Site-specific Cleavage of BMP4 by Furin, PC6, and PC7
Site-specific Cleavage of BMP4 by Furin, PC6, and PC7
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DOI：
10.1074/jbc.m109.028506
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发表时间：
2009-10-02
影响因子：
4.8
通讯作者：
Christian, Jan L.
中科院分区：
文献类型：
--
作者：
Nelsen, Sylvia M.;Christian, Jan L.
骨形态发生蛋白（BMP）需要前蛋白转化酶（PC）家族成员的蛋白水解激活。Pro-BMP 4最初在与成熟配体结构域相邻的位点（S1）处裂解，然后在前结构域内的上游位点（S2）处裂解。在S2位点的裂解，似乎以组织特异性方式发生，调节成熟BMP 4的活性和信号传导范围。为了验证组织特异性切割pro-BMP 4是由位点特异性蛋白酶的差异表达调节的假设，我们鉴定了在体内切割每个位点的PC。在非洲爪蟾卵母细胞中，弗林蛋白酶和PC6的功能冗余地切割pro-BMP 4的S1和S2位点，如反义介导的基因敲除和使用弗林蛋白酶和PC6选择性抑制剂alpha（1）-PDX的结果所证明的。相比之下，α（1）-PDX阻断了胚胎中pro-BMP 4的S2位点而不是S1位点的切割，表明存在发育调节的S1位点特异性转化酶。基于其所需的底物切割基序和对α（1）-PDX的抗性的知识，该蛋白酶可能是PC 7。与这一预测一致，除了弗林蛋白酶和PC6之外，针对PC 7工程化的α（1）-PDX变体完全抑制卵母细胞和胚胎中BMP 4的切割。进一步的研究表明，PC 7的成绩单的表达和聚腺苷酸化，和PC 7前体蛋白在卵母细胞和胚胎进行有效的自催化激活。这些结果表明，PC 7，或转化酶具有类似的底物特异性，功能选择性切割前BMP 4的S1位点的发育调节的方式。
Bone morphogenetic proteins (BMPs) require proteolytic activation by members of the proprotein convertase (PC) family. Pro-BMP4 is initially cleaved at a site adjacent to the mature ligand domain (S1) and then at an upstream site (S2) within the prodomain. Cleavage at the S2 site, which appears to occur in a tissue-specific fashion, regulates the activity and signaling range of mature BMP4. To test the hypothesis that tissue-specific cleavage of pro-BMP4 is regulated by differential expression of a site-specific protease, we identified the PCs that cleave each site in vivo. In Xenopus oocytes, furin and PC6 function redundantly to cleave both the S1 and S2 sites of pro-BMP4, as evidenced by the results of antisense-mediated gene knockdown and the use of the furin-and PC6-selective inhibitor alpha(1)-PDX. By contrast, alpha(1)-PDX blocked cleavage of the S2 but not the S1 site of pro-BMP4 in embryos, suggesting the existence of a developmentally regulated S1 site-specific convertase. This protease is likely to be PC7 based on knowledge of its required substrate cleavage motif and resistance to alpha(1)-PDX. Consistent with this prediction, an alpha(1)-PDX variant engineered to target PC7, in addition to furin and PC6, completely inhibited cleavage of BMP4 in oocytes and embryos. Further studies showed that pc7 transcripts are expressed and polyadenylated, and that the PC7 precursor protein undergoes efficient autocatalytic activation in both oocytes and embryos. These results suggest that PC7, or a convertase with similar substrate specificity, functions to selectively cleave the S1 site of pro-BMP4 in a developmentally regulated fashion.