cytochrome-c-t and Virus-Diseases

Nervous necrosis virus (NNV)-induced, host cell apoptosis mediates secondary necrosis by an ill-understood process. In this study, redspotted grouper nervous necrosis virus (RGNNV) is shown to induce mitochondria-mediated necrotic cell death in GL-av cells (fish cells) via cytochrome c release, and anti-apoptotic proteins are shown to protect these cells from death. Western blots revealed that cytochrome c release coincided with disruption of mitochondrial ultrastructure and preceded necrosis, but did not correlate with caspases activation. To identify the mediator(s) of this necrotic process, a protein synthesis inhibitor (cycloheximide; CHX; 0.33 microg/ml) was used to block cytochrome c release as well as PS exposure and mitochondrial membrane permeability transition pore (MMP) loss. CHX (0.33 microg/ml) completely blocked viral protein B2 expression, and partly blocked protein A, protein alpha, and a pro-apoptotic death protein (Bad) expression. Overexpression of B2 gene increased necrotic-like cell death up to 30% at 48 h post-transfection, suggesting that newly synthesized protein (B2) may be involved in this necrotic process. Finally, necrotic death was prevented by overexpression of Bcl-2 family proteins, zfBcl-x(L) and xfMcl-1a. Thus, new protein synthesis and release of cytochrome c are required for RGNNV-induced necrotic cell death, which can be blocked by anti-apoptotic Bcl-2 members.

Topics: Animals; bcl-X Protein; Caspases; Cell Line; Cycloheximide; Cytochromes c; Enzyme Activation; Fishes; Liver; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Models, Biological; Myeloid Cell Leukemia Sequence 1 Protein; Necrosis; Neoplasm Proteins; Nodaviridae; Phosphatidylserines; Protein Biosynthesis; Proto-Oncogene Proteins c-bcl-2; Viral Proteins; Virus Diseases

Natural killer (NK) cells play an important role in innate immunity against virally infected or transformed cells as the first defense line. Granzyme M (GzmM) is an orphan granzyme that is constitutively highly expressed in NK cells and is consistent with NK cell-mediated cytolysis. We recently demonstrated that GzmM induces caspase-dependent apoptosis with DNA fragmentation through direct cleavage of inhibitor of caspase-activated DNase (ICAD). However, the molecular mechanisms for GzmM-induced apoptosis are unclear. We found GzmM causes mitochondrial swelling and loss of mitochondrial transmembrane potential. Moreover, GzmM initiates reactive oxygen species (ROS) generation and cytochrome c release. Heat shock protein 75 (HSP75, also known as TRAP1) acts as an antagonist of ROS and protects cells from GzmM-mediated apoptosis. GzmM cleaves TRAP1 and abolishes its antagonistic function to ROS, resulting in ROS accumulation. Silencing TRAP1 through RNA interference increases ROS accumulation, whereas TRAP1 overexpression attenuates ROS production. ROS accumulation is in accordance with the release of cytochrome c from mitochondria and enhances GzmM-mediated apoptosis.

Topics: Apoptosis; Caspases; Cytochromes c; DNA Fragmentation; Granzymes; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Immunity, Innate; Killer Cells, Natural; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species; RNA Interference; Virus Diseases

Topics: Animals; Cytochromes c; Disease; Ectromelia; Extremities; Leukemia; Mice; Rabbits; Syndrome; Vaccination; Virus Diseases