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Studying regulation of programmed cell death through C.elegans model

Mechanisms of cell death activation in C. elegans

The activation of the cell death program is a complex and poorly understood biochemical process. In C. elegans, a sequential protein interaction cascade regulates the activation of apoptosis. The death-initiating EGL-1 (a BH3-only protein) binds to the death-inhibitory CED-9 (a Bcl-2 homologue), triggering the release of CED-4 (an Apaf-1 homologue) from the CED-4/CED-9 complex tethered on the surface of mitochondria and the subsequent activation of the CED-3 protease precursor. We are interested in understanding the biochemical and structural basis of these events, and at the same time, isolating small molecular compounds that may modulate these events in vivo. We address these issues from several directions. First, we set up a cell-free system to dissect different steps of CED-3 activation and to identify components important for CED-3 activation. Second, we have carried out several genetic screens to look for mutations that perturb the release of CED-4 or its translocation to the nuclear membrane, or the activation of CED-3. Third, we have assisted in the determination of three-dimensional structures of two protein complexes crucial for CED-3 activation, in collaboration with Dr. Yigong Shi, an outstanding X-ray crystallographers at Princeton University. Finally, we used a PCR-based in vitro selection method (SELEX) and a novel oil-based chemical delivery protocol to isolate small RNA molecules (aptamers) and chemical compounds that can bind or modify key cell death regulators (EGL-1, CED-9, CED-4 and CED-3) and thus affect cell death activation.

Thus far, we have reconstituted both the early steps (EGL-1 binding to CED-9 and release of CED-4 from the CED-4/CED-9 complex; Parrish et al., Proc. Natl. Acad. Sci. USA, 2000) and the later steps of CED-3 activation in vitro (release of CED-4 tetramers from the CED-4/CED-9 complex and activation of the CED-3 precursor by CED-4 tetramers; Yan et al., Nature, 2005). We participated in the determination of the structures of the EGL-1/CED-9 complex and the CED-4/CED-9 complex. Our studies suggest that the binding of EGL-1 to CED-9 induces a drastic CED-9 conformational change, which triggers the dissociation of the CED-4 dimer from the 2:1 CED-4/CED-9 complex and the formation of CED-4 tetramers that are sufficient for the activation of the CED-3 precursors in vitro (Yan et al., Mol. Cell, 2004; Yan et al., Nature, 2005). We have conducted SELEX screens and isolated five RNA aptamers that bind CED-9 with high affinity and specificity and that can potently induce cell death in C. elegans (Yang et al., J. Biol. Chem., 2006). Theseaptamers were also used to probe the regions of CED-9 important for binding to EGL-1 and CED-4, respectively. In a parallel study, we have developed a novel oil-based chemical delivery method that allows us to screen hydrophobic compounds in C. elegans. Using this new drug delivery protocol, we found that two major components of mothballs (naphthalene and para-dichlorobenzene) and a subclass of related benzenoidchemicals can inhibit apoptosis in C. elegans. Using a combination of genetic, biochemistry, and mass spectrometry analysis, we found that naphthalene (likely other related benzenoid chemicals) inhibits apoptosis in C.elegans (and likely in mammals) by oxidizing and thus inhibiting the activities of caspases through its quinone metabolites (Kokel et al., Nature Chemical Biology, 2006 and Kokel and Xue, ChemBioChem, 2006). Naphthalene and para-dichlorobenzene are the first small-molecule apoptosis inhibitors identified in C. elegans. Our findings suggest a cellular mechanism by which naphthalene and para-dichlorobenzene, which are known nongenotoxic carcinogens, may promote tumorigenesis in mammals. Our study also suggest that the power of C. elegans molecular genetics, in combination with the possibility of carrying out large-scale chemical screens, makes C. elegans an attractive and economic animal model for drug screens and subsequent target identification. We are now carrying out large-scale drug screens to identify compounds that can induce apoptosis or compounds that can inhibit apoptosis or necrosis so that they may be used as leads to develop drugs to treat human diseases caused by necrosis or abnormal apoptosis.

Publications:

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. ( and )

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. ( and )

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. ( and )

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. ( and )

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. ( and ). News in , , , , , , 

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

Geng, X., Shi, Y., Nakagawa, A., Yoshina, S., Mitani, S., Shi, Y., and Xue, D. (2008). Inhibition of CED-3 zymogen activation and apoptosis in Caenorhabditis elegans by a caspase homolog CSP-3. Nature Structural & Molecular Biology 15, 1094-1101. ( and ). 

