Characterization of proteases involved in cell death pathways
1)听Identification and characterization of downstream pathways of the CED-3 cell death protease
Analysis of downstream pathways of important enzymatic biomolecules (e.g., kinases and proteases) that have multiple substrates has always been a difficult challenge. Targets of these enzymes could be difficult to identify through conventional genetic screens, since elimination of one of the multiple targets of an enzyme may fail to cause any visible phenotype that can be scored in genetic screens.听听Enzymatic targets could also be difficult to identify through commonly used biochemical approaches such as protein interaction screens that require stable interaction rarely seen between an enzyme and a substrate.听听In fact, to date there have been no good methods available that can systematically and effectively address the problem of substrate identification.
During apoptosis,听a family of aspartate-specific cysteine proteases named听caspases听are听activated听proteolytically听to execute the apoptotic program.听听Little is known about the identities of their听in vivo听targets or their downstream pathways that mediate the killing functions.听听We have developed a novel, GFP-based genetic screen to identify downstream targets of the听颁.听别濒别驳补苍蝉听caspase, CED-3.听听In this screen, the green fluorescent protein (GFP) and a constitutively activated version of CED-3 were co-expressed in听颁.听别濒别驳补苍蝉听mechanosensory听neurons and GFP was used as a sensitive cell existing marker to isolate mutations that either partially suppress or delay ectopic neuronal deaths caused by the activated CED-3 (Figure 1).听听These听CED-3听protease听suppressors (we named these听cpsmutations) likely affect genes that act downstream of, or in parallel to,听ced-3听to mediate various cell-killing processes.听听We have screened approximately 150,000听颁.听别濒别驳补苍蝉听haploid genomes and isolated more than 80听cps听mutations.听听听Phenotypic analyses of these听cps听mutants suggest that these mutations not only suppress CED-3-induced ectopic neuronal deaths but also affect normal programmed cell death in听颁.听别濒别驳补苍蝉, indicating that they are true cell-death mutants.听Genetic analyses of these听cps听mutations indicate that they affect at least fourteen new cell death genes (cps-1听迟辞听cps-14) and one previously identified gene (ced-1), which acts downstream of听ced-3听to remove apoptotic cell corpses.听听Phenotypic analyses of these听cps听mutants suggest that they can be categorized into three major groups: 1) mutations that weakly suppress cell deaths in听颁.听别濒别驳补苍蝉听(cps-1听补苍诲听肠辫蝉-2), 2) mutations that delay the normal progression of apoptosis (cps-3, 4, 5, 6, 7, 8, 10,听补苍诲听11), and 3) mutations that may cause defect in the removal of cell corpses (cps-9, 12, 13, 14).听听These 14听cps听genes may encode important components that act downstream of the CED-3 protease to regulate or execute different aspects of the cell disassembly and removal process (Figure 2).听听Indeed, using the TUNEL assay听(an assay used to detect DNA breaks generated during apoptosis), we found that听cps-3, 4, 6, 7, 8, 10, 11, 13听补苍诲听14听genes are involved in the chromosome fragmentation process, one of the key steps and a hallmark of apoptosis.听听In addition, using an听annexin听V staining assay (used to detect surface-exposed听phosphatidylserine), we found that three genes (cps-12, cps-13,听补苍诲听肠辫蝉-14) affect externalization of听phosphatidylserine听(PS) in apoptotic cells.听听PS normally is restricted to the inner leaflet of the plasma membrane but is exposed or flipped out in apoptotic cells and has been suggested to serve as an engulfment signal for phagocytosis.听听How PS is flipped out in dying cells during apoptosis has not been clear and is a topic of intense studies.听听Molecular characterization of听cps-12, cps-13听补苍诲听cps-14听will likely provide important insights听towards addressing this fundamental biological question.
We recently cloned the听cps-6听gene and found that听cps-6听encodes a homologue of human mitochondrial endonuclease G (endoG).听听In collaboration with Dr.听Xiaodong听Wang's group at the University of Texas Southwestern Medical Center, we showed that the CPS-6 protein localizes to听颁.听别濒别驳补苍蝉听mitochondria and possesses an endonuclease activity that is capable of inducing the generation of apoptotic DNA ladders in isolated听Hela听cell nuclei.听听Furthermore, the mouse听EndoG听can substitute for the functions of听cps-6听in听颁.听别濒别驳补苍蝉, suggesting that CPS-6 and听EndoG听define an evolutionarily conserved DNA degradation pathway.听听Our study also demonstrates for the first time that mitochondria is important for apoptosis in invertebrates and is a conserved regulator of apoptosis听(听补苍诲听PDF).
