Erythroid heme synthesis regulation via the ferrochelatase [2Fe-2S] cluster

描述（由申请人提供）：拟定研究的目的是鉴定和表征亚铁螯合酶（血红素生物合成的末端酶）的[2Fe- 2S]簇在红系血红素合成过程中的作用。我们已经被刺激到接近这个问题，最近的发现与斑马鱼pinotage突变，其特点是深刻的低色素，小细胞性贫血，但没有积累的游离原卟啉。未发表的数据已经确定，导致这种缺陷的突变是在编码Atpif 1的基因中，Atpif 1是一种与血红素生物合成不直接相关的蛋白质，但确实对红系细胞合成血红素的能力有影响。该突变表型通过用来自酿酒酵母的缺乏[2Fe-2S]簇的亚铁螯合酶替换具有[2Fe-2S]簇的天然亚铁螯合酶来抑制。我们推测，亚铁螯合酶[2Fe-2S]簇调节血红素合成的传感和响应的线粒体pH值和/或膜电位，被发现在pinotage斑马鱼被改变。这种假定的作用在化学和生物学上是可行的，并且在某些方面类似于大肠杆菌转录调节子SoxR的[2Fe-2S]簇所起的作用。该提案的具体目标是确定：1）改变的[2Fe-2S]簇中点电位对体内亚铁螯合酶活性的影响，2）[2Fe-2S]簇所在的充满溶剂的通道是否对簇的调节功能是必需的，3）如果簇的推定调节作用对于具有红系细胞的生物体的亚铁螯合酶是独特的，和4）如果[2Fe-2S]簇通过直接影响催化的蛋白质内作用和/或通过亚铁螯合酶与线粒体铁蛋白1和/或原卟啉原氧化酶之间的蛋白质间相互作用发挥其功能。对于这些研究中产生和使用的所有亚铁螯合酶变体，将确定动力学和生物物理参数，并通过X射线晶体学验证蛋白质的结构完整性。事实上，pinotage突变体具有降低的亚铁螯合酶活性，但不积累游离原卟啉，如在红细胞生成性原卟啉症（EPP）中发现的，这表明亚铁螯合酶活性的丧失本身不一定导致EPP，但在某些情况下可能导致贫血。该研究结果将对红细胞生成领域产生重大影响，因为它们将在红细胞血红素合成中建立新的调控机制。此外，新的研究领域将开放，目前尚未确定的红细胞综合征和疾病的分子基础可能会被揭示。

DESCRIPTION (provided by applicant): The goal of the proposed research is to identify and characterize the role that the [2Fe- 2S] cluster of ferrochelatase, the terminal enzyme of heme biosynthesis, plays during erythroid heme synthesis. We have been stimulated to approach this question by recent discoveries with the zebrafish pinotage mutant, which is characterized by a profound hypochromic, microcytic anemia, but without accumulation of free protoporphyrin. Unpublished data have determined that the mutation causing this defect is in the gene encoding Atpif1, a protein not directly associated with heme biosynthesis, but one that does have an impact on the erythroid cell's ability to synthesize heme. This mutant phenotype is suppressed by replacement of the native ferrochelatase, which possesses a [2Fe-2S] cluster, with ferrochelatase from Saccharomyces cerevisiae, which lacks a [2Fe-2S] cluster. We hypothesize that the ferrochelatase [2Fe-2S] cluster modulates heme synthesis by sensing and responding to the mitochondrial pH and/or membrane potential that were found to be altered in the pinotage zebrafish. Such a postulated role is chemically and biologically feasible, and similar in some regards to the role played by the [2Fe-2S] cluster of the Escherichia coli transcriptional regulato SoxR. The specific aims of the proposal are to determine: 1) the impact of altered [2Fe-2S] cluster midpoint potential on in vivo ferrochelatase activity, 2) if the solvent-filled channel in which the [2Fe-2S] cluster resides is essential to the regulatory function of the cluster, 3) if th putative regulatory role of the cluster is unique to ferrochelatases of organisms that possess erythroid cells, and 4) if the [2Fe-2S] cluster exerts its function via an intra-protein action tha directly impacts catalysis and/or via inter-protein interactions between ferrochelatase and mitoferrin1 and/or protoporphyrinogen oxidase. For all ferrochelatase variants produced and utilized in these studies, kinetic and biophysical parameters will be determined and the structural integrity of the proteins validated by x-ray crystallography. The fact that pinotage mutants have diminished ferrochelatase activity, but do not accumulate free protoporphyrin as one finds in erythropoietic protoporphyria (EPP), indicates that loss of ferrochelatase activity pe se does not necessarily result in EPP, but in some circumstances can cause anemia. The study results will impact the field of erythropoiesis significantly since they will establish a new regulatory player in red cell heme synthesis. Additionally, new areas of investigation will open and the molecular basis of currently undefined red cell-based syndromes and diseases may be revealed.