FUNCTION AND REGULATION OF GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE 2

3-磷酸​​甘油酰基转移酶2的功能和调控

• 批准号: 7940097
• 负责人: Maria R. Gonzalez Baro
• 金额: $ 5.29万
• 依托单位: NATIONAL UNIVERSITY OF LA PLATA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7940097
• 关键词: 1,2-diacylglycerol; Acylation; Adipose tissue; Biological Assay; Brain; Cancer Cell Growth; Cell Proliferation; Cell physiology; Cells; Characteristics; Chinese Hamster Ovary Cell; Collaborations; Commit; Complement; Complex; Contraceptive methods; Data; Detection; Diglycerides; Enzymes; Esterification; Fatty Acids; Funding; Genes; Glycerol-3-phosphate acyltransferase; Glycerolipid Metabolism Pathway; Glycerophospholipids; Goals; Growth; Hepatic; Hormones; Human; In Vitro; Infertility; Insulin Resistance; Kinetics; Knowledge; Lipids; Liver; Location; Lysophospholipids; Malignant Neoplasms; Malignant neoplasm of brain; Malignant neoplasm of testis; Mammalian Cell; Mass Spectrum Analysis; Membrane; Messenger RNA; Metabolic; Mitochondria; Molecular; Mus; North Carolina; Nuclear Receptors; Nutrient; Organ; Outcome; Pathway interactions; Peripheral; Pharmaceutical Preparations; Phosphatidic Acid; Phospholipids; Physiological; Play; Polyunsaturated Fatty Acids; Production; Promoter Regions; Property; Protein Isoforms; Recombinants; Regulation; Reporter Genes; Reproduction; Research; Research Personnel; Role; S-Phase Fraction; Seminiferous tubule structure; Sexual Development; Sexual Maturation; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; Sperm Maturation; Spermatogenesis; Spermatogenic Cell; Testing; Testis; Testis Brain; Tissues; Transcriptional Regulation; Triglycerides; Universities; alpha-glycerophosphoric acid; base; cancer cell; cell motility; design; energy balance; gel electrophoresis; global health; human FRAP1 protein; improved; in vivo; insulin sensitivity; lipid biosynthesis; lipid metabolism; lysophosphatidic acid; mTOR Signaling Pathway; novel; nutrition; overexpression; oxidation; parent grant; public health relevance; research study; sperm cell; tumor

DESCRIPTION (provided by applicant): Spermatogenic cells are characterized by the presence of long-chain polyunsaturated fatty acids in their glycerolipids and hormone-coordinated cellular proliferation. The first and committed step in glycerolipid synthesis is the acylation of glycerol-3-phosphate, catalyzed by the enzyme glycerol-3-phosphate acyltransferase (GPAT). In mammalian cells, four GPAT isoforms have been cloned and characterized, each one with different kinetic properties, tissue and cell locations. The long-term goal of both groups collaborating in this project is to determine the cellular and metabolic functions of each GPAT isoform. Mitochondrial GPAT2 is expressed primarily in testis and secondarily in brain and some cancer cells. GPAT2-expressing tissues, like brain and cancer cells share some of the characteristics of spermatogenic cells, including an increased content of polyunsaturated fatty acids and rapid proliferation, respectively. The hypothesis underlying this project is that GPAT2 contributes to glycerophospholipid synthesis in tissues like testis and brain that have a high content of PUFA and that it catalyzes the synthesis of a signaling lipid molecule that helps to coordinate cellular proliferation. The specific aims of this project are: Aim 1: Determine whether the function and regulation of GPAT2 is related to the preferential esterification of polyunsaturated fatty acids in glycerolipids. Aim 2: Determine the function of GPAT2 in testis and cancer cells related to cellular proliferation and its mechanism. To test these hypotheses, recombinant mouse GPAT2 will be expressed in cultured mammalian cells, and glycerolipid synthesis will be assessed with different fatty acid substrates. Transcriptional regulation of GPAT2 will be determined in cells exposed to spermatogenesis-associated hormones, and by expressing the GPAT2 promoter region in a reporter-gene construct. The nuclear receptors involved in GPAT2 regulation will be determined by super-shift-gel electrophoresis assays. Experiments proposed to study GPAT2 activity related to cellular proliferation includes the mass-spectrometry quantification of lysophosphatidic acid, synthesized de novo as a signaling molecule, and the detection of activated intermediates of the mTOR signaling pathway in GPAT2 overexpressing cells. The physiological function of GPAT2 activity will be studied by post- transcriptionally silencing the enzyme's expression both in vivo and in vitro. Using GPAT2-targeted small- interfering RNA in sexually developing mice, sperm production and motility will be determined. The effect of GPAT2 silencing on the tumor-producing capability of cancer cells will also be determined. These studies will improve our understanding of the molecular mechanisms that govern sperm maturation and cellular proliferation, which are important to both reproduction and the proliferation of cancer cells. This research will be done in collaboration with NIH-funded investigator Dr. Rosalind A. Coleman, Department of Nutrition, University of North Carolina at Chapel Hill, USA. PUBLIC HEALTH RELEVANCE: This project is related to two major global health problems: human reproduction and cancer. Increasing our knowledge about the enzymes and molecular mechanisms involved in normal sperm maturation and in cancer growth and proliferation may improve the design of new inhibiting drugs to treat cancer or be used as a contraceptive method, or as activating drugs to treat infertility.