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2)听Characterization of the听颁.听别濒别驳补苍蝉听mitochondrial cell death pathways
The findings that apoptotic regulators such as cytochrome c,听endoG, and apoptosis-inducing-factor (AIF) are released from mitochondria to mediate different aspects of apoptosis indicate that听mitochondria plays听an important role in mammalian apoptosis.听听A family of proteins containing a unique听Bcl-2听homology听3听motif (BH3) are involved in releasing these mitochondrial听apoptogenic听factors.听听The finding that听cps-6听encodes a mitochondrial endonuclease prompted us to examine whether there are additional mitochondrial proteins involved in听C.elegans听apoptosis.听听We characterized the听颁.听别濒别驳补苍蝉听homologue of AIF, a human mitochondrial听oxidoreductase听that is released from mitochondria during apoptosis to induce chromosome condensation and fragmentation.听听Intriguingly, the听oxidoreductase听activity of AIF is dispensable for its听apoptogenic听functions and AIF does not possess a nuclease activity, raising a major question of how AIF induces apoptosis.听听We cloned the听worm听AIF听homologue,听wah-1,听补苍诲 used RNA interference (RNAi) to study its functions in听颁.听别濒别驳补苍蝉听apoptosis.听听We found that reduction of the听wah-1听activity in听C.elegans听delays the normal progression of apoptosis, results in accumulation of TUNEL-positive DNA breaks, and enhances the defects of other cell death mutants, indicating that听颁.听别濒别驳补苍蝉听AIF does play an important role in regulating听颁.听别濒别驳补苍蝉听apoptosis.听听Interestingly,听wah-1(RNAi)听results in cell death phenotypes that are similar to those of the听cps-6听mutant and fails to enhance the cps-6听cell death defects, indicating that听wah-1听补苍诲听cps-6听function in the same cell death pathway.听听Indeed, we found that WAH-1, which also localizes to听颁.听别濒别驳补苍蝉听mitochondria, can associate and cooperate with CPS-6听in vitro听to promote DNA degradation.听听In vivo, co-expression of听wah-1听补苍诲听cps-6听can synergistically induce cell killing.听听These findings indicate that CPS-6/EndoG听is likely a target or an effector that WAH-1/AIF interacts with to induce chromosome fragmentation during apoptosis and that CSP-6/EndoG听补苍诲 WAH-1/AIF define a single evolutionarily conserved cell death pathway initiated from mitochondria.听听Importantly, WAH-1 can be released from mitochondria by EGL-1, a听颁.听别濒别驳补苍蝉听BH3-domain containing cell death activator, in a manner similar to the release of cytochrome c or听EndoG听from mitochondria by mammalian BH3-domain containing proteins such as Bid or听Bim.听听However, this EGL-1-induced release of WAH-1 is dependent on the activity of the CED-3 protease, indicating that worm AIF functions in a听caspase-dependent manner.听听These observations strongly suggest that the mitochondrial cell death pathway is conserved between nematodes and humans (听补苍诲听PDF).
We听are currently focusing on identifying additional mitochondrial factors that are important for the regulation and execution of programmed cell death in听颁.听别濒别驳补苍蝉听using both genetic and biochemical approaches.听 We are particularly interested in understanding how mitochondrial听apoptogenic听factors are released from mitochondria to affect various aspects of apoptosis.听 We hope to identify most components functioning in听颁.听别濒别驳补苍蝉听mitochondrial cell death pathways.
