SAO-1 与 DLC-1 相互作用调控秀丽隐杆线虫生殖腺细胞凋亡的分子机制

细胞凋亡是可调控细胞死亡的主要形式之一，指当细胞受到内、外部环境因素刺激时由基因控制自发而有序的死亡。生物组织通过细胞凋亡可以维持内部环境的稳定性。细胞凋亡功能障碍涉及包括癌症在内的许多复杂疾病的病理过程。因此，对细胞凋亡的研究和理解对于凋亡相关疾病的治疗和预防有重要意义。细胞凋亡的核心机制在秀丽隐杆线虫和哺乳动物细胞中是高度保守的。在线虫中，当细胞凋亡信号启动时，上游BH3结构域（BH3-only）蛋白EGL-1转移到线粒体外膜，与CED-9/Bcl-2 结合，由此产生的CED-9/Bcl-2构象变化导致CED-4/Apaf-1从线粒体释放并易位至核膜，最后激活CED-3/Caspase，启动细胞凋亡。越来越多的研究表明CED-4/Apaf-1释放后转移到核膜是细胞凋亡至关重要的步骤，但是参与CED-4/Apaf-1易位的蛋白质和分子机制仍不清楚。本文以秀丽隐杆线虫为模式生物首次揭示了SAO-1通过C末端无序片段与动力蛋白轻链DLC-1相互作用，调控线虫生殖腺细胞中CED-4/Apaf-1的核膜定位，从而通过核心机制调控生殖腺细胞凋亡。

本文首先研究SAO-1的表达和定位及其在生殖腺细胞凋亡中的作用。SAO-1（Suppressor of aph-1）含有一个高度保守的GYF结构域（glycine–tyrosine-phenylalanine）、两个低复杂度结构域以及C末端无序片段。通过观察荧光标记的SAO-1转基因线虫，揭示了SAO-1蛋白在原始生殖细胞和生殖腺细胞中的表达模式；通过RNA干扰敲减和CRISPR/Cas9敲除sao-1，发现了SAO-1促进生殖腺细胞凋亡的功能。SAO-1功能的丧失导致线虫生殖腺细胞凋亡减少。进一步研究发现，SAO-1通过核心细胞凋亡途径调控线虫生殖腺细胞凋亡，在核心凋亡蛋白CED-9/Bcl-2功能丧失突变体ced-9(n1653) 中敲减sao-1，可以显著抑制ced-9(n1653) 突变体生殖腺细胞的过度凋亡。

本研究进一步发现SAO-1和动力蛋白轻链1（DLC-1）相互作用调控生殖腺细胞凋亡。本文首先通过蛋白质谱分析的方法获得了与SAO-1相互作用的蛋白数据库，进一步分析筛选和验证发现，SAO-1抑制DLC-1在生殖腺细胞质的降解。SAO-1功能的缺失导致线虫生殖腺细胞质的 DLC-1表达显著降低。通过荧光标签可视化蛋白的表达和定位，发现了SAO-1和DLC-1在线虫生殖腺细胞质中共定位表达。进一步通过荧光寿命成像技术和蛋白质体外结合实验，揭示了SAO-1和DLC-1在线虫生殖腺中存在直接相互作用。本研究还揭示了DLC-1通过核心凋亡途径调控生殖腺细胞凋亡。在功能丧失突变体ced-9(n1653) 中敲减dlc-1，可以显著抑制ced-9(n1653) 生殖腺细胞的过度凋亡。本文的遗传学研究进一步表明，SAO-1和DLC-1促进核心通路中的促凋亡蛋白CED-4/Apaf-1在生殖腺细胞核膜的积累。SAO-1和DLC-1缺失导致CED-4/Apaf-1在生殖腺细胞核膜上的聚集下降。

本文最后通过晶体学进一步解析了分辨率为2.4 Å的SAO-1（残基 182-205）和DLC-1复合物的晶体结构：SAO-1与DLC-1在晶体结构中形成2:4复合物；每个SAO-1肽采用扩展构象与两个DLC-1二聚体相互作用，从而在 SAO-1中产生两个相似的 DLC-1结合位点（187 VAT 189和199 CQT 201）。破坏SAO-1和DLC-1的相互作用位点后，DLC-1在线虫生殖腺细胞质的表达显著降低，CED-4/Apaf-1在生殖腺细胞核膜的聚集被抑制，且线虫生殖腺细胞凋亡减少。综上所述，本文首次发现了SAO-1通过C末端的保守位点（187 VAT 189）和（199 CQT 201）与DLC-1相互作用，调控DLC-1在线虫生殖腺细胞质中的表达、CED-4/Apaf-1在线虫生殖腺核膜定位，从而通过核心机制调控生殖腺细胞凋亡的分子机制。