描述（由申请人提供）：精子生成细胞的特征是其甘油脂质中存在长链多不饱和脂肪酸和激素达到的细胞增殖。甘油合成的第一个和致力的步骤是甘油-3-磷酸甘油的酰化，由甘油-3-磷酸酶酰基转移酶（GPAT）催化。在哺乳动物细胞中，已经克隆和表征四个GPAT同工型，每个GPAT同工型具有不同的动力学特性，组织和细胞位置。两组在该项目中的长期目标是确定每个GPAT同工型的细胞和代谢功能。线粒体GPAT2主要在睾丸中表达，其次在脑和一些癌细胞中表达。表达GPAT2的组织，例如脑和癌细胞，具有精子细胞的某些特征，包括分别增加多不饱和脂肪酸和快速增殖。该项目的基本假设是，GPAT2有助于睾丸和大脑等组织中的甘油磷脂合成，这些组织具有较高的PUFA含量，并且它催化了信号传导脂质分子的合成，从而有助于协调细胞增殖。该项目的具体目的是：目标1：确定GPAT2的功能和调节是否与甘油中多不饱和脂肪酸的优先酯化有关。 AIM 2：确定GPAT2在睾丸和癌细胞中与细胞增殖及其机制有关的功能。为了检验这些假设，将在培养的哺乳动物细胞中表达重组小鼠GPAT2，并将用不同的脂肪酸底物评估甘油合成。 GPAT2的转录调节将在暴露于与精子发生相关激素的细胞中确定，并通过在报告基因构建体中表达GPAT2启动子区域。参与GPAT2调节的核受体将由超移 - 凝胶电泳测定法确定。建议研究与细胞增殖有关的GPAT2活性的实验包括溶血磷酸酸的质谱定量定量，从头合成为信号分子，以及检测GPAT2过度表达细胞中MTOR信号途径的激活中间体。 GPAT2活性的生理功能将通过在体内和体外进行转录后的沉默来研究酶的表达。将确定精子的产生和运动性，使用靶向GPAT2的小型干扰RNA。还将确定GPAT2沉默对癌细胞产生肿瘤能力的影响。这些研究将提高我们对控制精子成熟和细胞增殖的分子机制的理解，这对于繁殖和癌细胞的增殖都很重要。这项研究将与NIH资助的研究员Rosalind A. Coleman博士合作，美国北卡罗来纳大学Chapel Hill的营养系Rosalind A. Coleman博士。 公共卫生相关性：该项目与两个主要的全球健康问题有关：人类繁殖和癌症。提高我们对正常精子成熟涉及的酶和分子机制以及癌症生长和增殖的了解可能会改善新的抑制药物治疗癌症或用作避孕方法，或者用作治疗不孕症的药物。