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. ( and ) 

Geng, X., Zhou, Q.H., Kage-Nakadai, E., Shi, Y., Yan, N., Mitani, S., and Xue, D. (2009). Caenorhabditis elegans caspase homolog CSP-2 inhibits CED-3 autoactivation and apoptosis in germ cells. Cell Death & Differentiation 16, 1385-1394. ( and )

Chen, X.D.*, Wang, Y.*, Chen, Y.Z.*, Harry, Brian, L.H., Nakagawa, A., Lee, E.S., Guo, H.Y., and Xue, D. (2016). Regulation of CED-3 caspase localization and activation by C. elegans nuclear membrane protein NPP-14. Nature Structural & Molecular Biology 23, 958-964 ( and . *Equal contribution.

Smith, C.E., Soti, S., Jones, T.A., Nakagawa, A., Xue, D., and Yin, H. (2017).  Non-steroidal Anti-inflammatory Drugs Are Caspase Inhibitors. Cell Chem. Biol. 24, 281-292. ( and )

Regulation of sexually dimorphic apoptosis in C. elegans

How a cell responds to internal or external cues to activate the cell death program is another vitally important question. We decided to focus on studying the regulation of sexually dimorphic apoptosis, an ancient and conserved developmental process, in which sex-specific cells or organs (e.g. female-specific Mullerian Duct in mammals) are eliminated in the opposite sex by apoptosis. Misregulation of sex-specific cell deaths could result in severe sexual disorders (e.g. persistent Mullerian duct syndrome). In C. elegans, two sets of neurons undergo sex-specific deaths. Two HSN motor neurons control egg laying in hermaphrodite animals but undergo apoptosis in males where they are not needed. In contrast, four male-specific CEM neurons are speculated to mediate chemotaxis of males towards the hermaphrodites during courtship and are programmed to die in hermaphrodites where they are dispensable. The sexually dimorphic deaths of HSNs and CEMs present an excellent paradigm for studying the regulation of cell death activation and death signaling pathways. Several genes have been found to be involved in regulating sex-specific deaths, including two sex-determining genes (her-1 and tra-2) and a key cell death gene (egl-1) (mutations in these genes cause abnormal HSN or CEM deaths). These genes delineate a death-signaling pathway in which the sex differentiation pathway mediated by a male-promoting secreted protein (HER-1) and its receptor (TRA-2)integrates into the cell-death pathway through EGL-1 to control HSN/CEM deaths. In order to identify additional components in this death-signaling pathway, we have carried out several genetic screens to look for mutations that alter sex-specific HSN and CEM cell deaths in C. elegans. From these screens, we have isolated more than 30 mutations. These include more than a dozen mutations affecting general sex differentiation, 6 mutations in three key cell-death genes (ced-3, ced-4 and egl-1), and 5 mutations in four genes (rsd-1, rsd-4, rsd-5, and rsd-6; regulators of sex-specific death) that affect only the deaths of HSN or CEM or both but not any other cell death. For example, several semi-dominant mutations in egl-41 cause inappropriate HSN death and improper CEM survival in hermaphrodites (but have no effect on HSN/CEM deaths in males), which can be suppressed by a loss-of-function mutation (sm151) in the rsd-4 gene. We cloned the egl-41 gene and found that it is identical to the sel-10 gene, which was previously identified by Iva Greenwald's laboratory to be important for cell signaling during vulval development. However, a role for sel-10 in apoptosis has not been observed before. The SEL-10 protein contains a F-box motif and seven WD repeats and is likely a component of a Skp1-Culin-F box (SCF) E3 ubiquitin ligase complex that may target the degradation of important cell death regulators in a sex-specific manner. Interestingly, rsd-4encodes a C. elegans Skp1 homologue, SKR-1 (Skp1 related protein 1), which is another component of the SCF E3 ligase complex (Killian et al., Developmental Biology, 2008). We also cloned rsd-5 and rsd-6, two genes that regulate CEM-specific cell deaths in hermaphrodites and in males, respectively. rsd-5 appears to encode a regulatory factor of the SCFSEL-10 E3 ligase complex. rsd-6 encodes a BarH homeodomainprotein, which is identical to the ceh-30 gene (Peden et al., Genes & Development, 2007). We also cloned a new gene, tra-5, which encodes a novel protein and appears to affect general sex determination. Overall, our studies of sexually dimorphic apoptosis in nematodes have provided important insights into how sexual dimorphism, an ancient reproducing mechanism, is regulated and achieved by appropriate apoptosis and how cell death signaling is regulated in general.

Publications:

 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. ( and ).

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

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 SCFSEL-10 complex,regulates sex-determination and LIN-12/Notch signaling in C. elegans. Developmental Biology 322, 322-331. ( and )

Mapes, J., Chen, J.T., Yu, J.S., and Xue, D. (2010). Somatic sex determination in C. elegans is modulated by SUP-26 repression of tra-2 translation. Proc. Natl. Acad. Sci. USA 107:18022-18027. ( and )