解析线粒体参与细胞器互作的蛋白网络及分子机制

Mitochondria are essential for many biological processes to happen in cells including apoptosis. Inter-organelle communication plays a vital role in maintaining mitochondrial function, consequently, it is likely this process also contributes to apoptosis. Despite the fast growth of this field, the mechanisms of many mitochondria-organelle communications remain to be elucidated; and we lack the full spectrum of protein-protein interaction network which mediates these biological processes. In order to construct this network and analysis how Inter-organelle communication impact apoptosis, we will first identify proteins localized on the surface of organelles and illustrate their binding topology by using novel chemical cross-linkers and new mass spec method. Then, through a systematic CRISPR/Cas9 screen focused on proteins in this network, we intend to identify proteins in this network which plays a role in apoptosis. We will further decipher how mitochondria-organelle communication stabilize mitochondria and hinder apoptosis under cellular stresses. In previous studies, we created several chemical cross-linkers and constructed a pioneering network. Through analysis of this network, we identified a novel protein interaction between mitochondrial protein PHB1 and endoplasmic reticulum (ER) protein Erlin2. Moreover, disrupting this protein interaction altered ER and mitochondrial morphology, future render cells more sensitive to apoptosis inducing reagent. We will use proposed approach to identify novel mitochondria-organelle communications and illustrate how these biological processes regulate programmed cell death. This study will illustrate how mitochondria-organelle communication impact apoptosis through regulation of mitochondria; and has the potential to provide a blueprint for future study of inter-organelle communications.

线粒体不仅是能量代谢的核心细胞器，也在细胞命运决定中起重要作用。线粒体与其他细胞器的互作是其行使功能和维持稳态的重要因素，之前的研究提示这种稳态维持作用也影响着细胞命运决定。蛋白在介导细胞器互作中起重要作用，通过前期的研究，我们不仅为鉴定互作蛋白开发了部分新材料和新方法，也通过预实验发现了参与线粒体与内质网互作的蛋白：PHB1和Erlin2，且影响两者互作会导致线粒体和内质网形态和结构的改变，并增加细胞对凋亡诱导药物的敏感性。我们将进一步系统的鉴定参与线粒体-细胞器互作的蛋白，并在此基础上寻找影响细胞凋亡的线粒体-细胞器互作机制。该课题的开展将为全面的阐述线粒体-细胞器互作在维持线粒体稳态、影响细胞命运决定中的作用；也将为细胞器互作的分子机制研究提供线粒体相关部分的蓝图。

细胞器互作对线粒体功能至关重要，线粒体通过与其他细胞器的互作维持其结构的动态变化和行使部分生物学功能。细胞器互作是一个高度动态的过程，且调节和维持这一过程依赖蛋白与蛋白之间的相互作用。我们从细胞器互作的这两个关键点入手，开发新的实验方法以期系统和定量的研究线粒体与其他细胞器互作的过程并勾画互作的蛋白质基础。我们首先确定了可能参与细胞器互作的线粒体表面蛋白。为了探索这些蛋白的直接相互作用蛋白，我们开发了用于细胞内进行蛋白交联，且可用于三级质谱分析的交联剂，并对其进行了提高溶解性、提高穿膜能力、提高交联效率以及提高质谱检测效率的改良。我们也开发了用于动态和定量分析线粒体与其他细胞器互作的基于化学发光能量转移（BRET)原理的检查方法，通过该方法我们发现改变线粒体钙的含量影响线粒体与内质网的互作，并初步探索了在这种情况下影响线粒体与内质网的互作的分子机制。该研究致力于开发系统性研究细胞器互作的工具和手段，有望对细胞器互作领域的研究带来新方法、新技术和新思路。