream components of the sphingosine biosynthetic pathway; 2) map the domains important for catalysis, heterodimerization, regulated Sptlc2p stability, and determine the topology of Sptlclp and Sptlc2p, and establish whether relocalization of SPT regulates activity; 3) elucidate the mechanism by which the HSN1 dominant negative mutations decrease SPT activity; and 4) construct transgenic mice expressing the SPTLC1-HSN1 dominant negative allele and an SPTLC1 "knock-out" mouse. SPT complexes will be immunopurified and their components identified by MALDI-TOF mass spectrometry. Two hybrid screens will be used to identify Sptlclp and Sptlc2p interacting proteins. Mapping of domains, determination of topology, intracellular localization, and characterization of dominant negative mutants will be performed using a combination of yeast genetics and expression of normal and mutant proteins in mammalian cells in which siRNAs are used to ablate endogenous gene expression. Mice transgenic for wildtype or mutant SPTLC1 will be characterized for behavioral, morphological, biochemical, and electrophysiological changes. Parallel analysis will be completed on SPTLC1 conditional knock-out mice. Taken together these studies will illuminate fundamental issues underlying the biology of sphingolipids, their regulation, and the disease process of HSN1.