MEMBRANE DEFECT IN THE SLO SYNDROME

SLO 综合征中的膜缺陷

• 批准号: 6781828
• 负责人: Thomas N. Tulenko
• 金额: $ 27.12万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-12 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6781828
• 关键词: Smith Lemli Opitz syndrome; calcium flux; cell proliferation; cellular pathology; cholesterol; clinical research; cyclodextrins; enzyme deficiency; fibroblasts; folate; human tissue; inborn lipid /lipoprotein disorder; inositol phosphates; laboratory mouse; lipid biosynthesis; membrane biogenesis; membrane permeability; membrane structure; molecular pathology; oxidized lipid; sodium potassium exchanging ATPase; steroid biosynthesis

DESCRIPTION (provided by applicant): The Smith-Lemli-Opitz (SLOS) syndrome is an often lethal, autosomal recessive birth defect characterized by widespread neurological, skeletal and anatomical abnormalities including cleft palate in afflicted subjects. This disorder has been shown to be caused by an inheritable metabolic defect in which the enzyme that catalyzes the final step in cholesterol biosynthesis, DHCR7, is genetically deficient. In SLOS, blood and tissue levels of cholesterol are greatly suppressed while the levels of the principle precursor to cholesterol synthesis, 7 dehydrocholesterol (7-DHC) are greatly elevated. Because cholesterol is absolutely required for the normal biosynthesis of cell membranes as well as steroid and sex hormones, it is thought that the suppressed cholesterol synthesis in SLOS patients underlies the widespread tissue and organ malformations. While the metabolic defect underlying SLOS has been defined, its impact on cell function has not been determined. In skin fibroblasts obtained from SLOS patients, we have generated preliminary data demonstrating that membrane calcium permeability is markedly augmented while membrane-bound Na+/7K+ATPase activity, folate uptake and IP3 signaling is markedly suppressed. Furthermore, employing X-ray diffraction analyses in synthetic membranes prepared to mimic SLOS membranes, we observed a highly atypical membrane structure. We have developed the hypothesis that in SLOS patients, the deficiency in cholesterol biosynthesis leads to the production of cell membranes that are defective in their composition, dynamics and function, and that this membrane defect contributes to the cellular pathobiology in these patients. In this study, Aim 1 will determine whether defects exist in composition, structure and fluidity of the cell plasma membrane of skin fibroblasts obtained from SLOS patients and DHCR7 transgene knockout (k/o) mice. Aim 2 will determine whether these membrane defects correlate with impaired membrane function (folate uptake, Ca++ permeability, Na+/K+ATPase activity and IP3 signaling) and cell proliferation. Aim 3 will determine the degree to which cell and membrane functions can be corrected by restoring the membrane sterols back to normal levels. This project employs biophysical, biochemical and cell biology approaches to study fibroblasts obtained from SLOS patients and DHCR7 k/o mice in an attempt to shed light on the cellular basis underlying this profoundly debilitating disease which has no cure. By defining the membrane defects in SLOS we will advance our understanding of cell and molecular basis of this disease. In addition, we expect to validate the DHCR7 k/o mouse model of SLOS and thereby provide greater opportunity for us and others to study SLOS in the hope of providing clinical relief to patients faced with this disease.

描述(申请人提供)：Smith-Lemli-Opitz(SLOS)综合征是一种通常致命的常染色体隐性出生缺陷，其特征是广泛的神经、骨骼和解剖学异常，包括受试者的腭裂。这种疾病被证明是由一种遗传的代谢缺陷引起的，在这种缺陷中，催化胆固醇生物合成最后一步的酶DHCR7是遗传缺陷的。在SLOS中，血液和组织中的胆固醇水平大大受到抑制，而胆固醇合成的主要前体7-脱氢胆固醇(7-DHC)的水平则大幅上升。由于胆固醇是细胞膜以及类固醇和性激素正常生物合成所必需的，因此人们认为SLOS患者胆固醇合成受到抑制是广泛的组织和器官畸形的基础。虽然SLOS潜在的代谢缺陷已经确定，但其对细胞功能的影响尚未确定。在SLOS患者的皮肤成纤维细胞中，我们产生的初步数据表明，膜钙通透性显著增强，而膜结合的Na+/7K+ATPase活性、叶酸摄取和IP3信号显著抑制。此外，在模拟SLOS膜制备的合成膜中使用X射线衍射分析，我们观察到了高度非典型的膜结构。我们提出的假设是，在SLOS患者中，胆固醇生物合成的缺陷导致细胞膜的产生在其组成、动力学和功能方面存在缺陷，并且这种膜缺陷导致了这些患者的细胞病理生物学。在这项研究中，目的1将确定SLOS患者和转基因敲除(k/o)小鼠皮肤成纤维细胞的细胞膜成分、结构和流动性是否存在缺陷。目的2将确定这些膜缺陷是否与膜功能受损(叶酸摄取、钙通透性、Na+/K+ATPase活性和IP3信号)和细胞增殖有关。目标3将确定通过将膜类固醇恢复到正常水平可以纠正细胞和膜功能的程度。该项目使用生物物理、生化和细胞生物学方法研究从SLOS患者和KhR7 k/o小鼠获得的成纤维细胞，试图揭示这种无法治愈的严重虚弱疾病背后的细胞基础。通过定义SLOS的膜缺陷，我们将加深对该病的细胞和分子基础的理解。此外，我们还希望验证SLOS的DHCR7 k/o小鼠模型，从而为我们和其他人提供更多研究SLOS的机会，希望能为面临这种疾病的患者提供临床缓解。