Programmed cell death is a naturally-occurring cellular process in which cells self-destruct by activation of an intrinsic suicide program. Like cell proliferation, cell death is an essential aspect of animal development and homeostasis. Both processes are tightly controlled so that cell numbers in tissues and organs are maintained at appropriate levels. Misregulation of programmed cell death may underlie many human diseases including cancers, autoimmune disorders, neurodegenerative disorders and immunodeficiency diseases.

We use the nematode Caenorhabditis elegans as a model system to study how programmed cell death is regulated, activated and executed, because C. elegans is uniquely amenable to both molecular genetic and biochemical analyses and because the cell death pathway is conserved between nematodes and mammals. The study of cell death in nematodes can provide crucial information towards understanding cell death mechanisms in humans, and ultimately, may identify means to combat human diseases caused by inappropriate apoptosis.

Genetic studies in C. elegans have identified many genes that are important for five sequential steps of programmed cell death (see Figure below): the decision-making step of which cells should die, the activation of the cell death program, the execution of the cell-killing process, the engulfment of cell corpses, and finally, the degradation of cellular debris. Several genes have been cloned and found to encode proteins homologous to factors involved in mammalian cell death, including two transcription factors (ces-1 and ces-2), a BH3-domain-containing cell-death initiator (egl-1), a cysteine protease that executes the suicide program (ced-3), a putative activator of the CED-3 death protease (ced-4, a homolog of mammalian death protease activator Apaf-1), and a cell-death inhibitor similar to the mammalian cell-survival factor Bcl-2 (ced-9). The CED-3 death protease is particularly interesting. CED-3 is first synthesized as an inactive proenzyme and later is activated through proteolysis, which then triggers the activation of the cell-death program and a series of cellular and morphological changes in the dying cells. Thus the study of how CED-3 is activated and how it acts to cause cell death offers a great paradigm for studying the regulation and execution of programmed cell death.

A genetic pathway of programmed cell death in the nematode Caenorhabditis elegans

We focus on addressing three key issues about the CED-3 death protease: 1) how is CED-3 expressed and activated in the right cell and at the right time? 2) what are the substrates of the CED-3 death protease? and 3) how do proteolytic cleavages of these substrates by CED-3 elicit the cellular and morphological changes that lead to the demise of a cell?

Using a combination of genetic, molecular biological, biochemical and structural biological approaches, we are examining the regulation and the expression of the ced-3 gene in C. elegans, the interactions of the CED-3 protein with other proteins in the cell death pathway, and the mechanism of CED-3 activation. We have performed both genetic and biochemical screens to identify proteins that regulate the activation or activity of the CED-3 death protease and proteins that are substrates of the CED-3 death protease. So far, we have identified at least fourteen new genes (cps-1 to cps-14; CED-3 protease suppressors) that act downstream of, or in parallel to, the CED-3 death protease to cause cell death and at least eight new genes that function to activate the CED-3 death protease in specific subsets of cells (please see detailed description). We have also initiated biochemical and structural analysis of crucial protein interactions and mechanisms that are involved in the activation of the CED-3 protease (please see detailed description). Molecular genetic characterization and biochemical and structural analysis of these new cell-death genes and their interactions with other cell death factors in C. elegans will help elucidate how programmed cell death is regulated, activated and executed in general.

We recently cloned the cps-6 gene and found that cps-6 encodes a homologue of human mitochondrial endonuclease G (endoG) (Abstract and PDF). In collaboration with Dr. Xiaodong Wang’s group at the University of Texas Southwestern Medical Center, we showed that the CPS-6 protein localizes to C. elegans mitochondria and possesses an endonuclease activity that is capable of inducing the generation of apoptotic DNA ladders in isolated Hela cell nuclei. Furthermore, we found that the mouse EndoG can substitute the functions of cps-6 in C. elegans, suggesting that CPS-6 and EndoG define an evolutionarily conserved DNA degradation pathway. Our study demonstrated for the first time that the mitochondria is important for apoptosis in invertebrates and is a conserved regulator of apoptosis.

