Establishing Signaling and Biological Function of Novel Sphingolipids in Yeast

建立酵母中新型鞘脂的信号传导和生物学功能

• 批准号: 8841600
• 负责人: Nadia A. Rana
• 金额: $ 1.4万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2015-07-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8841600
• 关键词: Address; Alanine; Amino Acids; Anabolism; Apoptosis; Autophagocytosis; Biological Process; Calibration; Candidate Disease Gene; Catalytic Domain; Cells; Ceramides; Complex; Coupled; Data; Deaminase; Detection; Development; Developmental Process; Diabetes Mellitus; Disease; Dose; Enzymes; Exhibits; Fatty Acids; Fumonisins; Gene Expression Regulation; Generations; Genes; Genetic; Glycine; Goals; Growth; Heating; Hereditary Sensory Neuropathy; Human body; Hydro-Lyases; Inflammatory Response; Ions; Label; Laboratories; Lead; Lipids; Malignant Neoplasms; Measures; Mediator of activation protein; Mentors; Messenger RNA; Metabolic; Metabolic syndrome; Metabolism; Methodology; Methods; Monitor; Neuropathy; Palmitoyl Coenzyme A; Parents; Pathway interactions; Physical condensation; Point Mutation; Production; Radiolabeled; Reaction; Regulation; Regulatory Pathway; Relative (related person); Resolution; Role; Saccharomyces cerevisiae; Serine; Signal Transduction; Sphingolipids; Sphingosine; Stearates; Substrate Specificity; System; Techniques; Testing; Time; Transferase; Up-Regulation; Work; Yeasts; alanylglycine; amino acid metabolism; aminoacid biosynthesis; base; cysteinylglycine; dosage; high standard; human disease; inhibitor/antagonist; instrumentation; mass spectrometer; meetings; method development; multiple reaction monitoring; mutant; novel; public health relevance; radiotracer; research study; response; screening; sensor; sphinganine; stearoyl-coenzyme A; thermozymocidin; tool; uptake

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.

描述(申请人提供)：丝氨酸棕榈酰转移酶(SPT)是执行丝氨酸和棕榈酰Co-A之间的缩合反应的酶，该缩合反应被称为鞘磷脂生物合成的关键步骤。通过其下游的鞘磷脂代谢产物，这一重要途径的调节失调与多种人类疾病有关，包括糖尿病、代谢综合征、神经病变和癌症，以及几个关键的调节途径，如细胞凋亡、炎症反应和自噬。包括导师实验室在内的几个实验室的最新发现表明，SPT表现出与其他非规范氨基酸和脂肪酸的底物混杂，这导致了这种混杂的新的鞘脂代谢产物的发现，如脱氧鞘氨酸和脱氧脱甲基鞘氨酸.该项目的长期目标是在酿酒酵母中建立新型鞘磷脂的信号和生物学功能。我们现在已经开发了基于质谱学的新方法，以允许对这一新兴类别的鞘脂进行高灵敏度和精确度的检测和定量。初步数据表明，鞘磷脂和氨基酸利用率之间可能存在调节关系，这种关系从典型的丝氨酸和棕榈酰Co-A延伸到其他氨基酸，如丙氨酸和甘氨酸，以及包括硬脂酸在内的其他脂肪酸。这项建议的主要重点将集中在表征新的鞘磷脂代谢物和探索SPT协调氨基酸代谢的能力，并将解决以下目标：1)开发用于检测和定量新型SLS的MS检测方法。我们将使用三四元质谱仪上的多反应监测，结合高质量的正宗SL标准和新型SLS的类似SILAC的代谢标记来识别和测量新型SL代谢物的相对水平。2)研究非丝氨酸氨基酸对这些新型SLS的代谢和调节作用。我们将检查新代谢物的产生对氨基酸剂量的响应，并进行时间进程研究，以确定这些代谢物的合成速度。高剂量的氨基酸，伴随着SL合成抑制剂的治疗，将被用来追踪它们进入SLS和SLS的潜在代谢 神经酰胺。3)明确新的SLS在氨基酸代谢调节中的作用。我们将利用微阵列方法来确定参与氨基酸代谢的基因，这些基因受到这些新的SL代谢物的调节。使用氨基酸加成、加热或豆蔻菌素处理，我们将验证这些基因中的哪些子集也参与SL代谢。这些结果的融合将有助于确定新的SLS及其在信号转导中的潜在作用，并确定它们在酵母中的生物学功能。这些方法，包括MS定量技术，以及代谢特征，可以在以后推断并应用于探索哺乳动物系统中的相似之处。