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3) Functional genomic analysis of the apoptotic DNA degradation process in听颁.听别濒别驳补苍蝉
The observations that multiple genes (cps-3, 4, 6, 7, 8, 10, 11, 13, 14听补苍诲听wah-1)听are involved in the apoptotic DNA degradation process in听颁.听别濒别驳补苍蝉听indicate that this is likely a rather complicated and tightly regulated process.听听To identify all the nucleases involved in听颁.听别濒别驳补苍蝉听apoptosis, we conducted a candidate-based, genome-wide听RNAi听screen to systematically search for genes important for apoptotic DNA degradation in听颁.听别濒别驳补苍蝉.听听We used听RNAi听to individually inhibit the expression of 77听颁.听别濒别驳补苍蝉听genes that encode nucleases or nuclease-related proteins and have identified nine candidate apoptotic nucleases,听including two previously known apoptotic nucleases (CPS-6 and NUC-1).听听We named these new听cell death-related听nucleases as CRN nucleases.听听Molecular genetic analyses of these听crn听genes indicate that these nine apoptotic nucleases comprise at least two independent pathways that contribute to cell killing by degrading chromosomal DNA, with听cps-6, crn-1, crn-4, crn-5,听and听crn-7听acting together in one pathway and听crn-2/crn-3听in another pathway (Figure 2).听听nuc-1听补苍诲听crn-6听appear to act at later stages of apoptotic DNA degradation.听听Interestingly, several听crn听genes have human homologues that play important roles in RNA processing and splicing, protein folding, DNA replication and damage repair, suggesting that these CRN proteins may play dual roles in both cell survival and cell death.听听The identification of seven听crn听genes will allow systematic deciphering of the mechanisms of apoptotic DNA degradation, which remain a poorly understood biological process听(听补苍诲听PDF).
As a first step towards the understanding of how apoptotic nucleases interact to promote apoptotic DNA degradation, we initiated biochemical and mechanistic studies of CRN-1,听a homologue of the human Fen-1 endonuclease that plays important roles in DNA replication and repair.听听We found that CRN-1 localizes to nuclei and can associate and cooperate with CPS-6 to promote stepwise DNA fragmentation, utilizing the endonuclease activity of CPS-6 and both the 5'-3'听exonuclease听activity and a novel gap-dependent endonuclease activity of CRN-1.听听Our results suggest that CRN-1/FEN-1 may play a critical role in switching the state of cells from DNA replication/repair to DNA degradation during apoptosis听(听补苍诲听听PDF).
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4) Identification and characterization of genes involved in exposure or recognition of 'eat-me' signals during removal of apoptotic cell corpses
Phagocytosis of apoptotic cells is an integral part of cell death execution and an important event in tissue remodeling, suppression of inflammation, and regulation of immune responses.听During apoptosis, 'eat-me' or engulfment signals are exposed or released from the dying cells to trigger the phagocytic events by neighboring cells or macrophages.听听Very little is known about what these 'eat-me' signals are or how they are exposed or released from the dying cells.听听One of the candidate 'eat-me' signals is听phosphatidylserine听(PS), which normally is restricted to the inner leaflet of the plasma membrane but is exposed on the surface of apoptotic cells.听听It is unclear what regulates the externalization of PS in apoptotic cells and how phagocytes recognize PS and subsequently initiate the phagocytic events.听听From our听cps听screens, we have identified three genes (cps-12, cps-13听补苍诲听肠辫蝉-14) that affect the externalization of PS in apoptotic cells.听Molecular characterization of these three genes will provide important insights on how PS externalization is regulated and executed in apoptotic cells.
Recently, a putative听phosphatidylserine听receptor (PSR) was identified and proposed to mediate PS recognition and phagocytosis.听听Interestingly,听颁.听别濒别驳补苍蝉听contains a gene (F29B9.4) that shares 56% sequence identity with human PSR.听听To investigate whether the听颁.听别濒别驳补苍蝉听PSR homologue (named听psr-1) affects cell corpse engulfment, we isolated a deletion allele (tm469) in the听psr-1听locus and analyzed its mutant phenotypes.听听We found that the听psr-1(tm469)听mutation does result in a defect in cell corpse engulfment.听听Interestingly,听psr-1听appears to act in the same cell corpse engulfment pathway as听ced-2, ced-5, ced-10听and听ced-12.听听Genetic bypass experiments indicate that听psr-1听acts upstream of听ced-2, ced-5, ced-10听补苍诲听ced-12听to control cell corpse engulfment.听听In vitro听颁.听别濒别驳补苍蝉听PSR-1 behaves like human PSR and binds preferentially PS or cells with exposed PS.听听Interestingly, the intracellular domain of PSR-1 interacts with CED-5 and CED-12, suggesting that PSR-1 may transduce engulfment signal through CED-5 and CED-12.听听Our findings suggest that PSR-1 is likely an upstream receptor for the signaling pathway containing CED-2, CED-5, CED-10 and CED-12 proteins and plays an important role in recognizing听phosphatidylserine听during phagocytosis (听补苍诲听PDF).
