Two-step processing of human frataxin by mitochondrial processing peptidase - Precursor and intermediate forms are cleaved at different rates
Two-step processing of human frataxin by mitochondrial processing peptidase - Precursor and intermediate forms are cleaved at different rates
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DOI：
10.1074/jbc.m006539200
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发表时间：
2000-12-29
影响因子：
4.8
通讯作者：
Isaya, G
中科院分区：
文献类型：
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作者：
Cavadini, P;Adamec, J;Isaya, G
我们以前已经证明，人类Frataxin前体(p-FXN)的成熟涉及线粒体加工肽酶(MPP)的两次切割，然而另一组人没有证实这一观察结果，他们只报告了一次切割。在这里，我们最终证明了MPP分两个连续的步骤切割p-FXN，产生18,826-Da的中间体(i-FXN)和17,255-Da的成熟(m-FXN)形式，后者对应于人体组织中的内源性Frataxin。这两个裂解发生在残基41-42和55-56之间，都与MPP共识序列RX向下箭头(X/S)匹配。重组大鼠和酵母MPP催化p-->；I步的速度分别是I-->；m步的4倍和40倍。在分离的大鼠线粒体中，p-FXN经历了p-->；i-->；m-->；d(1)-->；d(2)，其中d(1)和d(2)代表一种未知的蛋白酶产生的m-rM的两个C-末端片段，i-->；m步长是有限的，p-->；m的总切割速率不超过m-->；d(1)->；D(2)，使得m-FXN的水平在孵育过程中长达3小时不会改变。致病的Frataxin突变(W173G)抑制I-gt；m步骤导致I-FXN的非特异性降解，因此，MPP催化的两个加工步骤中的第二个步骤限制了线粒体内成熟Frataxin的水平。
We showed previously that maturation of the human frataxin precursor (p-fxn) involves two cleavages by the mitochondrial processing peptidase (MPP), This observation was not confirmed by another group, however, who reported only one cleavage. Here, we demonstrate conclusively that MPP cleaves p-fxn in two sequential steps, yielding a 18,826-Da intermediate (i-fxn) and a 17,255-Da mature (m-fxn) form, the latter corresponding to endogenous frataxin in human tissues. The two cleavages occur between residues 41-42 and 55-56, and both match the MPP consensus sequence RX down arrow (X/S). Recombinant rat and yeast MPP catalyze the p --> i step 4 and 40 times faster, respectively, than the i --> m step. In isolated rat mitochondria, p-fxn undergoes a sequence of cleavages, p --> i --> m --> d(1) --> d(2), with d(1) and d(2) representing two C-terminal fragments of m-Rm produced by an unknown protease, The i --> m step is limiting, and the overall rate of p --> i --> m does not exceed the rate of m --> d(1) --> d(2), such that the levels of m-fxn do not change during incubations as long as 3 h. Inhibition of the i --> m step by a disease-causing frataxin mutation (W173G) leads to nonspecific degradation of i-fxn, Thus, the second of the two processing steps catalyzed by MPP limits the levels of mature frataxin within mitochondria.