Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein

Although the function of metallothionein (MT), a 6- to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca(2+) act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT(−/−)) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed.

尽管分子量为6~7 kDa的富含半胱氨酸的金属结合蛋白——金属硫蛋白（metallothionein，MT）的功能仍未明确，但体外研究已提示，MT是细胞内氧化还原信号通路的重要组成部分，同时也是一氧化氮（nitric oxide，NO）的作用靶点。为直接在活细胞中研究MT与NO的相互作用，我们构建了一种融合蛋白：MT被夹在两个突变型绿色荧光蛋白（green fluorescent proteins，GFPs）之间。针对该嵌合体（FRET-MT）开展的体外研究证实，荧光共振能量转移（fluorescent resonance energy transfer，FRET）可用于追踪MT释放金属离子时伴随的构象变化。对活体细胞内皮细胞的成像实验显示，可升高细胞质钙离子浓度的试剂，会通过内源性生成的NO快速且持续地从MT中释放金属离子。肠系膜动脉的肌源性反射在金属硫蛋白基因敲除小鼠（MT⁻/⁻）中缺失，而阻断内源性NO合成则可恢复该反射，这一发现为该相互作用在完整组织中的功能提供了实验依据。本研究首次将基于绿色荧光蛋白（GFP）的分子内荧光共振能量转移技术应用于天然蛋白，并证实FRET-MT可作为细胞内NO生成的替代指示剂。此外，本研究还揭示了MT的金属硫醇盐簇在血管组织NO信号通路中的重要作用。