Abstract
Pus10 is a pseudouridine synthase present in Archaea and Eukarya, but not in Bacteria and yeast. It has been suggested that the human PUS10 (DOBI) gene is needed during TRAIL-induced apoptosis. We analyzed the role of PUS10 in TRAIL-induced apoptosis by immunofluorescence, immunoblotting and several indicators of apoptosis. We examined several TRAIL-sensitive cell lines and we also examined some resistant cell lines after treatment with cycloheximide. PUS10 is mainly present in the nucleus. Early during apoptosis, PUS10 translocates to mitochondria via CRM1-mediated export with the concurrent release of cytochrome c and SMAC. Caspase-3 is required for PUS10 translocation, which reciprocally amplifies the activity of caspase-3 through the intrinsic/mitochondrial pathway. This suggests that in addition to cytoplasmic factors, nuclear factors also have a direct role in the major apoptosis pathways. However, p53 is not involved in TRAIL-induced PUS10 movement. The caspase-3-mediated movement of PUS10 and the release of mitochondrial contents enhancing caspase-3 activity creates a feedback amplification loop for caspase-3 action. Therefore, any defect in the movement or interactions of PUS10 would reduce the TRAIL sensitivity of tumor cells.
Highlights
Apoptosis is a genetically determined mode of cell death that relies on energy-dependent cascade of caspase-mediated molecular events.[1,2,3,4,5] It is important for natural cell turnover during development and aging, and in the elimination of virusinfected and damaged cells
RNAi-mediated phenotypic screen of HeLa cells suggested that the human PUS10 (DOBI, FLJ32312) is required for the TRAIL-induced apoptotic signal to progress through the intrinsic pathway.[6]
We used PC3 cells, which are p53 null,[23] to determine the role of human PUS10 (HuP10) in TRAIL-induced apoptosis. This was done to avoid any effects of p53 in apoptosis. Both immunofluorescence (IF) of cells and immunoblot (IB) analyses of nuclear and cytoplasmic fractions using a commercially available anti-HuP10 antibody determined that HuP10 is normally present in the nucleus. (This antibody recognizes HuP10 in both IB and IF analyses; see Supplementary Figures S1C–S1I) The granular appearance of the signal suggests that HuP10 may be concentrated in certain areas within the nuclei, it does not seem to be present in the nucleoli
Summary
Apoptosis is a genetically determined mode of cell death that relies on energy-dependent cascade of caspase-mediated molecular events.[1,2,3,4,5] It is important for natural cell turnover during development and aging, and in the elimination of virusinfected and damaged cells. The intrinsic pathway can be initiated, independent of BID, by the oligomerization of pro-apoptotic proteins, for example, BAK, BAX and so on, which permeabilize the outer mitochondrial membrane and release cytochrome c, SMAC, OMNI and so on.[1,4,12] The effector caspases cleave several proteins that lead to DNA fragmentation, chromatin condensation, cell membrane blebbing, cell shrinkage, formation of apoptotic bodies and cell death.[7] The activities of some caspases are redundant and overlap, for example, initiator caspase-8 with caspase-10, and effector caspase-3 with caspase-6 and -7. The crystal structure of human PUS10 (HuP10) shows that the protein has two domains: an N-terminal (Met1-His285)
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