Novel Mechanisms for Heme-dependent Degradation of ALAS1 Protein as a Component of Negative Feedback Regulation of Heme Biosynthesis
Novel Mechanisms for Heme-dependent Degradation of ALAS1 Protein as a Component of Negative Feedback Regulation of Heme Biosynthesis
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DOI：
10.1074/jbc.m116.719161
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发表时间：
2016-09-23
影响因子：
4.8
通讯作者：
Furuyama, Kazumichi
中科院分区：
文献类型：
--
作者：
Kubota, Yoshiko;Nomura, Kazumi;Furuyama, Kazumichi
在真核细胞中，通过负反馈调节非特异性5-氨基酮戊酸合成酶(ALAS1)，血红素的合成受到血红素自身的严格控制，ALAS1是血红素生物合成的限速酶。然而，推动线粒体中ALAS1蛋白依赖于血红素的降解的机制在很大程度上尚不清楚。在目前的研究中，我们提供了证据表明，线粒体ATP依赖的蛋白酶ClpXP是CLPX和CLPP的异构体，参与了线粒体中依赖于血红素的ALAS1的降解。我们发现ALAS1以一种血红素依赖的方式与ClpXP形成复合体，并且siRNA介导的抑制CLPX或CLPP表达诱导了ALAS1在人肝癌细胞系中的积聚。我们还发现，ALAS1上特定的血红素结合基序位于成熟蛋白的N端，是形成依赖于血红素的蛋白质复合体所必需的。此外，氯化高铁血红素介导的ALAS1氧化修饰导致线粒体基质中另一种依赖于ATP的蛋白酶LONP1重新进入ALAS1蛋白复合体。值得注意的是，成熟ALAS1蛋白N-末端的血红素结合位点也是ALAS1依赖于血红素的氧化所必需的。这些结果表明，在血红素与该蛋白上的血红素结合基序结合后，ALAS1经历了构象变化。ALAS1结构的这种变化可能会促进ALAS1与线粒体中ATP依赖的蛋白酶之间形成复合体，从而加速ALAS1蛋白的降解，以维持适当的细胞内血红素水平。
In eukaryotic cells, heme production is tightly controlled by heme itself through negative feedback-mediated regulation of nonspecific 5-aminolevulinate synthase (ALAS1), which is a rate-limiting enzyme for heme biosynthesis. However, the mechanism driving the heme-dependent degradation of the ALAS1 protein in mitochondria is largely unknown. In the current study, we provide evidence that the mitochondrial ATP-dependent protease ClpXP, which is a heteromultimer of CLPX and CLPP, is involved in the heme-dependent degradation of ALAS1 in mitochondria. We found that ALAS1 forms a complex with ClpXP in a heme-dependent manner and that siRNA-mediated suppression of either CLPX or CLPP expression induced ALAS1 accumulation in the HepG2 human hepatic cell line. We also found that a specific heme-binding motif on ALAS1, located at the N-terminal end of the mature protein, is required for the heme-dependent formation of this protein complex. Moreover, hemin-mediated oxidative modification of ALAS1 resulted in the recruitment of LONP1, another ATP-dependent protease in the mitochondrial matrix, into the ALAS1 protein complex. Notably, the heme-binding site in the N-terminal region of the mature ALAS1 protein is also necessary for the heme-dependent oxidation of ALAS1. These results suggest that ALAS1 undergoes a conformational change following the association of heme to the heme-binding motif on this protein. This change in the structure of ALAS1 may enhance the formation of complexes between ALAS1 and ATP-dependent proteases in the mitochondria, thereby accelerating the degradation of ALAS1 protein to maintain appropriate intracellular heme levels.