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Related Publications:
Parrish, J., Li, L., Klotz, K.,听Ledwich, D., Wang, X.D., and Xue, D. (2001).听听颁.听别濒别驳补苍蝉听mitochondrial endonuclease G is important for apoptosis.听听Nature听412, 90-94. (听补苍诲听PDF).
Wang, X.C., Yang, C.L., Cai, J.J., Shi, Y.G., and Xue, D. (2002).听Mechanisms of AIF-mediated apoptotic DNA degradation in听Caenorhabditis elegans.听Science听298, 1587-1592.听(听补苍诲听PDF).听听(PDF)听
Parrish, J. and Xue, D. (2003).听Functional genomic analysis of apoptotic DNA degradation in听C. elegans.听Mol. Cell听11, 987-996. (听补苍诲听PDF)
Parrish, J., Yang, C.L., Shen, B.H., and Xue, D. (2003). CRN-1, a听Caenorhabditis elegans听FEN-1听homologue,听cooperates with CPS-6/EndoG to promote apoptoticDNA degradation.听EMBO. J.听22, 3451-3460. (听补苍诲听PDF)听Science SKTE Review (PDF)听
Wang, X.C., Wu, Y.C., Fadok, V.,听Lee, M.C.,听Gengyo-Ando, K.,听Cheng, L.C.,听Ledwich, D., Hsu, P.K., Chen, J.Y., Chou, B.K.,听Henson, P., Mitani, S., and Xue, D. (2003). Cell Corpse Engulfment听Mediated by听C. elegans听Phosphatidylserine Receptor Through CED-5 and CED-12.听Science听302, 1563-1566.(听补苍诲听PDF).听Science Persepectives (PDF)听补苍诲听Science SKTE Review (PDF)
Wang, X.C., Wang, J., Gengyo-Ando, K., Gu, L.C., Sun, C.L., Yang, C.L., Shi, Y., Kobayashi, T., Shi, Y.G., Mitani, S., Xie, X.S., and Xue, D. (2007). "颁.听别濒别驳补苍蝉听mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1".听Nature Cell Biology听9, 541-549.听(听补苍诲听PDF).听听(PDF)
Darland-Ransom, M., Wang, X.C., Sun, C.L., Mapes, J., Gengyo-Ando, K., Mitani, S. and Xue, D. (2008).听Role of听C. elegans听TAT-1 protein in maintaining plasma membrane phosphatidylserine asymmetry.听Science听320, 528-531.听(听补苍诲听PDF).听Science Perspectives (PDF)
Breckenridge, D., Kang, B.H.,听Kokel, D.,听Mitani, S.,听Staehelin, A.L., and Xue, D. (2008).听Caenorhabditis听elegans听drp-1听补苍诲听fis-2听regulate distinct cell death execution pathways downstream of听ced-3and independent of听ced-9.听听Mol. Cell听31 586-597.听(听补苍诲听)
Hsiao, Y.Y., Nakagawa, A., Shi, Z., Mitani, S., Xue, D. and Yuan, H. S. (2009). Crystal structure of CRN-4: implications for domain function in apoptotic DNA degradation.听Mol. Cell.听Biol.听29, 448-457.听(听补苍诲听PDF)
Lai, H.J., Lo, S.Z.,听Kage-Nakadai, E.,听Mitani, S., and Xue, D. (2009).听The roles and acting mechanism of听Caenorhabditis听elegans听DNase听II genes in apoptotic DNA degradation and development.听PLoS听One听4, e7348.听(听补苍诲听PDF)
Nakagawa, A.*, Shi, Y.*, Kage-Nakadai, E., Mitani, S., and Xue, D. (2010).听Caspase-Dependent Conversion of Dicer Ribonuclease into a Death-Promoting Deoxyribonuclease.听Science听328, 327-334.听*Equal contributions (听补苍诲听PDF). Research Article featured on the听听of听Science听补苍诲听Science Perspectives,听Developmental Cell Preview,听Highlights in Nature Structural and Molecular Biology,听Nature Reviews Molecular Cell Biology,听Disease Models and Mechanisms, and听Faculty of 1000
Wang, X.C., Li W., Zhao, D.F., Liu, B., Shi, Y., Chen, B.H., Yang, H.W., Guo, P.F., Geng, X., Shang, Z.H., Peden, E., Kage-Nakadai, E., Mitani, S., and Xue, D. (2010).听颁.听别濒别驳补苍蝉听transthyretin-like protein TTR-52 mediates recognition of apoptotic cells by the CED-1 phagocyte receptor.听Nature Cell Biology听12, 655-664.听(听补苍诲听PDF)听Nature Cell Biology News and Views
Lin, J.L., Nakagawa, A., Lin, C.L., Hsiao, Y.Y., Yang, W.Z., Wang, Y.T., Doudeva, L.G., Skeen-Gaar, R.R., Xue, D., and Yuan, H.S. (2012).听Structural insights into apoptotic DNA degradation by CED-3 Protease Suppressor-6 (CPS-6) from听Caenorhabditis elegans.听Journal of Biological Chemistry听287:7110-7120听(听补苍诲听).
