Thrombospondin type 1 repeats interact with matrix metalloproteinase 2 - Regulation of metalloproteinase activity
Thrombospondin type 1 repeats interact with matrix metalloproteinase 2 - Regulation of metalloproteinase activity
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DOI：
10.1074/jbc.m003834200
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发表时间：
2000-10-13
影响因子：
4.8
通讯作者：
Simons, M
中科院分区：
文献类型：
--
作者：
Bein, K;Simons, M
血栓反应蛋白被认为是血管生成的抑制剂，然而，这种活性的机制尚不清楚。在这项研究中，我们使用酵母双杂交系统鉴定了与血小板反应蛋白1 （TSP1）和2 （TSP2）特性蛋白样1型重复序列（TSR）相互作用的蛋白，其中鉴定出与两个TSR相互作用的蛋白之一是基质金属蛋白酶2 （MMP2）。分离得到的MMP2 cDNA克隆编码237-633个氨基酸残基，其中包括催化中心两侧的纤维连接蛋白样明胶结合区和羧基血红素样区。对该克隆的进一步测试表明，TSR与含有催化结构域的MMP2末端区域WH相互作用。使用纯化的完整的TSP1、TSP2、MMP2和MMP9，通过免疫沉淀和Western blotting进一步证实了在酵母中观察到的蛋白质相互作用。尽管MMP2通过其明胶结合域或紧密定位位点与TSP1和TSP2相互作用，但在体外实验中，TSP1和TSP2均未被MMP2降解。组织培养和体外实验表明，纯化的TSR和完整的TSP1的存在导致MMP活性的抑制。TSP1抑制mmp3依赖性的pro-MMP3激活和凝血酶诱导的pro-MMP2激活的能力表明，TSP1可能通过阻止MMP2和MMP9酶原的激活来抑制MMP活性。
Thrombospondins are thought to function as inhibitors of angiogenesis, However, the mechanism(s) of this activity is not well understood. In this study, we have used the yeast two-hybrid system to identify proteins that interact with the thrombospondins 1 (TSP1) and 2 (TSP2) properdin-like type 1 repeats (TSR), One of the proteins identified that interacted with both TSR was matrix metalloproteinase 2 (MMP2). The isolated MMP2 cDNA clone encoded amino acid residues 237-633, which include the fibronectin-like gelatin binding region flanking the catalytic center and the carboxyl hemopexin-like region. Further testing of this clone demonstrated that the TSR interacted with the WH,terminal region of the MMP2 that contains the catalytic domain. The protein interaction observed in yeast was further demonstrated by immunoprecipitation and Western blotting using purified intact TSP1, TSP2, MMP2, and MMP9. Although MMP2 interacted with TSP1 and TSP2 via its gelatin-binding domain or a closely mapping site, neither TSP1 nor TSP2 was degraded by MMP2 in vitro. Tissue culture and in vitro assays demonstrated that the presence of purified TSR and intact TSP1 resulted in inhibition of MMP activity. The ability of TSP1 to inhibit MMP3-dependent activation of pro-MMP3 and thrombin-induced activation of pro-MMP2 suggests that the TSPs may inhibit MMP activity by preventing activation of the MMP2 and MMP9 zymogens.