Gene Discovery and Heparan Sulfate Biogenesis

描述(申请人提供)：基因发现和硫酸乙酰肝素生物发生。动物细胞表达多种硫酸乙酰肝素蛋白多糖(HSPGs)，它由不同的蛋白核心和一条或多条硫酸乙酰肝素链组成。HSPG结合了大量的生长因子、细胞因子、细胞外基质成分、酶和酶抑制剂，它们作为细胞信号、细胞附着和内吞的受体或辅助受体。这些相互作用在很大程度上取决于硫酸乙酰肝素链上糖醛酸的组成和排列，而这又取决于各种生物合成酶和调节因子的底物专一性。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、生化研究，并在细胞或模式生物中发生突变。相反，关于引起硫酸乙酰肝素不同组成和结合特性的机制的信息很少。这项赠款的中心假设是，除了那些编码生物合成酶的基因外，还有其他基因存在，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状RNA的稳定表达的基因沉默技术来寻找这些调控因子。为了实现这一目标，我们将(1)采用高通量筛选方法来鉴定对其作用依赖于HSPGs(FGF2-Saporin和白喉毒素)的细胞毒剂产生抗性的shRNA；以及(2)通过扩增和测序相关shRNA克隆来鉴定调节HSPGs组成的基因。通过这种方式识别的基因将根据预测或已知的功能被分类，个别候选基因将在不同的细胞系中进行测试。还将进行HS的详细结构研究和与测试配体的结合研究。这项工作的意义在于有可能发现与HSPG表达有关的新的调节因子。这些实验的结果有一个更广泛的目标，那就是将该领域带入新的研究领域。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的项目和一个候选基因，可以解释HSPG表达出错的疾病，如癌症、炎症和动脉粥样硬化。深入了解细胞用来调节硫酸肝素成分的机制也可能导致治疗与硫酸肝素形成变化相关的人类疾病的新药物靶点，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与硫酸肝素形成有关的新的调节因素。通过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，候选基因可能会出现，可以解释硫酸肝素形成错误的疾病，如癌症、炎症和动脉粥样硬化，这反过来可能为药物开发定义新的靶点。

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.