Metabolic Pathways and Defects in Fructose Metabolism

果糖代谢的代谢途径和缺陷

• 批准号: 7209541
• 负责人: Dean R. TOLAN
• 金额: $ 2.76万
• 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/7209541
• 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.

说明（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）。缺乏对果糖代谢位点和基因型-表型关系的了解仍然存在，反映了对正常果糖代谢的不完全了解。两个主要问题的答案将提供新的信息。首先，除了肝脏和肾脏，还有哪些组织在果糖代谢中起作用？第二，能否找到小分子来稳定HFI中的主要缺陷酶，这些酶含有A149P取代（ap醛缩酶）？拟议的研究将包括：1)结合生物信息学和分子方法确定果糖同化和利用的位点；2)确定ap醛dolase的高分辨率结构，并通过基于结构的配体设计（sld）和化学文库的高通量筛选，用它来寻找稳定的小分子配体；3)使用基因靶向技术创建HFI动物模型；4)在不同的美国人群中识别HFI突变。特别是西班牙裔、非裔美国人和其他尚未被很好地表征的种族群体，并将这些发现与这些种族群体的任何特定表型联系起来。研究人员将利用大型表达序列标签数据库（dbEST）分析小鼠和人类中GLUT5、GLUT2、酮己糖激酶、醛缩酶、己糖激酶和三糖激酶的重叠表达谱，以预测果糖代谢的替代位点。这些全球预测的验证和表征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来完成。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 -醛缩酶的热稳定性试验结合sld筛选大的小分子文库（由组合化学产生），将确定恢复酶功能的小分子。如果基因靶向动物模型模拟人类HFI病理，果糖代谢的代谢谱和部位将被确定。如果动物模型无症状，则将果糖代谢能力的差异与正常小鼠和人类先前确定的果糖代谢位点和途径进行比较。最后，非裔美国人和西班牙裔美国人HFI受试者的血液样本将通过直接DNA测序来鉴定基因缺陷，从而为这些美国人提供可靠的非侵入性诊断方法。