Research for the relationship of hereditary sideroblastic anemia with enzyme complex formation in mitochondria

1. 为了研究红系特异性5-氨基乙酰酸合成酶（ALAS-E）和atp特异性琥珀酰辅酶a合成酶（A-SCS）能否在体外形成功能酶复合物，我们利用基于baco -virus的表达系统表达了这两种酶（ALAS-E、SCS-α、SCS-βA和SCS-βG）组分的重组蛋白。在本实验中，我们成功纯化了具有酶活性的ALAS-E重组蛋白，但纯化组分（SCS-α和SCS-βA = A-SCS，或SCS-α和SCS-βG = G-SCS）的任何组合都无法检测到SCS的酶活性。这些结果表明，具有酶活性的SCS可能需要磷酸化或糖基化，这仅发生在哺乳动物细胞中。另外，哺乳动物SCS可能需要特定的辅助因子来维持其催化活性。如果存在这样的辅助因子，则辅助因子的减少可能导致铁母细胞性贫血。为了确定ala - e与SCS-βA相互作用的特定区域，制备了几个ala - e蛋白缺失突变体。然后，利用酵母双杂交系统测定突变体ala - e蛋白与SCS-βA蛋白的相互作用。结果表明，ALAS-E的n端缺失147个氨基酸或c端缺失50个氨基酸，破坏了ALAS-E与SCS-βA蛋白的相互作用。由于这些ALAS-E的缺失突变体破坏了ALAS-E的同型二聚体的形成，因此ALAS-E的同型二聚体的形成可能对线粒体中ALAS-E和A-SCS的酶复合物的形成很重要。

1. To examine whether Erythroid specific 5-aminolevulinate synthase (ALAS-E) and ATP-specific succinyl CoA synthethase (A-SCS) form functional enzyme complex in vitro, we have expressed recombinant proteins of components of each enzyme (ALAS-E, SCS-α, SCS-βA and SCS-βG) using Baclo-virus based expression system. In this experiment, we have successfully purified enzymatically active ALAS-E recombinant protein, however, enzymatic activity of SCS could not be detected in any combination of purified component (SCS-α and SCS-βA = A-SCS, or SCS-α and SCS-βG = G-SCS). These results suggest that phosphorilation or glycosilation SCS might be needed for enzymatically active SCS, which specifically occur in only mammalian cells. Alternatively, mammalian SCS may request specific co-factor(s) for its catalytic activity. If such co-factor(s) exist, decrease amount of the co-factor may cause sideroblastic anemia.2. To determine the specific region for interaction of ALAS-E and SCS-βA, several deletion mutants of ALAS-E protein were made. Then, the interaction of such mutant ALAS-E proteins and SCS-βA protein were determined using yeast two hybrid system. As a results, 147 amino acid deletion of N-terminal or 50 amino acid deletion of C-terminal of ALAS-E disrupt the interaction of ALAS-E and SCS-βA protein. Since these deletion mutant of ALAS-E results the disruption of homo-dimer formation of ALAS-E, homodimer formation of ALAS-E might be important for enzyme complex formation of ALAS-E and A-SCS in mitochondria.