MOLECULAR ANALYSIS OF CYSTATHIONE BETA SYNTHASE DISORDERS IN HUMAN DISEASE

该提案的长期目标是确定胱硫醚β-合酶（CBS）基因的异常表达如何在人类疾病中起作用，特别是高同型半胱氨酸尿症和由于高同型半胱氨酸血症引起的血管闭塞性疾病。CBS是硫代谢分支的中心酶，是63 kDa亚基的四聚体;该酶结合两种底物，同型半胱氨酸和丝氨酸，以及三种配体，PLP，PLMet和血红素。它在人类21号染色体上编码;它的遗传缺陷导致最常见的同型胱氨酸尿症。我们已经分离了酵母、小鼠、大鼠和人CBS基因的cDNA和基因组克隆。我们最近获得了人类基因的完整的28-kbp DNA序列，包括启动子区上游约5 kbp的序列。我们将人CBS cDNA克隆到几个融合表达载体中，并开发了纯化大量全长和截短酶的方法。我们证明了CBS是一种血红素，是一种含血红素的酶，卟啉是PLP结合和活性所必需的。到目前为止，我们与我们的合作者一起确定了100种同型胱氨酸尿症突变。我们的具体目标是1）研究市售的CBS敲除小鼠模型，以确定CBS基因被破坏的纯合子动物早期致死的原因，并通过治疗提高其存活率。如果存活率可以得到充分改善，我们将研究动物是否出现高胱氨酸尿症的临床信号; 2）彻底表征CBS的转录调节; 3）研究CBS的翻译后调节; 4）对人体中CBS的组织特异性调节进行详细分析; 5）确定酵母是否是研究血红素在CBS中作用的合适模型系统; 6）继续优化CBS的表达、纯化和结晶条件，以通过X射线衍射解析CBS的结构。这个项目的作用是扩大我们的知识的初级到三级酶的结构，并调查的可能性，CBS的异常调节是一个因素，对心血管，脑血管和神经退行性疾病的易感性。

The long-term objective of this proposal are to define how abnormal expression of the cystathionine beta-synthase (CBS) gene plays a role in human disease, in particular, homocystinuria and vascular occlusive disease due to hyperhomocysteinemia. CBS, a central enzyme at a branch pint in sulfur metabolism, is a tetramer of 63 kDa subunits; the enzyme binds two substrates, homocysteine and serine, and three ligands, PLP, AdoMet, and heme. It is encoded in humans on chromosome 21; it's inherited deficiency causes the most common form of homocystinuria. We have isolated cDNA and genomic clones for yeast, mouse, rat, and human CBS genes. We have recently obtained the full 28-kbp DNA sequence of the human gene including approximately 5kbp of sequence upstream of the promoter region. We cloned the human CBS cDNA into several fusion expression vectors and developed methods to purify large amounts of the full-length and truncated enzyme to homogeneity. We demonstrated that CBS is a heme is a heme-containing enzyme, and that the porphyrin is required for both PLP binding and activity. We have, together with our collaborators, identified 100 mutations in homocystinuria so far. Our specific aims are 1) To study the commercially available CBS-knockout mouse model to identify the cause of early lethality in animals homozygous for the disrupted CBS gene and to improve their survival by treatment. If survival can be sufficiently improved we will investigate if the animals develop the clinical signals of homocystinuria; 2) to thoroughly characterize the transcriptional regulation of CBS; 3) to investigate the post-translational regulation of CBS; 4) to perform a detailed analysis of the tissue specific regulation of CBS in humans; 5) to determine if yeast is a suitable model system in which to investigate the role of heme in CBS; 6) to continue to optimize the expression, purification, and the crystallization conditions for solving the structure of CBS by X-ray diffraction. The role of this project is to extend our knowledge of the primary to the tertiary enzyme structure and to investigate the possibility that aberrant regulation of CBS is a factor in predisposition towards cardiovascular, cerebrovascular and neurodegenerative disease.