| 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 tissue s 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-1regulated 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-1expression 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-1interacts 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-1interacts 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. |
