Serine Palmitoyl Transferase and Hereditary Neuropathy

描述(由申请人提供)： 丝氨酸棕榈酰基转移酶(SPT)催化鞘磷脂合成中的关键和限速步骤。SPTLC1基因突变编码SPT的Sptlclp亚单位，导致遗传性感觉神经病1型(HSN1)，这是最常见的遗传性神经病。因此，神经鞘脂的异常合成可能与该病的病理生理学有关。Spt含有至少一个额外的亚基Sptlc2p，它与Sptlclp形成杂二聚体。HSN1突变体Sptlclp蛋白是SPT活性的显性负抑制因子，因为它们能够形成催化失活的稳定的异源二聚体。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。使用一组适用于遗传和生化操作的模型系统，我们将确定SPT突变如何对HSN1的病理生理学做出贡献。具体地说，我们建议：1)分离和鉴定哺乳动物SPT复合体，并鉴定鞘氨醇生物合成途径的下游成分；2)绘制对催化、异二聚、调节Sptlc2p稳定性至关重要的结构域，并确定Sptlclp和Sptlc2p的拓扑结构，并建立SPT的重新定位是否调节活性；3)阐明HSN1显性负突变降低SPT活性的机制；以及4)构建表达SPTLC1-HSN1显性负等位基因的转基因小鼠和SPTLC1“敲除”小鼠。SPT络合物将被免疫纯化，并用MALDI-TOF质谱仪鉴定其组分。将使用两个杂交筛选来鉴定Sptlclp和Sptlc2p相互作用的蛋白质。结构域的绘制、拓扑结构的确定、细胞内定位和显性负突变的鉴定将结合酵母遗传学和哺乳动物细胞中正常和突变蛋白的表达，其中siRNAs用于去除内源基因表达。转基因野生型或突变型SPTLC1的小鼠将在行为、形态、生化和电生理方面发生变化。平行分析将在SPTLC1条件性基因敲除小鼠上完成。综上所述，这些研究将阐明鞘脂的生物学基础、它们的调节以及HSN1的疾病过程。

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.