Metabolic Pathways and Defects in Fructose Metabolism

描述(申请人提供)：长期摄入会导致糖尿病和肥胖症。最严重和最常见的果糖代谢遗传性疾病是遗传性果糖不耐受(HFI)。这种疾病仍然缺乏果糖代谢部位和基因-表型关系的知识，反映了对正常果糖代谢的不完全了解。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，能否找到稳定HFI中主要缺陷酶的小分子，这种酶含有A149P取代(AP-醛缩酶)？拟议的研究将，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-醛缩酶的高分辨率结构，并利用它通过基于结构的配体设计(SBLD)和高通量筛选化学文库来寻找稳定的小分子配体，3)使用基因打靶技术创建HFI的动物模型，以及4)识别不同美国人群中的HFI突变，特别是西班牙裔、非裔美国人和其他尚未得到很好特征的种族群体，并将这些发现与这些种族中的任何特定表型相关联。将对表达序列标签的大型数据库(DBEST)进行分析，以寻找小鼠和人类中GLUT5、GLUT2、酮已糖激酶、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聚合酶链式反应(Q-PCR)、RNA原位杂交(RISH)和鉴定放射性果糖氧化过程中的代谢中间产物来完成。对于第二种假设，AP-醛缩酶的高分辨结构将由大分子X射线结晶学确定。使用AP-醛缩酶的热稳定性分析结合SBLD筛选大的小分子文库(通过组合化学产生)将识别恢复酶功能的小分子。如果以基因为靶标的动物模型模拟人类HFI的病理，将确定果糖代谢的代谢概况和位置。如果动物模型没有症状，则将果糖代谢能力的差异与之前在正常小鼠和人类中确定的果糖代谢部位和途径进行比较。最后，非裔美国人和西班牙裔美国人的血液样本将被用于通过DNA直接测序来识别基因缺陷，从而为这些美国人提供一种可靠的非侵入性诊断方法。

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.