Gene Discovery and Heparan Sulfate Biogenesis

描述（由申请人提供）：基因发现和硫酸肝素生物发生。动物细胞精心制作各种硫酸肝素蛋白聚糖（HSPGs），它们由不同的蛋白质核心和一个或多个硫酸肝素链组成。HSPGs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，它们作为细胞信号传导、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肝素链中硫酸糖和糖醛酸外显体的组成和排列，而硫酸肝素链又取决于各种生物合成酶和调节因子的底物特异性。大多数参与硫酸肝素生物生成的酶已经被鉴定、克隆、以重组蛋白表达、生物化学研究和在细胞或模式生物中突变。相比之下，关于引起硫酸肝素的可变组成和结合特性的机制缺乏信息。该基金的中心假设是，除了编码生物合成酶的基因外，还存在其他基因，这些基因的表达或调节酶的转录/翻译，或协调酶的作用，以实现在不同细胞类型中观察到的硫酸肝素的最终组成。我们的目标是通过基于短发夹rna在整个人类基因组中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对依赖于HSPGs （fgf2 -皂苷和白喉毒素）的细胞毒性药物产生抗性的shrna；(2)通过相关shRNA克隆的扩增和测序，鉴定和表征调控HSPGs组成的基因。以这种方式鉴定的基因将根据预测的或已知的功能分类，个别候选基因将在不同的细胞系中进行测试。详细的HS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将该领域推向新的研究领域的更广泛的目标。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，并提供了一个可能解释HSPG表达错误的疾病的候选基因，如癌症、炎症和动脉粥样硬化。对细胞用来调节硫酸肝素组成的机制的深入了解也可能导致新的药物靶点，用于治疗与硫酸肝素形成改变相关的人类疾病，如癌症、炎症和动脉粥样硬化。

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.