Apoptosis, one of the major forms of regulated cell death, which refers to the spontaneous and orderly death of cells controlled by genes when stimulated by internal and external environmental factors. Biological tissues maintain the stability of the internal environment through apoptosis. Apoptotic dysfunction is involved in the pathological process of many complex diseases, including cancer. Therefore, the research and understanding of apoptosis are important for the treatment and prevention of apoptosis-related diseases. The core mechanism of apoptosis is highly conserved in Caenorhabditis elegans (C. elegans) and Mammalian cells. In C. elegans, when apoptosis is activated, the upstream BH3-only protein EGL-1 is transferred to the outer membrane of mitochondria and bound with CED-9/Bcl-2. The resultant conformational change in CED-9/Bcl-2 leads to the release of CED-4/Apaf-1 from mitochondria and its subsequent translocation to the nuclear membrane. Finally, the CED-3/Caspase protein is activated, and cell death ensues. An increasing number of studies have suggested that the translocation of CED-4/Apaf-1 from mitochondria to the nuclear membrane is a crucial step in apoptosis. However, the protein and molecular mechanisms involved in CED-4/Apaf-1 translocation remain unclear. This study uses C. elegans as the model organism and revealed that SAO-1 C-terminal interacts with dynein light chain (DLC-1) and regulates the accumulation of CED-4/Apaf-1 in the nuclear membrane, thus promoting germ cell apoptosis through the core apoptotic pathway.

This study first investigated the expression and localization of SAO-1 and its role in the apoptosis of germ cells. SAO-1 (Suppressor of aph-1) is a protein containing a highly conserved GYF (glycine–tyrosine-phenylalanine) domain, two low complexity domains, and C-terminal disordered fragments in C. elegans. The fluorescence-labeled SAO-1 transgenic worms created by CRISPR/Cas9 revealed the expression pattern of SAO-1 protein in primordial germ cells and germ cells. Knocking down SAO-1 using RNAi or knocking knock out sao-1 by CRISPR/Cas9, revealed that SAO-1 promotes apoptosis, and the loss function of SAO-1 resulted in reduced apoptosis in C. elegans germ cells. Further studies showed that SAO-1 regulated the apoptosis of C. elegans germline through the core apoptotic pathway. It was found that the number of apoptotic corpses were significantly reduced in the loss-of-function mutant ced-9(n1653) worms after knocked down of sao-1.

Further study suggested that SAO-1 interacts with DLC-1 to regulate germ cell apoptosis. Firstly, the protein database of proteins interacting with SAO-1 was obtained by protein mass spectrometry. Further analysis found that SAO-1 inhibits DLC-1 degradation, and loss of SAO-1 resulted in a significant reduction of DLC-1 expression in the cytoplasm of C. elegans germline. Subsequently, the expression and localization of the protein were visualized by fluorescence microscope, it was found that SAO-1 and DLC-1 co-localized in the cytoplasm of C. elegans germline. Fluorescence lifetime imaging and Pull down experiments showed that SAO-1 interacts with DLC-1 directly in C. elegans germline. Further studies showed that DLC-1 regulated the apoptosis of C. elegans germline through the core apoptotic pathway. it was found that the number of apoptotic corpses was significantly reduced in the loss-of-function mutant ced-9(n1653) worms after DLC-1 depletion. The genetic studies further showed that SAO-1 and DLC-1 regulate apoptosis by regulating the accumulation of CED-4/Apaf-1 in the nuclear envelope of germ cells. Inactivation of SAO-1 and DLC-1 lead to decreased CED-4/Apaf-1 accumulation on the nuclear membrane of germ cells.

Finally, the high-resolution crystal structure analysis further revealed that SAO-1 interacts with DLC-1 to form a 2:4 complex: each of the two β-sheets in the SAO-1 peptide interacted with two DLC-1 dimers. Each SAO-1 peptide adopts an extended conformation to interact with two DLC-1 dimers, resulting in two similar DLC-1 binding sites in SAO-1 (187 VAT 189 and 199 CQT 201). Destroying the interaction site of SAO-1 and DLC-1 leads to reduce of the DLC-1 expression in the cytoplasm, the CED-4/Apaf-1 accumulation in the nuclear membrane, and the apoptotic corpses of the germline. In summary, this study found that SAO-1 interacts with DLC-1 and regulates the expression of DLC-1 in the germline cytoplasm of C. elegans via its C-terminal (187 VAT 189) and (199 CQT 201) and regulates the accumulation of CED-4/Apaf-1 in the nuclear membrane, thus promoting germ cell apoptosis through the core apoptotic pathway for the first time.

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