No abstract

During apoptosis, phosphatidylserine (PS), normally restricted to the inner leaflet of the plasma membrane, is exposed on the surface of apoptotic cells and serves as an “eat-me” signal to trigger phagocytosis. It is poorly understood how PS exposure is activated in apoptotic cells. Here we report that CED-8, a C. elegans protein implicated in controlling the kinetics of apoptosis and a homolog of the XK family proteins, is a substrate of the CED-3 caspase. Cleavage of CED-8 by CED-3 activates its proapoptotic function and generates a carboxyl terminal cleavage product, acCED-8, that promotes PS externalization in apoptotic cells and can induce ectopic PS exposure in living cells. Consistent with its role in promoting PS externalization in apoptotic cells, ced-8 is important for cell corpse engulfment in C. elegans. Our finding identifies a crucial link between caspase activation and PS externalization, which triggers phagocytosis of apoptotic cells.

Radiation-induced bystander effects (RIBE) refer to a unique process, in which factors released by irradiated cells or tissues exert effects on other parts of the animal not exposed to radiation, causing genomic instability, stress responses, and altered apoptosis or cell proliferation1–3. Despite important implications in radioprotection, radiation safety and radiotherapy, the molecular identities of RIBE factors and their mechanisms of action remain elusive. Here we use C. elegans as an animal model to study RIBE and have identified a cysteine protease CPR-4, a human cathepsin B homolog, as the first RIBE factor in nematodes. CPR-4 is secreted from animals irradiated with ultraviolet (UV) or ionizing gamma rays (IR) and is the major factor in the conditioned medium that leads to inhibition of cell death and increased embryonic lethality in unirradiated animals. Moreover, CPR-4 causes these effects and stress response at unexposed sites distal to the irradiated tissue. The activity of CPR-4 is regulated by the p53 homolog cep-1 in response to radiation and CPR-4 appears to act through the insulin-like growth factor receptor, DAF-2, to exert RIBE. Our study provides critical insights into the elusive RIBE and will facilitate identification of additional RIBE factors and their mechanisms of action.

Systems biology is increasingly being applied in nanosafety research for observing and predicting the biological perturbations inflicted by exposure to nanoparticles (NPs). In the present study, we used a combined transcriptomics and proteomics approach to assess the responses of human monocytic cells to Au-NPs of two different sizes with three different surface functional groups, i.e., alkyl ammonium bromide, alkyl sodium carboxylate, or poly(ethylene glycol) (PEG)-terminated Au-NPs. Cytotoxicity screening using THP-1 cells revealed a pronounced cytotoxicity for the ammonium-terminated Au-NPs, while no cell death was seen after exposure to the carboxylated or PEG-modified Au-NPs. Moreover, Au-NR3+ NPs, but not the Au-COOH NPs, were found to trigger dose-dependent lethality in vivo in the model organism, Caenorhabditis elegans. RNA sequencing combined with mass spectrometry-based proteomics predicted that the ammonium-modified Au-NPs elicited mitochondrial dysfunction. The latter results were validated by using an array of assays to monitor mitochondrial function. Au-NR3+ NPs were localized in mitochondria of THP-1 cells. Moreover, the cationic Au-NPs triggered autophagy in macrophage-like RFP-GFP-LC3 reporter cells, and cell death was aggravated upon inhibition of autophagy. Taken together, these studies have disclosed mitochondria-dependent effects of cationic Au-NPs resulting in the rapid demise of the cells.

The conserved phosphatidylserine receptor (PSR) was first identified as a receptor for phosphatidylserine, an "eat-me" signal exposed by apoptotic cells. However, several studies suggest that PSR may also act as an arginine demethylase, a lysyl hydroxylase, or an RNA binding protein through its N-terminal JmjC domain. How PSR might execute drastically different biochemical activities, and whether they are physiologically significant, remain unclear. Here we report that a lysine-rich motif in the extracellular domain of PSR-1, the Caenorhabditis elegans PSR, mediates specific phosphatidylserine binding in vitro and clearance of apoptotic cells in vivo. This motif also mediates phosphatidylserine-induced oligomerization of PSR-1, suggesting a mechanism by which PSR-1 activates phagocytosis. Mutations in the phosphatidylserine-binding motif, but not in its Fe(II) binding site critical for the JmjC activity, abolish PSR-1 phagocytic function. Moreover, PSR-1 enriches and clusters around apoptotic cells during apoptosis. These results establish that PSR-1 is a conserved, phosphatidylserine-recognizing phagocyte receptor.

Programmed cell clearance is a highly regulated physiological process of elimination of dying cells that occurs rapidly and efficiently in healthy organisms. It thus ensures proper development as well as homeostasis. Recent studies have disclosed a considerable degree of conservation of cell clearance pathways between nematodes and higher organisms. The externalization of the anionic phospholipid phosphatidylserine (PS) has emerged as an important “eat-me” signal for phagocytes and its exposition on apoptotic cells is controlled by phospholipid translocases and scramblases. However, there is mounting evidence that PS exposure occurs not only in apoptosis, but may also be actively expressed on the surface of cells undergoing other forms of cell death including necrosis; PS is also expressed on the surface of engulfing cells. Additionally, PS may act as a “save-me” signal during axonal regeneration. Here we discuss mechanisms of PS exposure and its recognition by phagocytes as well as the consequences of PS signaling in nematodes and in mammals.

Caspases are cysteine proteases with critical roles in apoptosis. The Caenorhabditis elegans caspase CED-3 is activated by autocatalytic cleavage, a process enhanced by CED-4. Here we report that CED-3 zymogen localizes to the perinuclear region in C. elegans germ cells and that CED-3 autocatalytic cleavage is held in check by C. elegans nuclei and activated by CED-4. The nuclear pore protein NPP-14 interacts with the prodomain of CED-3 zymogen, colocalizes with CED-3 in vivo, and inhibits CED-3 autoactivation in vitro. Several missense mutations in the CED-3 prodomain result in stronger association with NPP-14 and reduced CED-3 activation by CED-4 in the presence of nuclei or NPP-14, leading to cell death defects. Those same mutations enhance autocatalytic cleavage of CED-3 in vitro and increase apoptosis in vivo in the absence of npp-14. Our results reveal a critical role for nuclei and nuclear membrane proteins in regulating activation and localization of CED-3.

The Caenorhabditis elegans aminophospholipid translocase TAT-1 maintains phosphatidylserine (PS) asymmetry in the plasma membrane and regulates endocytic transport. Despite these important functions, the structure-function relationship of this protein is poorly understood. Taking advantage of the tat-1 mutations identified by the C. elegans million mutation project, we investigated the effects of 16 single amino acid substitutions on the two functions of the TAT-1 protein. Two substitutions that alter a highly conserved PISL motif in the fourth transmembrane domain and a highly conserved DKTGT phosphorylation motif, respectively, disrupt both functions of TAT-1, leading to a vesicular gut defect and ectopic PS exposure on the cell surface, whereas most other substitutions across the TAT-1 protein, often predicted to be deleterious by bioinformatics programs, do not affect the functions of TAT-1. These results provide in vivo evidence for the importance of the PISL and DKTGT motifs in P4-type ATPases and improve our understanding of the structure-function relationship of TAT-1. Our study also provides an example of how the C. elegans million mutation project helps decipher the structure, functions, and mechanisms of action of important genes.