Syntaxin 12通过介导线粒体囊泡融合及铁转运影响心脏发育的机制研究

Iron homeostasis of cells is tightly regulated, and mitochondria play a central role in cell metabolism and iron homeostasis regulation. SNARE family proteins are involved in vesicle recognition and fusion of different membranous organelles, however, the specific SNARE proteins and complexes responsible for mitochondrial vesicle fusion are still unclear. Our previous work indicated that Syntaxin 12, a SNARE family member, co-located with mitochondria, and Syntaxin 12 knockout mice had abnormal mitochondrial morphology and cardiac function. Meanwhile, Syntaxin 12 knocked out mice showed iron deficiency in the heart. Therefore, it was hypothesized that Syntaxin 12 was involved in mitochondrial vesicular fusion and iron transport, which further affected heart development. We intend to use ultra-high resolution real-time monitoring of Syntaxin 12 vesicles to determine whether it is involved in mitochondrial vesicle fusion. Proteins that interact with Syntaxin 12 and regulate mitochondrial vesicular fusion were screened out by means of immunoprecipitation and mass spectrometry, and the regulatory mechanism was elucided. We will further clarify how Syntaxin 12 regulates mitochondrial iron transport and heart development, providing a theoretical basis for the study of development defects and human diseases caused by iron disorders.

细胞铁稳态受到严密调控，而线粒体在细胞代谢及铁稳态调控中居于核心地位，通过囊泡向线粒体输送铁是一种高效安全的方式。SNARE家族参与不同膜性细胞器囊泡识别与融合，而专一性负责线粒体囊泡融合的SNARE蛋白及其复合体仍然未知。我们前期工作表明SNARE家族蛋白Syntaxin12与线粒体存在共定位，Syntaxin12敲除小鼠心肌线粒体形态异常及心脏功能异常。同时，Syntaxin12敲除小鼠心脏铁缺乏。因此提出假说，Syntaxin12参与线粒体囊泡融合及铁转运，进一步影响心脏发育。我们拟利用超高分辨实时监测Syntaxin12囊泡，明确其是否参与线粒体囊泡融合；通过免疫共沉淀与质谱结合的方式筛出与Syntaxin12互作且调控线粒体囊泡融合的蛋白，阐明其作用机制。我们将进一步明确Syntaxin12调控线粒体铁转运及心脏发育的方式，为研究铁紊乱导致的发育异常及人类疾病提供理论基础。

本项目在分子、细胞、生化、动物水平等多空间尺度上研究Syntaxin 12调控线粒体膜泡融合与铁转运，进而影响心脏发育，通过活细胞观察、质谱与蛋白组学、免疫共沉淀等方法系统研究Syntaxin 12调控线粒体囊泡融合与铁转运的分子机制，明确影响心脏发育的原因，取得了一系列成果：（1）首先，本项目发现在小鼠心脏组织中，Syntaxin 12与SNAP25及VAMP2互作，调控线粒体囊泡融合过程。这也是首次阐明了介导线粒体囊泡融合的关键蛋白。（2）铁对于线粒体的功能维持至关重要，线粒体呼吸链需要铁离子的参与。我们的研究结果也证实Syntaxin 12可与转铁蛋白受体TFR1共定位并影响其周转，从而调控线粒体的铁转运。我们由此证明Syntaxin 12参与向线粒体输送铁离子的囊泡转运并阐明了相关分子机制。（3）心肌细胞线粒体的正常运转离不开铁离子。本项目发现，Syntaxin 12基因敲除小鼠的心脏发育异常，出现心衰表型。我们进一步的研究揭示Syntaxin 12敲除小鼠心脏中铁缺乏。我们构建了Syntaxin 12敲低后的斑马鱼模型，同样发现了心脏发育的异常。相关动物实验结果证实了Syntaxin 12可能影响心肌细胞线粒体的铁转运，从而导致心脏异常。（4）本项目首次发现GTPBP3蛋白的关键突变位点并对其影响线粒体功能的表型进行验证。上述理论及机制研究成果部分已发表于Oxidative Medicine and Cellular Longevity等杂志上。通过本课题的实施，我们揭示了Syntaxin 12参与心脏发育调节机制及相关的关键蛋白，阐明了介导线粒体囊泡转运的关键SNARE复合体成员，明确了Syntaxin 12通过与铁转运受体TFR1互作而完成线粒体铁转运进而影响心脏发育的分子机制。本项目提示Syntaxin 12有可能作为心脏相关疾病治疗的潜在靶点。在SCI收录杂志论文发表发表标注本基金号的研究论文8篇，培养硕士研究生1人，达到或超过了课题的预期目标。