MOLECULAR GENETIC STUDIES ON METHYLMALONYL MUTASE

这项申请是我们第一次颁发相同奖项的延续 头衔。我们的进展报告描述了旨在 人和小鼠甲基丙二酰辅酶A基因的克隆与鉴定 突变酶(MCM)cDNA，编码MCM的基因组基因座(mut，mut)，以及 突变导致MCM缺乏症的独特表型。我们有 建立了将基因转移到原代成纤维细胞和S. Ceriviseae，并使用这些系统来表征功能缺陷 突变型基因产物及其对过表达的生物学反应 MCM。这项建议的重点是应用分子方法来回答 我们正在进行的关于MCM的能动作用的工作提出的问题 代谢流、动态平衡和疾病。这项工作将重点放在 三个问题。MCM基因突变是如何影响酶活性的？ 突变分析已经描绘出两个独立的功能结构域 MCM：一个与钴胺结合结构域有关，另一个与 参与等位基因互补的待定功能。 我们将识别特定的突变以评估其位置和功能 MCM中的其他域，并探索 MCM缺乏症的基因分型和生化或临床表型。 什么机制调节酶的活性水平？初步 研究已经确定了几种可能决定水平的机制 对MCM活动的影响。我们将进一步评估组织特异性的模式 MCM基因的表达及序列在启动子中的作用 和可能影响转录或翻译调控的c DNA。 MCM。MCM中MCM酶活性和突变的调节 通过MCM影响代谢流量？我们已经证明了这个水平 MCM酶活性的变化通常不会限制代谢产物的流量 丙酸通过成纤维细胞中的MCM。我们将研究这一关系 肝癌组织中MCM酶活性与MCM代谢通量的关系 过表达重组MCM和抑制内源性酶对细胞的影响 带有反义结构的表达。我们将调查MCM是否 与氨基酸相比，在代谢流量中具有不同的动力学作用 丙酸或丙二酸甲酯。最后，我们将调查 观察到某些突变等位基因抑制丙酸的机制 在存在高于正常水平的MCM时通过MCM的通量 酶活性；一种表明MCM可能是一种 较大的多酶复合体的组成部分，在 测定丙酸代谢能力。这些研究是 旨在提供对MCM在以下方面的作用的动态理解 代谢流量和动态平衡以及MCM的后果 缺乏症。

This application is a continuation of our FIRST award with the same title. Our PROGRESS REPORT describes molecular genetic studies aimed at cloning and characterizing cDNAs for human and mouse methylmalonyl CoA mutase (MCM) cDNAs, the genomic loci (MUT, mut) encoding MCM, and mutations causing distinctive phenotypes of MCM deficiency. We have established methods for gene transfer into primary fibroblasts and S. ceriviseae and used these systems to characterize functional defects in mutant gene products and the biological response to overexpression of MCM. This proposal focuses on applying molecular methods to answer questions raised by our ongoing work concerning the dynamic role of MCM in metabolic flux, homeostasis, and disease. This work will focus on three questions. How do mutations in MCM affect enzyme activity? Mutation analysis has delineated two independent functional domains in MCM: one involved in cobalamin binding domain, and a second with undetermined function which participates in interallelic complementation. We will identify select mutations to assess the location and function of other domains within MCM and explore the relationship between the genotype and the biochemical or clinical phenotype of MCM deficiency. What mechanisms regulate the level of enzyme activity? Preliminary studies have identified several mechanisms which may determine the level of MCM activity. We will further assess the pattern of tissue specific expression of MCM mRNA as well as the role of sequences in the promoter and cDNA which may affect transcriptional or translational regulation of MCM. How does regulation of MCM enzyme activity and mutations in MCM affect metabolic flux through MCM? We have demonstrated that the level of MCM enzyme activity does not normally limit flux of metabolites from propionate through MCM in fibroblasts. We will study the relationship between MCM enzyme activity and metabolic flux through MCM in hepatoma cells by overexpressing recombinant MCM and inhibiting endogenous enzyme expression with antisense constructs. We will investigate whether MCM has a different kinetic role in metabolic flux from amino acids compared to propionate or methylmalonate. Finally, we will investigate the mechanism for the observation that some mutant alleles inhibit propionate flux through MCM in the presence of higher than normal levels of MCM enzyme activity; an observation which suggests that MCM may be a component of a larger multi-enzyme complex with a critical role in determining the capacity for propionate metabolism. These studies are intended to provide a dynamic understanding of the role of MCM in metabolic flux and homeostasis as well as the consequences of MCM deficiency.