Establishing Signaling and Biological Function of Novel Sphingolipids in Yeast

描述（由申请人提供）：丝氨酸棕榈酰转移酶（SPT）是进行丝氨酸和棕榈酰辅酶A之间缩合反应的酶，该反应被称为鞘脂生物合成的关键步骤。通过其下游鞘脂代谢产物的这一重要途径的失调已经涉及多种人类疾病，包括糖尿病、代谢综合征、神经病和癌症，以及几种关键的调节途径，如细胞凋亡、炎症反应和自噬。包括导师在内的几个实验室的最新发现表明，SPT与其他非典型氨基酸以及脂肪酸都表现出底物混杂性，这导致发现了这种混杂性的新型鞘脂代谢物副产物，如脱氧二氢鞘氨醇和脱氧去甲基二氢鞘氨醇。该项目的长期目标是在酿酒酵母中建立新型鞘脂的信号传导和生物学功能。我们现在已经开发了新的基于质谱的方法，以允许高灵敏度和准确度的检测和定量这一新兴的新类别的鞘脂。初步数据表明，鞘脂和氨基酸可用性之间可能存在调节关系，该关系超出了标准丝氨酸和棕榈酰辅酶A，延伸到其他氨基酸，如丙氨酸和甘氨酸，以及其他脂肪酸，包括硬脂酸。该提案的主要重点将集中在新型鞘脂代谢物的表征和探索SPT协调氨基酸代谢的能力，并将实现以下目标：1）开发用于检测和定量新型SL的MS检测方法。我们将在三重四级质谱仪上使用多反应监测，结合高质量的真实SL标准品和新型SL的SILAC样代谢标记，以鉴定和测量新型SL代谢物的相对水平。2)表征这些新型SL通过非丝氨酸氨基酸的代谢和调节。我们将检查响应于氨基酸剂量的新型代谢物的产生，并进行时间过程研究以确定这些代谢物的合成速率。高剂量的氨基酸，同时使用SL合成抑制剂的抑制剂治疗，将用于追踪它们潜在的SL代谢， 神经酰胺3)明确新型SL在氨基酸代谢调节中的作用。我们将利用微阵列的方法来确定参与氨基酸代谢的基因，这些新的SL代谢物的调节。使用氨基酸添加，热，或多球壳菌素治疗，我们将验证这些基因的子集也参与SL代谢。这些结果的融合将有助于定义新的SL及其在信号传导中的潜在作用，并定义其在酵母中的生物学功能。这些方法，包括MS定量技术，以及代谢表征，可以外推和应用于探索哺乳动物系统中的相似之处。

DESCRIPTION (provided by applicant): Serine Palmitoyl Transferase (SPT) is the enzyme that carries out the condensation reaction between Serine and Palmitoyl Co-A that is known as the committed step of sphingolipid biosynthesis. Dysregulation of this important pathway through its downstream sphingolipid metabolites has been implicated in a variety of human diseases, including diabetes, metabolic syndrome, neuropathy, and cancer, as well as several key regulatory pathways such as apoptosis, inflammatory responses, and autophagy. Recent discoveries from several laboratories, including the mentor's, have demonstrated SPT exhibits substrate promiscuity, both with other non-canonical amino acids as well as fatty acids, which has led to the uncovering of novel sphingolipid metabolite by-products of this promiscuity such as deoxysphinganine and deoxydemethylsphinganine. The long-term goal of this project is to establish signaling and biological functions for novel sphingolipids in Saccharomyces cerevisiae. We have now developed novel mass spectral-based methods to allow for high sensitivity and accuracy detection and quantitation of this emerging new class of sphingolipids. Preliminary data suggests there may be a regulatory relationship between sphingolipids and amino acid availability that extends beyond the canonical Serine and Palmitoyl Co-A to other amino acids such as Alanine and Glycine, and other Fatty Acids including Stearate. This proposal's main focus will be centered on the characterization of the novel sphingolipid metabolites and exploration of SPT's ability to coordinate amino acid metabolism, and will address the following aims: 1) Development of MS detection methodology for detection and quantitation of novel SLs. We will use Multiple Reaction Monitoring on a triple quad mass spectrometer in conjunction with high quality authentic SL standards and SILAC-like metabolic labeling of novel SLs to identify and measure relative levels of novel SL metabolites. 2) To characterize the metabolism and regulation of these novel SLs by non-Serine amino acids. We will examine production of novel metabolites in response to amino acid dosage, and carry out time-course studies to determine rate of synthesis of these metabolites. High amino acid dose, concomitant with treatment with inhibitors of SL synthesis inhibitor, will be used to track their potential metabolism into SLs and ceramides. 3) To define roles for novel SLs in amino acid metabolism regulation. We will utilize microarray approaches to determine genes involved in amino acid metabolism that are regulated by these novel SL metabolites. Using amino acid addition, heat, or myriocin treatment, we will validate which subset of these genes are also involved in SL metabolism. The amalgamation of these results will serve to define novel SLs and their potential roles in signaling, and to define their biological functions in yeast. These methodologies, including MS quantitative techniques, as well as metabolic characterization, can later be extrapolated and applied to exploring parallels in mammalian systems.