The finding that cps-6 encodes a mitochondrial endonuclease prompted us to examine whether there are additional mitochondrial proteins involved in C. elegans apoptosis. We characterized the C. elegans homologue of AIF, a human mitochondrial oxidoreductase that is released from mitochondria during apoptosis to induce chromosome condensation and fragmentation. Intriguingly, the oxidoreductase activity of AIF is dispensable for its apoptogenic functions and AIF does not possess a nuclease activity, raising a major question of how AIF induces apoptosis. We cloned the worm AIF homologue, wah-1, and used RNA-mediated interference (RNAi) to study its functions in C. elegans apoptosis (Abstract and PDF). We found that reduction of the wah-1 activity in C. elegans 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 C. elegans AIF does play an important role in regulating C. elegans apoptosis. Interestingly, wah-1(RNAi) results in cell death phenotypes that are similar to those of the cps-6 mutant and fails to further enhance the cps-6 cell death defects, indicating that wah-1 and cps-6 function in the same cell death pathway. Indeed, we found that WAH-1 which also localizes to C. elegans mitochondria can associate and cooperate with CPS-6 in vitro to promote DNA degradation. In vivo, co-expression of wah-1 and cps-6 can synergistically induce cell killing. These findings indicate that CPS-6/EndoG is likely a target or effector that WAH-1/AIF interacts with to induce chromosome fragmentation during apoptosis and that CSP-6/EndoG and WAH-1/AIF define a single evolutionarily conserved cell death pathway initiated from mitochondria. Importantly, we found that WAH-1 can be released from mitochondria by EGL-1, a C. elegans BH-3-domain containing cell death activator, in a manner similar to the release of cytochrome c or EndoG from mitochondria by mammalian BH3-domain containing proteins such as Bid or Bim. Interestingly, 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 caspase-dependent manner. These observations strongly suggest that the mitochondrial cell death pathway is conserved between nematodes and humans.

We are now in the process of identifying additional mitochondrial factors that are important for the regulation and execution of programmed cell death in C. elegans using both genetic and biochemical approaches. We are particularly interested in understanding how mitochondrial apoptogenic factors are released from mitochondria to affect various aspects of apoptosis. We hope to identify most components functioning in C. elegans mitochondrial cell death pathways.

Recent Publications

1) Ledwich, D., Wu, Y.C., Driscoll, M, and Xue, D. (2000). Methods for the study of programmed cell death in the nematode Caenorhabditis elegans. Methods in Enzymology, 322, 76-88. (Abstract and PDF)

2) Parrish, J., Metters, H., Chen, L., and Xue, D. (2000). Demonstration of the in vivo interaction of key cell death regulators by structure-based design of second-site suppressors. Proc. Natl. Acad. Sci. USA. 97, 11916-11921. (Abstract and PDF)

3) Parrish, J., Li, L., Klotz, K., Ledwich, D., Wang, X.D., and Xue, D. (2001). C. elegans mitochondrial endonuclease G is important for apoptosis. Nature 412, 90-94. (Abstract and PDF). Nature Reviews Mol. Cell Biol.

4) Xue, D., Wu, Y.C., and Shah, M.M. (2001). Programmed cell death in C. elegans -- a genetic framework. Chapter II in "Apoptosis: The Molecular Biology of Programmed Cell Death". Page 23-55. Edited by M. Jacobson & N. McCarthy. Oxford University Press. (PDF)

5) Wang, X.C., Yang, C.L., Cai, J.J., Shi, Y.G., and Xue, D. (2002). Mechanisms of AIF-mediated apoptotic DNA degradation in Caenorhabditis elegans. Science 298, 1587-1592. (Abstract and PDF). Nature Reviews Molecular Cell Biology (PDF)

6) Parrish, J. and Xue, D. (2003). Functional genomic analysis of apoptotic DNA degradation in C. elegans. Mol. Cell 11, 987-996. (Abstract and PDF)

7) Parrish, J., Yang, C.L., Shen, B.H., and Xue, D. (2003). CRN-1, a Caenorhabditis elegans FEN-1 homologue, cooperates with CPS-6/EndoG to promote apoptoticDNA degradation. EMBO. J. 22, 3451-3460. (Abstract and PDF) Science STKE review (PDF)

8) Wu, Y.C. and Xue, D. (2003). Programmed cell death in C. elegans. Chapter IV in "Essentials of Apoptosis: A Guide for Basic and Clinical Research". Page 135-144. Edited by X.M. Yin & Z. Dong. The Humana Press Inc. (PDF)

9) Wang, X.C., Wu, Y.C., Fadok, V., Lee, M.C., Gengyo-Ando, K., Cheng, L.C., Ledwich, D., Hsu, P.K., Chen, J.Y., Chou, B.K., Henson, P., Mitani, S., and Xue, D. (2003). Cell Corpse Engulfment Mediated by C. elegans Phosphatidylserine Receptor Through CED-5 and CED-12. Science 302, 1563-1566. (Abstract and PDF). Science Perspectives (PDF) and Science STKE review (PDF)

