Functional characterization of sonic hedgehog mutations associated with holoprosencephaly
Functional characterization of sonic hedgehog mutations associated with holoprosencephaly
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DOI：
10.1074/jbc.m405161200
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发表时间：
2004-10-08
影响因子：
4.8
通讯作者：
Ruat, M
中科院分区：
文献类型：
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作者：
Traiffort, E;Dubourg, C;Ruat, M
发育基因Sonic hedgehog （SHH）的突变和SHH信号的改变与全前脑畸形（HPE）有关，HPE是一种罕见的疾病，其特征是大脑和颅面异常。基于小鼠n端和果蝇c端刺猬蛋白的晶体结构，我们开发了相应的人类蛋白质（sh - n, sh - c）的三维模型，使我们能够在这两个结构域中识别与HPE错义突变相关的关键区域。我们进一步描述了与其中11个突变相关的功能后果。在转染的HEK293细胞中，8个突变体（W117R、W117G、H140P、T150R、C183F、L271P、I354T、A383T）的活性sh - n片段的产生显著受损。在基于报告细胞的实验中，这些细胞培养的上清液显示没有显著的sh信号活性。两个突变体（G31R, D222N）与sh - n的产生和信号活性降低有关。最后，一个携带A226T突变的突变体显示出与野生型蛋白相当的活性。这项工作表明，分析的大多数与hpe相关的SHH突变对SHH- n的可用性及其生物活性具有有害影响。然而，由于shh相关突变的基因型和表型之间缺乏相关性，我们的研究表明，其他因素干预了HPE异常谱的发展。
Mutations of the developmental gene Sonic hedgehog (SHH) and alterations of SHH signaling have been associated with holoprosencephaly (HPE), a rare disorder characterized by a large spectrum of brain and craniofacial anomalies. Based on the crystal structure of mouse N-terminal and Drosophila C-terminal hedgehog proteins, we have developed three-dimensional models of the corresponding human proteins (SHH-N, SHH-C) that have allowed us to identify within these two domains crucial regions associated with HPE missense mutations. We have further characterized the functional consequences linked to 11 of these mutations. In transfected HEK293 cells, the production of the active SHH-N fragment was dramatically impaired for eight mutants ( W117R, W117G, H140P, T150R, C183F, L271P, I354T, A383T). The supernatants from these cell cultures showed no significant SHH-signaling activity in a reporter cell-based assay. Two mutants (G31R, D222N) were associated with a lower production of SHH-N and signaling activity. Finally, one mutant harboring the A226T mutation displays an activity comparable with the wild-type protein. This work demonstrates that most of the HPE-associated SHH mutations analyzed have a deleterious effect on the availability of SHH-N and its biological activity. However, because of the lack of correlation between genotype and phenotype for SHH-associated mutations, our study suggests that other factors intervene in the development of the spectrum of HPE anomalies.