Dissecting the interaction between tissue inhibitor of metalloproteinases-3 (TIMP-3) and low density lipoprotein receptor-related protein-1 (LRP-1): Development of a "TRAP" to increase levels of TIMP-3 in the tissue.
Dissecting the interaction between tissue inhibitor of metalloproteinases-3 (TIMP-3) and low density lipoprotein receptor-related protein-1 (LRP-1): Development of a "TRAP" to increase levels of TIMP-3 in the tissue.
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DOI：
10.1016/j.matbio.2016.07.004
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发表时间：
2017-05
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通讯作者：
S. D. Scilabra;Kazuhiro Yamamoto;Martina Pigoni;K. Sakamoto;S. Müller;Alkmini A. Papadopoulou;S. Lichtenthaler;L. Troeberg;H. Nagase;K. Kadomatsu
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作者：
S. D. Scilabra;Kazuhiro Yamamoto;Martina Pigoni;K. Sakamoto;S. Müller;Alkmini A. Papadopoulou;S. Lichtenthaler;L. Troeberg;H. Nagase;K. Kadomatsu
金属蛋白酶组织抑制剂3（TIMP-3）是细胞外基质转换的关键调节因子，具有抑制基质金属蛋白酶（MMPs）、类金刚烷酶金属蛋白酶（亚当斯）和血小板反应蛋白基序的亚当斯（ADAMTSs）的能力。TIMP-3是一种分泌性蛋白，其细胞外水平通过低密度脂蛋白受体相关蛋白-1（LRP-1）的内吞作用进行调节。在这项研究中，我们开发了一种能够在细胞外“捕获”TIMP-3的分子，从而增加其组织生物利用度。LRP-1含有四个配体结合簇。为了研究LRP-1上的TIMP-3结合位点，我们产生了含有四个不同结合簇或每个簇的部分的可溶性小受体（sLRP）。我们使用了一系列生物化学方法来研究TIMP-3与不同sLRP的结合。我们发现，TIMP-3结合的配体结合簇II的受体具有最高的亲和力和可溶性的minireceptor包含的N-末端的一半簇II特异性阻断TIMP-3的内化，而不影响营业额的金属蛋白酶。基于质谱的分泌组分析表明，这种名为T3 TRAP的小受体选择性地增加细胞外空间中的TIMP-3水平，并抑制许多细胞表面蛋白的组成性脱落。总之，T3 TRAP代表了一种生物学工具，可用于调节组织中TIMP-3水平，并可能被开发为治疗以TIMP-3缺陷为特征的疾病，包括关节炎。
Tissue inhibitor of metalloproteinases 3 (TIMP-3) is a key regulator of extracellular matrix turnover for its ability to inhibit matrix metalloproteinases (MMPs), adamalysin-like metalloproteinases (ADAMs) and ADAMs with thrombospondin motifs (ADAMTSs). TIMP-3 is a secreted protein whose extracellular levels are regulated by endocytosis via the low-density-lipoprotein receptor-related protein-1 (LRP-1). In this study we developed a molecule able to “trap” TIMP-3 extracellularly, thereby increasing its tissue bioavailability. LRP-1 contains four ligand-binding clusters. In order to investigate the TIMP-3 binding site on LRP-1, we generated soluble minireceptors (sLRPs) containing the four distinct binding clusters or part of each cluster. We used an array of biochemical methods to investigate the binding of TIMP-3 to different sLRPs. We found that TIMP-3 binds to the ligand-binding cluster II of the receptor with the highest affinity and a soluble minireceptor containing the N-terminal half of cluster II specifically blocked TIMP-3 internalization, without affecting the turnover of metalloproteinases. Mass spectrometry-based secretome analysis showed that this minireceptor, named T3TRAP, selectively increased TIMP-3 levels in the extracellular space and inhibited constitutive shedding of a number of cell surface proteins. In conclusion, T3TRAP represents a biological tool that can be used to modulate TIMP-3 levels in the tissue and could be potentially developed as a therapy for diseases characterized by a deficit of TIMP-3, including arthritis.