10) Friedman, J. and Xue, D. (2004). To live or die by the sword: the regulation of apoptosis by the proteasome. Developmental Cell 6, 460-461. (PDF)

11) Yang, N, Gu, L.C., Kokel, D., Han, A.D., Chen, L., Xue, D., and Shi, Y.G. (2004). Structural, Biochemical and Functional Analyses of CED-9 Recognition by the Pro-apoptotic Proteins EGL-1 and CED-4. Mol. Cell 15, 999-1006. (Abstract and PDF)

12) Breckenridge, D. and Xue, D. (2004). Regulation of mitochondrial membrane permeabilization by BCL-2 family proteins and caspases. Curr. Opin. Cell Biol. 16, 647-652. (Abstract and PDF)

13) Conradt, B. and Xue, D. (2005). Programmed cell death. WormBook, ed. The C. elegans Research Community. (Abstract and PDF)

14) Yan, N., Chai, J.J., Lee, E.S., Gu, L.C., Liu, Q., He, J.Q., Wu, J.W., Li, H.L., Hao, Q., Xue, D., and Shi, Y.G. (2005). Structure of the CED-4/CED-9 complex reveals insights into programmed cell death in Caenorhabditis elegans. Nature 437, 831-837. (Abstract and PDF)

15) Fadeel, B. and Xue, D. (2005). PS externalization: from corpse clearance to drug delivery. Cell Death Differentiation 13, 360-362. (PDF)

16) Parrish, J., and Xue, D. (2005). Cuts can kill: the roles of apoptotic nucleases in cell death and animal development. Chromosoma 115, 89-97. (PDF)

17) Yang, C.L., Yan, N., Parrish, J., Wang, X.C., Shi, Y.G., and Xue, D. (2006). RNA aptamers targeting the cell death inhibitor CED-9 induce cell killing in C. elegans. Journal of Biological Chemistry 281, 9137-9144. (Abstract and PDF)

18) Kokel, D., Li, Y.H., Qin, J., and Xue, D. (2006). The non-genotoxic carcinogens naphthalene and para-dichlorobenzene suppress apoptosis in C. elegans. Nature Chemical Biology 2, 338-345. (Abstract and PDF). News in ScienceNOW, The Guardian, The Skottish Daily Record, Denver Post, Sina net (新浪网), NIGMS e-newsletter Biomedical Beat

19) Kokel, D. and Xue, D. (2006). A class of benzenoid chemicals suppresses apoptosis in C. elegans. ChemBioChem 7, 2010-2015. (Abstract and PDF)

20) 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). "C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1". Nature Cell Biology 9, 541-549. (Abstract and PDF). Nature Reviews Molecular Cell Biology (PDF)

21) Peden, E., Kimberly, E.L., Gengyo-Ando, K., Mitani, S., and Xue, D. (2007). Control of sex-specific apoptosis in C. elegans by the BarH homeodomain protein CEH-30 and the transcriptional repressor UNC-37/Groucho. Genes & Development 21, 2195-3207. (Abstract and PDF).

22) Darland-Ransom, M., Wang, X.C., Sun, C.L., Mapes, J., Gengyo-Ando, K., Mitani, S. and Xue, D. (2008). Role of C. elegans TAT-1 protein in maintaining plasma membrane phosphatidylserine asymmetry. Science 320, 528-531. (Abstract and PDF). Science Perspectives (PDF)

23) Breckenridge, D., Kang, B.H., Kokel, D., Mitani, S., Staehelin, A.L., and Xue, D. (2008). Caenorhabditis elegans drp-1 and fis-2 regulate distinct cell death execution pathways downstream of ced-3 and independent of ced-9. Mol. Cell 31 586-597. (Abstract and PDF)

24) Peden, E., Killian, D., and Xue, D. (2008). Cell death specification in C. elegans. Cell Cycle 7, 2479-2484. (Abstract and PDF)

25) Killian, D., Harvey, E., Johnson, P., Otori, M., Mitani, S., and Xue, D. (2008). SKR-1, a homolog of Skp1 and a member of the SCF^SEL-10 complex, regulates sex-determination and LIN-12/Notch signaling in C. elegans. Developmental Biology, in press. (Abstract and PDF)