Sequence features of substrates required for cleavage by GlpG, an Escherichia coli rhomboid protease
Sequence features of substrates required for cleavage by GlpG, an Escherichia coli rhomboid protease
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DOI：
10.1111/j.1365-2958.2007.05715.x
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发表时间：
2007-05-01
影响因子：
3.6
通讯作者：
Maegawa, Saki
中科院分区：
文献类型：
--
作者：
Akiyama, Yoshinori;Maegawa, Saki
菱形体是属于膜内切割蛋白酶的丝氨酸蛋白酶家族，其被认为催化膜内底物蛋白的蛋白水解。目前尚不清楚菱形蛋白酶的底物是否具有允许菱形特异性切割的共同序列特征。我们以前表明，GIPG，大肠杆菌菱形，可以切割I型模型膜蛋白的Bla-LY 2-MBP具有第二个跨膜区的乳糖通透酶（LY 2）在膜外区域在体内和体外，和蛋白水解的决定因素驻留在LY 2序列。在这里，我们的特点是在LY 2的序列功能，允许有效的切割GIPG和确定两个元素，一个亲水性区域，包括切割位点和螺旋不稳定的残基在下游疏水区。螺旋去稳定剂相对于切割位点的定位的重要性被建议。这两个元素似乎协同促进底物的GipG蛋白水解。最后，结合体内筛选的切割位点残基的随机诱变揭示GlpG分别偏好在P1和P1'位点具有小侧链和负电荷的残基。
Rhomboids are a family of serine proteases belonging to intramembrane cleaving proteases, which are supposed to catalyse proteolysis of a substrate protein within the membrane. It remains unclear whether substrates of the rhomboid proteases have a common sequence feature that allows specific cleavage by rhomboids. We showed previously that GIpG, the Escherichia coli rhomboid, can cleave a type I model membrane protein Bla-LY2-MBP having the second transmembrane region of lactose permease (LY2) at the extramembrane region in vivo and in vitro, and that determinants for proteolysis reside within the LY2 sequence. Here we characterized sequence features in LY2 that allow efficient cleavage by GIpG and identified two elements, a hydrophilic region encompassing the cleavage site and helix-destabilizing residues in the downstream hydrophobic region. Importance of the positioning of helix-destabilizers relative to the cleavage site was suggested. These two elements appear to co-operatively promote proteolysis of substrates by GIpG. Finally, random mutagenesis of the cleavage site residues in combination with in vivo screening revealed that GlpG prefers residues with a small side chain and a negative charge at the P1 and P1' sites respectively.