Department of Biological Sciences, Xian Jiaotong Liverpool University, 111 Ren Ai Road, Suzhou 215123, China.
Molecules. 2019 Jan 11;24(2):255. doi: 10.3390/molecules24020255.
Renal carcinoma cells express Membrane Type 1-Matrix Metalloproteinase (MT1-MMP, MMP-14) to degrade extracellular matrix components and a range of bioactive molecules to allow metastasis and cell proliferation. The activity of MT1-MMP is modulated by the endogenous inhibitors, Tissue Inhibitor of Metalloproteinases (TIMPs). In this study, we describe a novel strategy that would enable a "designer" TIMP-1 tailored specifically for MT1-MMP inhibition (V4A/P6V/T98L; 1.66 nM) to be targeted to the plasma membrane for more effective MT1-MMP inhibition. To achieve this, we fuse the designer TIMP-1 to the glycosyl-phosphatidyl inositol (GPI) anchor of the prion protein to create a membrane-tethered, high-affinity TIMP variant named "T1" that is predominantly located on the cell surface and co-localised with MT1-MMP. Confocal microscopy shows that T1 is found throughout the cell surface in particular the membrane ruffles where MT1-MMP is most abundant. Expression of T1 brings about a complete abrogation of the gelatinolytic activity of cellular MT1-MMP in HT1080 fibrosarcoma cells whilst in renal carcinoma cells CaKi-1, the GPI-TIMP causes a disruption in MMP-mediated proteolysis of ECM components such as fibronectin, collagen I and laminin that consequently triggers a downstream senescence response. Moreover, the transduced cells also suffer from an impairment in proliferation and survival in vitro as well as in NOD/SCID mouse xenograft. Taken together, our findings demonstrate that the GPI anchor of prion could be exploited as a targeting device in TIMP engineering for MT1-MMP inhibition with a potential in renal carcinoma therapy.
肾癌细胞表达膜型 1 基质金属蛋白酶（MT1-MMP，MMP-14），以降解细胞外基质成分和多种生物活性分子，从而促进转移和细胞增殖。MT1-MMP 的活性受内源性抑制剂组织金属蛋白酶抑制剂（TIMPs）调节。在这项研究中，我们描述了一种新策略，该策略可使专门针对 MT1-MMP 抑制的“设计” TIMP-1（V4A/P6V/T98L；1.66 nM）靶向质膜，从而更有效地抑制 MT1-MMP。为了实现这一目标，我们将设计的 TIMP-1 与朊病毒蛋白的糖基磷脂酰肌醇（GPI）锚定融合，创建一种膜结合、高亲和力的 TIMP 变体，命名为“T1”，主要位于细胞表面，并与 MT1-MMP 共定位。共焦显微镜显示，T1 存在于整个细胞表面，特别是在富含 MT1-MMP 的膜皱襞处。T1 的表达完全阻断了 HT1080 纤维肉瘤细胞中细胞 MT1-MMP 的明胶酶活性，而在肾癌细胞 CaKi-1 中，GPI-TIMP 导致 ECM 成分（如纤维连接蛋白、胶原 I 和层粘连蛋白）的 MMP 介导的蛋白水解中断，从而触发下游衰老反应。此外，转导细胞在体外增殖和存活以及 NOD/SCID 小鼠异种移植中也受到损害。总之，我们的研究结果表明，朊病毒的 GPI 锚可以作为 TIMP 工程中针对 MT1-MMP 抑制的靶向装置，在肾细胞癌治疗中具有潜在应用价值。
