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Gene Discovery and Heparan Sulfate Biogenesis

基因发现和硫酸乙酰肝素生物发生

基本信息

批准号：
8035999
负责人：
Jeffrey D Esko
金额：
$ 19.31万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-01 至 2012-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8035999
关键词：
Affect Animal Model Animals Area Atherosclerosis Binding Biogenesis Biological Assay Candidate Disease Gene Categories Cell Adhesion Cell Line Cell-Matrix Junction Cells Core Protein Cytotoxic agent Cytotoxin DNA Development Diphtheria Toxin Disease Drug Delivery Systems Endocytosis Ensure Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Extracellular Matrix FGF2 gene Family Fibroblast Growth Factor Future Gene Silencing Genes Genetic Genetic Transcription Glycoconjugates Goals Grant Growth Factor Harvest Heparan Sulfate Biosynthesis Heparan Sulfate Proteoglycan Heparitin Sulfate Human Genome Individual Inflammation Inorganic Sulfates Lead Lentivirus Vector Libraries Ligands Link Malignant Neoplasms Modification Mutate Outcome Pattern Plant Lectins Polysaccharides Property Proteins Recombinant Proteins Resistance Screening procedure Sorting - Cell Movement Source Substrate Specificity Techniques Testing Tissues Translations Unspecified or Sulfate Ion Sulfates Uronic Acids Work base cell type cytokine drug development gene discovery genome-wide glycosylation high throughput screening human disease inhibitor/antagonist insight intercellular communication novel overexpression public health relevance receptor research study small hairpin RNA small molecule sugar

项目摘要

DESCRIPTION (provided by applicant): Gene Discovery and Heparan Sulfate Biogenesis. Animal cells elaborate a variety of heparan sulfate proteoglycans (HSPGs), which consist of different protein cores and one or more heparan sulfate chains. The HSPGs bind numerous growth factors, cytokines, extracellular matrix components, enzymes and enzyme inhibitors, and they act as receptors or coreceptors for cell signaling, cell attachment, and endocytosis. These interactions depend to a large extent on the composition and the arrangement of sulfated sugars and epimers of uronic acids in the heparan sulfate chains, which in turn depend on the substrate specificity of various biosynthetic enzymes and regulatory factors. Most of the enzymes involved in heparan sulfate biogenesis have been identified, cloned, expressed as recombinant proteins, studied biochemically, and mutated in cells or model organisms. In contrast, a dearth of information exists regarding the mechanisms that give rise to the variable composition and binding properties of heparan sulfate. The central hypothesis of this grant is that genes exist other than those that encode the biosynthetic enzymes, whose expression either modulate the transcription/translation of the enzymes or orchestrate their action to achieve the final composition of heparan sulfate observed in different cell types. Our objective is to search for these regulatory factors through gene- silencing techniques based on stable expression of short hairpin RNAs directed to the whole human genome. Towards this goal, we will (1) adapt high throughput screening assays to identify shRNAs that induce resistance to cytotoxic agents whose action depend on HSPGs (FGF2-Saporin and diphtheria toxin); and (2) identify and characterize genes that modulate the composition of HSPGs by amplification and sequencing the relevant shRNA clones. Genes identified in this way will be sorted into categories based on predicted or known function, and individual candidates will be tested in different cell lines. Detailed structural studies of HS and binding studies with test ligands also will be performed. The significance of this work lies in the potential for discovery of novel regulatory factors involved in HSPG expression. The outcome of these experiments has the wider goal of moving the field into new areas of study. Each factor identified in this way provides a new project for future study and a candidate gene that might explain disorders in which HSPG expression goes awry, such as cancer, inflammation, and atherosclerosis. Insight into the mechanisms that cells use to regulate heparan sulfate composition also might lead to novel drug targets for treating human disease associated with alterations in heparan sulfate formation, such as cancer, inflammation and atherosclerosis. PUBLIC HEALTH RELEVANCE: The significance of this work lies in its potential of uncovering novel regulatory factors involved in heparan sulfate formation. Each factor identified in this way provides new projects for future study. Furthermore, candidate genes might emerge that could explain disorders in which heparan sulfate formation goes awry, such as cancer, inflammation, and atherosclerosis, which in turn could define novel targets for drug development.

描述（由申请人提供）：基因发现和硫酸乙酰肝素生物发生。动物细胞产生多种硫酸乙酰肝素蛋白聚糖（HSPG），它们由不同的蛋白质核心和一条或多条硫酸乙酰肝素链组成。 HSPG 结合多种生长因子、细胞因子、细胞外基质成分、酶和酶抑制剂，并且它们充当细胞信号传导、细胞附着和内吞作用的受体或辅助受体。这些相互作用在很大程度上取决于硫酸乙酰肝素链中硫酸化糖和糖醛酸差向异构体的组成和排列，而这又取决于各种生物合成酶的底物特异性和调节因素。大多数参与硫酸乙酰肝素生物合成的酶已被鉴定、克隆、表达为重组蛋白、进行生化研究，并在细胞或模型生物体中发生突变。相比之下，关于硫酸乙酰肝素的可变组成和结合特性的机制的信息缺乏。这项资助的中心假设是，除了那些编码生物合成酶的基因之外，还存在其他基因，这些基因的表达要么调节酶的转录/翻译，要么协调它们的作用，以实现在不同细胞类型中观察到的硫酸乙酰肝素的最终组成。我们的目标是通过基于针对整个人类基因组的短发夹RNA的稳定表达的基因沉默技术来寻找这些调节因子。为了实现这一目标，我们将 (1) 采用高通量筛选方法来鉴定能够诱导细胞毒性药物抗性的 shRNA，这些细胞毒性药物的作用依赖于 HSPG（FGF2-肥皂草素和白喉毒素）； (2) 通过扩增和测序相关 shRNA 克隆来鉴定和表征调节 HSPG 组成的基因。以这种方式识别的基因将根据预测或已知的功能进行分类，并且将在不同的细胞系中测试各个候选基因。 HS 的详细结构研究和与测试配体的结合研究也将进行。这项工作的意义在于发现参与 HSPG 表达的新调控因子的潜力。这些实验的结果具有更广泛的目标，即将该领域转移到新的研究领域。以这种方式确定的每个因素都为未来的研究提供了一个新项目，并提供了一个可能解释 HSPG 表达出错的疾病（例如癌症、炎症和动脉粥样硬化）的候选基因。对细胞调节硫酸乙酰肝素组成的机制的深入了解也可能会产生新的药物靶点，用于治疗与硫酸乙酰肝素形成改变相关的人类疾病，例如癌症、炎症和动脉粥样硬化。公共卫生相关性：这项工作的意义在于它有可能揭示硫酸乙酰肝素形成中涉及的新调节因素。以这种方式确定的每个因素都为未来的研究提供了新的项目。此外，可能会出现候选基因，这些基因可以解释硫酸乙酰肝素形成出错的疾病，例如癌症、炎症和动脉粥样硬化，这反过来又可以确定药物开发的新靶标。