Mapes, J., Chen, Y.Z., Kim, A., Mitani, S., Kang, B.H., and Xue, D. (2012). CED-1, CED-7, and TTR-52 act in a pathway to regulate exoplasmic phosphatidylserine expression on apoptotic and phagocytic cells.听Current Biology听22, 1267-1275.听(听补苍诲听PDF).听Faculty of 1000
Chen, Y.Z., Mapes, J., Lee, E.S. and Xue, D. (2013). Caspase-mediated activation of听Caenorhabditis elegans听CED-8 promotes apoptosis and PS externalization.听Nature Communications听4:2726 doi: 10.1038/ncomms3726.听(听补苍诲听PDF)
Ge, X., Zhao, X., Nakagawa, A., Gong, X., Skeen-Gaar, R., Shi, Y., Gong, H.P., Wang, X.Q., and Xue, D. (2013). A novel mechanism underlies caspase-dependent conversion of the dicer ribonuclease into a deoxyribonuclease during apoptosis.听Cell Research听24: 218-232听(听补苍诲听PDF).
Nakagawa, A., Sullivan, K., and Xue, D. (2014). Caspase-activated phosphoinositide binding by CNT-1 promotes apoptosis by inhibiting the AKT pathway.听Nature Structural & Molecular Biology听21, 1082-1090 (听补苍诲听PDF).
Yang, H.W.*, Chen, Y.Z.*, Zhang, Y.*, Wang, X.H., Zhao, X., Godfroy, J.I., Liang, L., Zhang, M., Zhang, T.Y., Yuan, Q., Royal, M.A., Driscoll, M.D., Xia, N.S., Yin, H., and Xue, D. (2015). A lysine-rich motif in the phosphatidylserine receptor PSR-1 mediates recognition and removal of apoptotic cells.听Nature Communications听, 6: 5717 doi: 10.1038/ncomms6717. *Equal contribution. (听补苍诲听PDF).听
Weaver, B.P., Zabinsky, R., Weaver, Y.M., Lee, E.S., Xue. D., and Han, M. (2014). CED-3 caspase acts with miRNAs to regulate non-apoptotic gene expression dynamics for robust development in听C. elegans.听eLife听doi: 10.7554/eLife.04265 (听补苍诲听PDF).
Neumann, B., Coakley, S., Giordano-Santini, R., Linton, C., Lee, E.S., Nakagawa, A., Xue, D., and Hilliard, M.A. (2015). EFF-1-mediated regenerative axonal fusion requires components of the apoptotic pathway.听Nature听517, 219鈥�222 (听补苍诲听PDF).
Sullivan, K.*, Nakagawa, A.*, Xue, D.#, and Espinosa, J.M.# (2015). Human ACAP2 is a homolog of听C. elegans听CNT-1 that promotes apoptosis in cancer cells.听Cell Cycle听14, 1771-1778. *Equal contribution. #Co-corresponding authors. (听补苍诲听PDF).
Lin, J.L.*, Nakagawa, A.*, Skeen-Gaar, R.R., Yang, W.Z., Zhao, P., Zhang, Z., Ge, X., Mitani, S., Xue, D.#, and Yuan, H.S.# (2016). Oxidative Stress Impairs Cell Death by Repressing the Nuclease Activity of Mitochondrial Endonuclease G.听Cell Reports听16, 279鈥�287. *Equal contribution. #Co-corresponding authors. (听补苍诲听PDF).
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Patents
Jay Parrish and Ding Xue, US patent 7368237 (Approved 05/06/2008). 听Entitled "Cell Death-Related Nucleases and Their Uses"