Molecular basis of inborn errors of ketone body metabolism:mainly tertiary structural changes of protein and abnormalities of splicing

对酮体代谢先天缺陷的分子基础进行了研究，特别是错义突变和异常剪接引起的三级结构变化。在与芬兰团队的合作下，我们首次展示了人类线粒体乙酰乙酰-辅酶A硫解酶(T2)同源四聚体的晶体结构。并揭示了POTS硫离子的活化机理。我们发现了T2缺乏症和琥珀酰辅酶A：3-酮酸辅酶A转移酶缺乏症的突变。我们对7个新的错义突变进行了瞬时表达分析，并对其温敏活性和稳定性进行了评价。然后讨论了突变体对三级结构的影响。在SCOT缺乏症的情况下，我们鉴定并鉴定了一种独特的剪接突变。在T2缺乏症中，我们还发现了一个外显子突变，它激活了其上游5个碱基的隐蔽剪接供体位点。通过微型基因剪接实验，我们明确地证明了该外显子突变是导致异常剪接的原因。我们还发现，ALU介导的基因组重排是某些患者T2缺陷的原因之一。

The study for molecular basis of inborn errors of ketone body metabolism, especially focused on tertiary structural changes by missense mutations and abnormal splicing, was performed. In collaboration with Finland group, we first showed crystal structure of human mitochondrial acetoacetyl-CoA thiolase (T2) homotetramer. and showed a mechanism of pots ssiurnion activation. We identified mutations in T2 deficiency and succinyl-CoA: 3-ketoacid CoA transferase deficiency. We analyzed 7 new missense mutations by transient expression analysis of cDNA and evaluated temperature sensitive activity and stability. Then we discussed the effect of mutant on the tertiary structure. In case of SCOT deficiency, we identified and characterized a unique splicing mutation. In T2 deficiency, we also identified an exonic mutation which activates cryptic splice donor site just its 5-base upstream. We dearly showed that this exonic mutation was responsible for the aberrant splicing by mini gene splicing experiment. We also showed that alu-mediated genomic rearrangement is one of the cause of T2 deficirncy in some patients.