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

基本信息

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
项目类别：
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

项目摘要

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）是进行丝氨酸和棕榈酰辅酶A之间的缩合反应的酶，该反应被称为鞘脂生物合成的关键步骤。这一重要途径通过其下游鞘脂代谢物的失调与多种人类疾病有关，包括糖尿病、代谢综合征、神经病和癌症，以及细胞凋亡、炎症反应和自噬等几个关键调节途径。包括导师在内的多个实验室的最新发现表明，SPT 与其他非规范氨基酸以及脂肪酸表现出底物混杂性，这导致发现了这种混杂性的新型鞘脂代谢副产物，例如脱氧二氢鞘氨醇和脱氧脱甲基二氢鞘氨醇。该项目的长期目标是建立酿酒酵母中新型鞘脂的信号传导和生物学功能。我们现在已经开发出基于质谱的新型方法，可以对这种新兴的新型鞘脂进行高灵敏度和准确度的检测和定量。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，这种关系超出了经典的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，以及包括硬脂酸在内的其他脂肪酸。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。我们将在三重四极杆质谱仪上使用多重反应监测，结合高质量的真实 SL 标准品和新型 SL 的类似 SILAC 代谢标记，以识别和测量新型 SL 代谢物的相对水平。 2) 表征非丝氨酸氨基酸对这些新型 SL 的代谢和调节。我们将检查新代谢物对氨基酸剂量的反应，并进行时间过程研究以确定这些代谢物的合成率。高氨基酸剂量，同时用 SL 合成抑制剂的抑制剂治疗，将用于追踪其潜在代谢为 SL 和神经酰胺。 3) 定义新型SL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，这些基因受这些新型 SL 代谢物的调节。通过添加氨基酸、加热或多球菌素处理，我们将验证这些基因的哪些子集也参与 SL 代谢。这些结果的结合将有助于定义新型 SL 及其在信号传导中的潜在作用，并定义它们在酵母中的生物学功能。这些方法，包括 MS 定量技术以及代谢表征，可以在以后进行推断并应用于探索哺乳动物系统中的相似之处。