MOLECULAR ANALYSIS OF CYSTATHIONE BETA SYNTHASE DISORDERS IN HUMAN DISEASE

本提案的长期目标是确定胱硫氨酸β -合成酶（CBS）基因的异常表达如何在人类疾病中发挥作用，特别是高同型半胱氨酸血症引起的同型半胱氨酸尿和血管闭塞性疾病。CBS是硫代谢分支上的中心酶，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， AdoMet和血红素。它在人类的21号染色体上编码；这种遗传缺陷导致了最常见的同型半胱氨酸尿。我们已经分离了酵母、小鼠、大鼠和人类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28 kbp DNA序列，其中包括启动子区域上游的约5kbp序列。我们将人CBS cDNA克隆到多个融合表达载体中，并开发了大量纯化全长和截断酶的方法，以达到均匀性。我们证明了CBS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，我们和我们的合作者已经确定了100个同型半胱氨酸尿的突变。我们的具体目标是：1)研究市售的CBS基因敲除小鼠模型，以确定被破坏的CBS基因纯合的动物早期死亡的原因，并通过治疗提高它们的存活率。如果存活率能够得到充分提高，我们将研究这些动物是否会出现同型半胱氨酸尿的临床信号；2)深入表征CBS的转录调控；3)研究CBS翻译后的规制；4)对人体CBS的组织特异性调控进行详细分析；5)确定酵母是否是研究血红素在CBS中的作用的合适模型系统；6)继续优化用x射线衍射法求解CBS结构的表达、纯化和结晶条件。该项目的作用是将我们对初级酶结构的认识扩展到三级酶结构，并研究CBS异常调节是心脑血管和神经退行性疾病易感性因素的可能性。

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.