MUTATIONS AFFECTING LIPOPROTEIN METABOLISM

影响脂蛋白代谢的突变

• 批准号: 6564845
• 负责人: Karen Reue
• 金额: $ 23.48万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564845
• 关键词: blood lipoprotein metabolism; calnexin; enzyme deficiency; fatty acid metabolism; gene mutation; genetic mapping; genetic models; genetic regulation; genetic screening; genetic strain; human genetic material tag; hypertriglyceridemia; inborn lipid /lipoprotein disorder; insulin sensitivity /resistance; laboratory mouse; lipid transport; lipoprotein lipase; molecular pathology; phenotype; protein structure function; quantitative trait loci; tissue /cell culture; transcription factor

Naturally occurring mutations in the mouse represents a rich for identification of novel genes affecting lipid metabolism. In this project, we will isolate the genes for two mouse mutations affecting triglyceride metabolism, fatty liver dystrophy (fld) and combined lipase deficiency (cld). The fld/fld mutant mice exhibit a transient hypertriglyceridemia and fatty liver during neonatal development. We recently determined that these animals subsequently develop insulin resistance and are grossly deficient in stores of white adipose tissue, and hypothesize that the fld mutation affects a component of the insulin signal transduction pathway. The hallmark feature of cld/cld mice is hypertriglyceridemia that is fatal within 2-3 days of birth, caused by the virtual absence of lipoprotein lipase and hepatic lipase activities. Recently we have determined that the basis for the defect is misfolding of newly synthesized lipase molecules in the endoplasmic reticulum. Our two aims will focus on isolation of the fld and cld genes and characterization of the cellular processes disrupted by the mutations. In aim 1 we will identify the two genes using positional and functional cloning strategies. In the case of fld, we have narrowed the critical region to approximately kb using genetic and physical strategies. In the case of fld, we have narrowed the critical region to approximately kb using genetic and physical mapping; analysis of this region via exon trapping has identified an fld gene candidate that appears to be rearranged in the fld genome and is not expressed in tissues from the mutant mouse. We will now confirm this candidate by sequence analysis; additional candidate genes that we have isolated from this region by exon trapping will be analyzed if needed. In the case of cld, the gene critical region has been isolated as a series of over-lapping yeast and bacterial artificial chromosome clones spanning a distance of approximately 2 megabases. We are now poised to identify the clone containing the cld gene by complementation in tissue culture cell line, followed by gene identification using methods that have proved successful for the fld mutation. In Aim 2, key questions concerning the molecular basis of the mutant phenotypes will be investigated. For fld these include determining the underlying defect in impaired glucose uptake by fld tissues, utilizing the isolated gene to explore protein function and evaluating the role of this gene in human diseases characterized by insulin resistance (e.g., FCHL, diabetes, atherosclerosis). For cld, we will investigate whether reduced levels of a specific chaperone, calnexin, contribute to the lipase deficiency that is the hallmark of this mutation. Once the gene is isolated, studies will focus on cld protein function and its potential role in human subjects exhibit combined lipase deficiency.

小鼠中自然发生的突变代表了对影响脂质代谢的新基因的丰富鉴定。在这个项目中，我们将分离两种影响甘油三酯代谢的小鼠突变基因，脂肪肝营养不良（fld）和联合脂肪酶缺乏症（cld）。fld/fld突变小鼠在新生儿发育过程中表现出短暂的高血脂和脂肪肝。我们最近确定这些动物随后发展为胰岛素抵抗，并且严重缺乏白色脂肪组织的储存，并假设fld突变影响胰岛素信号转导途径的一个组成部分。cld/cld小鼠的标志性特征是在出生后2-3天内致命的高脂血症，这是由于脂蛋白脂肪酶和肝脂肪酶活性的实际缺乏引起的。最近，我们已经确定，缺陷的基础是错误折叠的新合成的脂肪酶分子在内质网。我们的两个目标将集中在fld和cld基因的分离和突变破坏的细胞过程的表征。在目标1中，我们将使用位置和功能克隆策略识别这两个基因。在fld的情况下，我们使用遗传和物理策略将关键区域缩小到大约kb。在fld的情况下，我们已经缩小了关键区域约kb的遗传和物理作图，通过外显子捕获分析这一地区已确定了一个fld基因候选人，似乎是重新排列的fld基因组中，并没有在组织中表达的突变小鼠。我们现在将通过序列分析确认该候选基因;如果需要，将分析我们通过外显子捕获从该区域分离的其他候选基因。在cld的情况下，基因关键区域已经被分离为一系列重叠的酵母和细菌人工染色体克隆，跨越大约2兆碱基的距离。我们现在正准备通过组织培养细胞系中的互补作用来鉴定含有cld基因的克隆，然后使用已证明对fld突变成功的方法进行基因鉴定。在目标2中，将研究关于突变表型的分子基础的关键问题。对于fld，这些包括确定fld组织葡萄糖摄取受损的潜在缺陷，利用分离的基因探索蛋白质功能，并评估该基因在以胰岛素抵抗为特征的人类疾病中的作用（例如，FCHL、糖尿病、动脉粥样硬化）。对于cld，我们将研究是否一个特定的伴侣，钙连接蛋白的水平降低，有助于脂肪酶缺乏，这是该突变的标志。一旦该基因被分离出来，研究将集中在cld蛋白的功能及其在联合脂肪酶缺乏症中的潜在作用。