Multiple mechanisms for the regulation of heme biosynthesis and catabolism.

血红素是几种酶的辅基，并且是需氧生物体所必需的。由于细胞中过量的血红素增加了活性氧，因此细胞血红素水平在其生物合成和催化平衡下受到严格调节。为了了解血红素的生物合成和催化是如何通过蛋白质-蛋白质相互作用的机制来调节的，我们进行了以下实验1。为了了解哪些蛋白参与血红素生物合成和催化的调控，我们使用非特异性氨基乙酰丙酸合酶、红系特异性氨基乙酰丙酸合酶（ALAS 2）、血红素加氧酶1或血红素加氧酶2（HO-2）作为诱饵蛋白筛选了人cDNA文库。结果发现，20 S蛋白酶体的一个组分可以与HO-2蛋白结合。由于该蛋白的过表达降低了HO-2蛋白的表达水平，因此该蛋白应该参与了HO-2蛋白降解的调节。2.使用RNA干扰 ...更多信息 利用RNA干扰技术，我们尝试建立铁粒幼细胞性贫血的模型细胞。我们首先制备了有效抑制ALAS 2表达的小干扰RNA（small interfering RNA，siRNA），然后制备了表达有效短发夹RNA（short hairpin RNA，shRNA）的表达载体。将该载体导入在转化生长因子β 1刺激后能够在细胞内产生血红蛋白的YN-1细胞中，用G418孵育这些细胞，以选择组成型表达针对ALAS 2的shRNA的细胞。选择ALAS 2低表达的YN-1细胞，命名为YN 1-ALAS 21 ow细胞。然而，即使在通过TGF-β 1的红系分化后也没有观察到环形铁粒幼细胞，其是铁粒幼细胞性贫血的标志。结果表明，YN 1-ALAS 21 ow细胞中ALAS 2 mRNA的表达水平低于对照细胞，提示YN 1-ALAS 21 ow细胞中ALAS 2的表达水平可能足以保护细胞免受线粒体内铁积累的影响。少

Heme is a prosthetic group of several enzymes, and essential for aerobic organisms. Since excess amount of heme in the cells increases reactive oxygen species, cellular heme level is strictly regulated under the balance of its biosynthesis and catabolism. To know how heme biosynthesis and catabolism is regulated by the mechanisms through protein-protein interaction, we have performed following experiments 1. To know what kind of proteins are involved in the regulation of heme biosynthesis and catabolism, we have screened human cDNA libraries using non-specific aminolevulinate synthase, erythroid-specific aminolevulinate synthase (ALAS2), heme oxygenase 1 or heme oxygenase 2 (HO-2) as a bait protein. As a result, we have found that one of the components of 20S proteasome could associate with HO-2 protein. Since over-expression of this protein reduced HO-2 protein expression level, this protein should be involved in the regulation of protein degradation of HO-2. 2. Using RNA interference … More (RNAi) technology, we have tried to establish the model cells for sideroblastic anemia. First, we have prepared effective small interfering RNA (siRNA) for suppression of ALAS2 expression, then, prepared expression vector, which express effective short-hairpin RNA (shRNA). This vector has been introduced into YN-1 cells, which is able to produce hemoglobin in the cells after the stimulation by transforming growth factor beta 1, and these cells were incubated with G418 to select the cells which constitutively express shRNA against ALAS2. YN-1 cells that express ALAS2 at low level were selected, and named as YN1-ALAS21ow cells. However, ringed sideroblast, which is the hallmark of the sideroblastic anemia, was not observed even after the erythroid differentiation by TGF-betal. We have confirmed that YN1-ALAS21ow cells expressed lower level of ALAS2 mRNA than control cells, YN1-ALAS21ow cells might expressed enough level of ALAS2 for protecting the cells from iron accumulation in mitochondria. Less