Regulation of erythroid differentiation by trnscription factors and heme

1)我们发现，在野生型MEL细胞中，二甲亚砜增加了红系转录因子NF-E2' DNA复合物的形成，部分原因是通过诱导p45基因的表达，并且NF-E2的DNA结合和反激活活性都受到丝氨酸/苏氨酸磷酸化如Ras信号级联的调节。一个Mafk同源二聚体可能抑制转录不仅通过竞争DNA结合位点，而且通过直接抑制转录。2)利用酵母GAL4-MafK融合蛋白双杂交筛选，我们鉴定出两个新的bZip转录因子Bach1和Bach2作为MafK的异源二聚化伙伴，这些Bach蛋白除了具有一个cnc型bZip结构域外，还具有另一个蛋白相互作用基元BTB结构域。Bach1的表达普遍存在，但Bach2的表达仅限于单核细胞和神经元细胞。Bach蛋白在体外通过与MafK形成异源二聚体结合到NF-E2结合位点。在成纤维细胞转染实验中，Bach1和Bach2具有转录抑制因子的功能，但在培养的红细胞中，它们分别具有转录激活因子和转录抑制因子的功能。3)一个短氨基酸序列，血红素调控基序（HRM），已被证明在体外参与血红素抑制蛋白质转运到线粒体。为了阐明HRM在体内5-氨基乙酰酸合成酶（ALAS）转运的家庭调节中的作用，我们构建了一系列ALAS非特异性异构体（ALAS- n）的突变体，其中HRMs内的特异性半胱氨酸残基被转化为丝氨酸。通过瞬时转染，在QT6成纤维细胞中表达野生型和突变型酶，然后分析酶的线粒体输入。在野生型ALAS-N中观察到的家抑制，当酶中的所有三个HRMs发生突变时，完全消失，这表明FIRM实际上是血红素调节ALAS-N在细胞内运输所必需的。相比之下，在相同的实验条件下，外源血红素不影响红系阿拉斯（ALAS- e）的进口。4)我们分析了日本男性吡哆醇难治性x - 1相关铁母细胞性贫血（XLSA）患者的ALAS2基因（ALAS2），并鉴定出ALAS2突变（D19OV）。突变对酶的体外活性没有影响。然而，骨髓细胞中酶的数量被发现减少到正常对照的大约5%。此外，在瞬时转染分析中，线粒体中发现了比成熟的ALAS-E更大的异常加工蛋白的积累。这些较大的产物在结合体外转录/翻译和前体导入分离线粒体的分析中可重复检测到。这些结果表明，引起pyridoine -refractory XLSA的突变可能会影响ALAS-E前体的加工，从而引起ALAS-E蛋白的不稳定性。少

1) We showed that dimethyl sulfoxide increased the eryThroid transcription factor NF-E2' DNA complex formation in the wild -type MEL cells, in part by the induction of p45 gene expression, and that both DNA binding and transactivation activity of NF-E2 are regulated by Ser/Thr phosphorylation suchas Ras siganling cascade. An Mafk homodimer may suppress transcription not only by competition for the DNA binding site, but also by directly inhibiting transcription.2) Using a yeast two-hybrid screen with a GAL4-MafK fusion protein, we identified two novel bZip transcription factors, Bach1 and Bach2, as heterodimerization partners of MafK, In addition to a CNC-type bZip domain, these Bach proteins possess another protein interaction motif, BTB domain. Expression of Bach1 appears ubiquitous, but that of Bach2 is restricted to monocytes and neuronal cells. Bach proteins bind in vitro to NF-E2 binding sites by forming heterodimers with MafK.Bach1 and Bach2 function as transcription repressors i … More n transfection assays using fibroblast cells, but they function as a transcription activator and repressor, respectively, in cultured erythroid cells.3) A short amino acid sequence, the heme regulatory motif (HRM), has been shown to be involved in the hemin inhibition of protein transport into mitochondria in vitro. To elucidate the role of HRM in the home regulation of 5-aminolevulinate synthase (ALAS) transport in vivo, we constructed a series of mutants of the non-specific isoform of ALAS (ALAS-N) in which the specific cysteine residues within the HRMs were converted to serines. Wild-type and mutant enzymes were expressed in QT6 fibroblasts through transient transfection, followed by analyses of the mitochondrial import of the enzymes. The home inhibition, which was observed in the wild-type ALAS-N, abolished completely when all the three HRMs in the enzyme were mutated, indicating that the FIRM is actually required for the heme regulation of ALAS-N transport within the cells. In contrast, exogenous hemin did not affect the import of the erythroid ALAS (ALAS-E) under the comparable experimental conditions.4) We analyzed ALAS-E gene (ALAS2) of Japanese males with pyridoxine-refractory X-l inked sideroblastic anemia (XLSA) and identified an ALAS2 mutation (D19OV). The mutation did not affect the enzyme activity in vitro. However, the amount of the enzyme in bone marrow cells were found to be reduced to approximately 5% of the normal control. Additionally, accumulation of aberrantly processed proteins, the sizes of which were larger than that of mature ALAS-E, was found in mitochondria in transient transfection analyses. These larger products were reproducibly detected in assays combining in vitro transcription/translation and precursor import into isolated mitochondria. These results indicate that the mutation causing pyridoxine-refractory XLSA may affect the processing of ALAS-E precursor, thus provoking instability of the ALAS-E protein. Less