Site specificity determinants for prelamin A cleavage by the zinc metalloprotease ZMPSTE24.
Site specificity determinants for prelamin A cleavage by the zinc metalloprotease ZMPSTE24.
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DOI：
10.1074/jbc.ra120.015792
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Michaelis S
中科院分区：
文献类型：
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作者：
Babatz TD;Spear ED;Xu W;Sun OL;Nie L;Carpenter EP;Michaelis S
整合膜锌金属蛋白酶ZMPSTE 24对人类健康和长寿具有重要意义。ZMPSTE 24在核纤层蛋白A前体（核支架蛋白核纤层蛋白A的法尼基化前体）的成熟中执行关键的蛋白水解步骤。编码前核纤层蛋白A或ZMPSTE 24的基因中阻止分裂的突变导致过早衰老疾病哈钦森-吉尔福德早衰综合征（HGPS）和相关的早衰症。ZMPSTE 24具有新颖的结构，具有七个跨膜跨度，形成一个大的充满水的膜室，其催化位点面向室内部。前体蛋白A是ZMPSTE 24唯一已知的哺乳动物底物;然而，这种特异性的基础仍然不清楚。为了确定ZMPSTE 24切割的序列要求，我们将前层蛋白A易裂键（TRSY↓LLGN）侧翼的8个残基诱变至所有其他19个氨基酸，产生152个变体的文库。我们还用来自两栖动物、鸟类和鱼类前核层蛋白A的假定ZMPSTE 24切割位点的序列替换了这8个残基。使用提供ZMPSTE 24加工效率的灵敏量度的体内酵母测定来评估前层蛋白A变体的切割。我们发现，在C-末端一侧的切割位点的残基是最敏感的变化。与其他锌金属蛋白酶（包括嗜热菌蛋白酶）一致，ZMPSTE 24优选在P1'位置（Leu 647）处的疏水残基，但另外，在P2'处显示类似的模式，尽管是沉默的。我们的研究结果开始定义ZMPSTE 24的共有序列，这有助于阐明这种生理上重要的蛋白酶的功能，并可能最终导致识别其他底物。
The integral membrane zinc metalloprotease ZMPSTE24 is important for human health and longevity. ZMPSTE24 performs a key proteolytic step in maturation of prelamin A, the farnesylated precursor of the nuclear scaffold protein lamin A. Mutations in the genes encoding either prelamin A or ZMPSTE24 that prevent cleavage cause the premature aging disease Hutchinson–Gilford progeria syndrome (HGPS) and related progeroid disorders. ZMPSTE24 has a novel structure, with seven transmembrane spans that form a large water-filled membrane chamber whose catalytic site faces the chamber interior. Prelamin A is the only known mammalian substrate for ZMPSTE24; however, the basis of this specificity remains unclear. To define the sequence requirements for ZMPSTE24 cleavage, we mutagenized the eight residues flanking the prelamin A scissile bond (TRSY↓LLGN) to all other 19 amino acids, creating a library of 152 variants. We also replaced these eight residues with sequences derived from putative ZMPSTE24 cleavage sites from amphibian, bird, and fish prelamin A. Cleavage of prelamin A variants was assessed using an in vivo yeast assay that provides a sensitive measure of ZMPSTE24 processing efficiency. We found that residues on the C-terminal side of the cleavage site are most sensitive to changes. Consistent with other zinc metalloproteases, including thermolysin, ZMPSTE24 preferred hydrophobic residues at the P1’ position (Leu647), but in addition, showed a similar, albeit muted, pattern at P2’. Our findings begin to define a consensus sequence for ZMPSTE24 that helps to clarify how this physiologically important protease functions and may ultimately lead to identifying additional substrates.