Metabolic Pathways and Defects in Fructose Metabolism

果糖代谢的代谢途径和缺陷

• 批准号: 6822269
• 负责人: Dean R. TOLAN
• 金额: $ 35.22万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6822269
• 关键词: X ray crystallography; aldehyde lyase; carbohydrate metabolism; clinical research; fructose; fructose phosphate; gene expression profiling; gene mutation; genetically modified animals; hereditary fructose intolerance; human genetic material tag; human population genetics; human subject; immunocytochemistry; in situ hybridization; laboratory mouse; molecular biology information system; polymerase chain reaction

DESCRIPTION (provided by applicant): Long-term ingestion has effects on diabetes and obesity. The most drastic and common genetic disorder of fructose metabolism is hereditary fructose intolerance (HFI). Lack of knowledge about sites of fructose metabolism and about genotype-phenotype relationships still exists for this disease and reflects the incomplete understanding of normal fructose metabolism. Answers to two major questions will provide new information. First, other than liver and kidney, what other tissues play a role in fructose metabolism? Second, can small molecules be found that stabilize the major defective enzyme in HFI, that harboring an A149P substitution (AP-aldolase)? The proposed investigations will, 1) define sites for fructose assimilation and utilization using a combination of bioinformatics and molecular approaches, 2) determine a high-resolution structure of AP-aldolase, and use it to find stabilizing small-molecule ligands by both structure-based ligand design (SBLD) and high-throughput screening of chemical libraries, 3) create animal models for HFI using gene-targeting techniques, and 4) identify HFI mutations in the diverse US population, in particular Hispanic, African-American, and other ethnic groups that have not been well characterized, and correlate these findings to any specific phenotypes in these ethnic groups. The large database of expressed sequence tags (dbEST) will be analyzed for overlapping expression profiles of the GLUT5, GLUT2, ketohexokinase, aldolase, hexokinase, and triose kinase in both mouse and humans to predict alternative sites of fructose metabolism. Verification and characterization of these global predictions will be done by quantitative reverse-transcriptase polymerase chain reaction (Q-PCR), RNA in situ hybridization (RISH), and identification of metabolic intermediates during the oxidation of radioactive fructose. For the second hypothesis, a high-resolution structure of AP-aldolase will be determined by macromolecular X-ray crystallography. Screening large libraries of small molecules (produced by combinatorial chemistry) using a thermal-stability assay of AP-aldolase in conjunction with SBLD will identify small molecules that restore enzyme function. Should the gene-targeted animal model mimic the human HFI pathology, the metabolic profiles and sites of fructose metabolism will be determined. Should the animal model be asymptomatic, differences in the ability to metabolize fructose will be compared to previously determined sites and pathways for fructose metabolism in normal mice and humans. Lastly, blood samples from African-American and Hispanic-American HFI subjects will be used for identification of gene defects by direct DNA sequencing, thus offering a reliable non-invasive diagnostic method to these Americans.

描述（由申请人提供）：长期摄入对糖尿病和肥胖有影响。果糖代谢最严重和最常见的遗传性疾病是遗传性果糖不耐受（HFI）。缺乏有关果糖代谢位点和基因型-表型关系的知识仍然存在这种疾病，反映了对正常果糖代谢的不完全理解。对两个主要问题的回答将提供新的信息。首先，除了肝脏和肾脏，还有哪些组织在果糖代谢中发挥作用？第二，能否找到小分子来稳定HFI中的主要缺陷酶，即携带A149 P取代（AP-醛缩酶）？这些研究将：1）结合生物信息学和分子生物学方法确定果糖同化和利用的位点，2）确定AP-醛缩酶的高分辨率结构，并通过基于结构的配体设计（SBLD）和化学文库的高通量筛选来寻找稳定的小分子配体，3）使用基因靶向技术建立HFI的动物模型，和4）鉴定不同美国人群，特别是西班牙裔、非洲裔美国人和其他尚未被很好表征的种族群体中的HFI突变，并将这些发现与这些种族群体中的任何特定表型相关联。将分析表达序列标签（dbEST）的大型数据库中小鼠和人类中GLUT 5、GLUT 2、己酮糖激酶、醛缩酶、己糖激酶和丙糖激酶的重叠表达谱，以预测果糖代谢的替代位点。将通过定量逆转录酶聚合酶链反应（Q-PCR）、RNA原位杂交（RISH）和放射性果糖氧化过程中代谢中间体的鉴定对这些全局预测进行验证和表征。对于第二个假设，AP-醛缩酶的高分辨率结构将通过大分子X射线晶体学来确定。使用AP-醛缩酶的热稳定性测定结合SBLD筛选大的小分子文库（通过组合化学产生）将鉴定恢复酶功能的小分子。如果基因靶向动物模型模拟人HFI病理，则将确定果糖代谢的代谢谱和位点。如果动物模型是无症状的，则将代谢果糖的能力的差异与先前确定的正常小鼠和人类中果糖代谢的位点和途径进行比较。最后，来自非洲裔美国人和西班牙裔美国人HFI受试者的血液样本将用于通过直接DNA测序鉴定基因缺陷，从而为这些美国人提供可靠的非侵入性诊断方法。