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

描述(申请人提供)：拟议研究的目标是确定和表征血红素生物合成的末端酶--铁络合酶的[2Fe-2S]簇在红系血红素合成过程中所起的作用。最近对斑马鱼Pinotage突变体的发现刺激我们探讨这个问题，该突变体的特征是严重的低色素性、小细胞性贫血，但没有积累游离原卟啉。未发表的数据已经确定，导致这种缺陷的突变存在于编码ATPIF1的基因中，ATPIF1是一种与血红素生物合成没有直接关联的蛋白质，但确实会影响红系细胞合成血红素的能力。用缺乏[2Fe-2S]簇的酿酒酵母铁络合酶取代天然的[2Fe-2S]簇的铁络合酶，可抑制该突变表型。我们推测，铁络合酶[2Fe-2S]簇通过感应和响应在斑马鱼中发现改变的线粒体pH和/或膜电位来调节血红素的合成。这种假定的作用在化学和生物学上是可行的，在某些方面类似于大肠杆菌转录调控基因SoxR的[2Fe-2S]簇所起的作用。该建议的具体目的是确定：1)改变[2Fe-2S]簇中点电位对体内铁络合酶活性的影响，2)[2Fe-2S]簇所在的充满溶剂的通道是否对该簇的调节功能至关重要，3)该簇的潜在调控作用是否为拥有红系细胞的生物的铁络合酶所特有，以及4)[2Fe-2S]簇是否通过蛋白质内的作用直接影响催化和/或通过铁络合酶与有丝分裂蛋白1和/或原植物蛋白原氧化酶之间的蛋白质间相互作用来发挥其功能。对于这些研究中产生和使用的所有铁络合酶变体，将确定动力学和生物物理参数，并通过X射线结晶学验证蛋白质的结构完整性。Pinotage突变体的铁络合酶活性降低，但没有像在红细胞生成性原卟啉病(EPP)中发现的那样积累游离原卟啉，这一事实表明，铁络合酶活性的丧失本身并不一定会导致EPP，但在某些情况下会导致贫血。这项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一个新的调节角色。此外，新的研究领域将打开，目前未确定的基于红细胞的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 Unpublished data have determined that the mutation causing this defect is in the gene encoding Atpif1, a protein not directly associated with heme biosynthesis, but one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.