Protein characterization of a candidate mechanism SNP for Crohn's disease: the macrophage stimulating protein R689C substitution.
Protein characterization of a candidate mechanism SNP for Crohn's disease: the macrophage stimulating protein R689C substitution.
复制标题
DOI：
10.1371/journal.pone.0027269
复制
发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Herzberg O
中科院分区：
文献类型：
--
作者：
Gorlatova N;Chao K;Pal LR;Araj RH;Galkin A;Turko I;Moult J;Herzberg O
高通量全基因组关联研究（GWAS）目前正在鉴定大量与人类常见疾病风险相关的基因组位点。每一个这样的位点都对确定相关的潜在机制提出了挑战。在这里，我们报告了一个拟议的因果单核苷酸多态性（SNP）的基因座相关的风险克罗恩病和溃疡性结肠炎的实验表征。SNP位于编码巨噬细胞刺激蛋白（MSP）的MST 1基因中，并导致MSP β链（MSPβ）内的R689 C氨基酸取代。MSP与罗恩受体酪氨酸激酶的结合激活参与炎症反应的信号传导途径。我们纯化了野生型和突变型MSPβ蛋白，并比较了可能影响MSP/罗恩信号通路的生化和生物物理特性。表面等离子体共振（SPR）结合研究表明MSPβ R689 C与罗恩的亲和力比野生型MSPβ低约10倍，差示扫描荧光分析（DSF）显示突变MSPβ的热稳定性比野生型MSPβ略低1.6 K。发现该取代不损害MSP活化所需的间质蛋白酶-1的特异性Arg 483-Val 484肽键切割，并且突变蛋白质的胰蛋白酶片段的质谱显示由R689 C突变引入的游离巯基不形成异常的二硫键。总之，这些研究表明，错义SNP通过降低其对罗恩的亲和力以及可能通过对由降低的热力学稳定性引起的体内浓度的次级效应而损害MSP功能，导致MSP/罗恩信号传导途径的下调。
High throughput genome wide associations studies (GWAS) are now identifying a large number of genome loci related to risk of common human disease. Each such locus presents a challenge in identifying the relevant underlying mechanism. Here we report the experimental characterization of a proposed causal single nucleotide polymorphism (SNP) in a locus related to risk of Crohn's disease and ulcerative colitis. The SNP lies in the MST1 gene encoding Macrophage Stimulating Protein (MSP), and results in an R689C amino acid substitution within the β-chain of MSP (MSPβ). MSP binding to the RON receptor tyrosine kinase activates signaling pathways involved in the inflammatory response. We have purified wild-type and mutant MSPβ proteins and compared biochemical and biophysical properties that might impact the MSP/RON signaling pathway. Surface plasmon resonance (SPR) binding studies showed that MSPβ R689C affinity to RON is approximately 10-fold lower than that of the wild-type MSPβ and differential scanning fluorimetry (DSF) showed that the thermal stability of the mutant MSPβ was slightly lower than that of wild-type MSPβ, by 1.6 K. The substitution was found not to impair the specific Arg483-Val484 peptide bond cleavage by matriptase-1, required for MSP activation, and mass spectrometry of tryptic fragments of the mutated protein showed that the free thiol introduced by the R689C mutation did not form an aberrant disulfide bond. Together, the studies indicate that the missense SNP impairs MSP function by reducing its affinity to RON and perhaps through a secondary effect on in vivo concentration arising from reduced thermodynamic stability, resulting in down-regulation of the MSP/RON signaling pathway.
登录
查看更多内容
影响因子：
30.8
作者：
Marchini, Jonathan;Howie, Bryan;Donnelly, Peter
通讯作者：
Donnelly, Peter
影响因子：
30.8
作者：
通讯作者：
--
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
5.6
作者：
Holmes, O.;Pillozzi, S.;Gherardi, E.
通讯作者：
Gherardi, E.
影响因子：
8
作者：
Goyette, P.;Lefebvre, C.;Rioux, J. D.
通讯作者：
Rioux, J. D.