Multiple mechanisms for the regulation of heme biosynthesis and catabolism.

血红素是几种酶的假体，是有氧生物所必需的。由于细胞中过量的血红素增加了活性氧，细胞血红素水平在其生物合成和分解代谢的平衡下受到严格的调节。为了了解血红素的生物合成和分解代谢是如何通过蛋白-蛋白相互作用的机制来调节的，我们进行了以下实验1。为了了解哪些蛋白质参与了血红素生物合成和分解代谢的调控，我们以非特异性氨酰丙酸合成酶、红细胞特异性氨酰丙酸合成酶（ALAS2）、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类cDNA文库。因此，我们发现20S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，因此该蛋白可能参与了HO-2蛋白降解的调控。2. 利用RNA干扰技术（RNAi）建立了铁母细胞性贫血的模型细胞。首先制备抑制ALAS2表达的有效小干扰RNA (small interfering RNA, siRNA)，然后制备表达有效短发夹RNA （short-hairpin RNA, shRNA）的表达载体。将该载体导入n -1细胞中，经转化生长因子β 1刺激后，细胞内可产生血红蛋白，与G418孵育这些细胞，选择组成性表达shRNA对抗ALAS2的细胞。选择低水平表达ALAS2的yn1细胞，命名为YN1-ALAS21ow细胞。然而，环状铁母细胞是铁母细胞性贫血的标志，即使在红细胞被tgf - β分化后也未观察到。我们已经证实，YN1-ALAS21ow细胞表达的ALAS2 mRNA水平低于对照细胞，YN1-ALAS21ow细胞可能表达足够的ALAS2水平以保护细胞免受线粒体铁积累。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