Baculovirus protein
p35 inhibits programmed cell death in such diverse species as nematodes, insects and mammals (1, 2), which suggests that
p35 may function by interacting with an evolutionarily conserved component of a pathway for cell death. Two such conserved components are the cell-death effectors represented by the C. elegans protein CED-3 and the mammalian cysteine protease interleukin-1B-converting enzyme (ICE) and the cell-death inhibitors represented by the C. elegans protein CED-9 and the product of the mammalian proto-oncogene
bcl-2 (3) . We examined the interaction of
p35 with CED-3, which like the pro-form of ICE can autocleave to generate a cysteine protease (4). We found that recombinant
p35 protein purified from bacteria efficiently inhibited CED-3 protease activity in vitro. Furthermore, 35S-methionine-labeled
p35 protein synthesized in vitro was cleaved by purified CED-3 protease to generate two products of 10K and 25K. We determined by microsequencing analysis that CED-3 cleaves
p35 between aspartate 87 and glycine 88. These results suggest that
p35 may prevent programmed cell death in C. elegans by directly inhibiting CED-3 activity. Three observations indicate that the CED-3 cleavage site in
p35 is important for the activity of
p35 in inhibiting cell death in C. elegans. 1) A
p35 mutation (D87E) that greatly reduced
p35 activity in vitro as a CED-3 substrate and inhibitor abolished
p35 activity in vivo in protecting against cell death in C. elegans. 2) Introduction of a second
p35 mutation (K127G) that created a new but weak CED-3 cleavage site (aspartate-glycine) partially restored the ability of
p35 to protect against cell death. 3) Introduction of the CED-3 cleavage site in
p35 into the cowpox virus protein crmA, which inhibits mammalian apoptosis (5) but not programmed cell death in C. elegans, caused crmA to block CED-3-mediated cell death. These results indicate that the CED-3 cleavage site in
p35 can confer to certain proteins the ability to protect against programmed cell death and suggest that
p35 may block cell death in C. elegans by acting as a competitive inhibitor of the CED-3 protease. We found that
p35 was also cleaved by four vertebrate cysteine protease activities of the CED-3 family -- mouse ICE, human NEDD-2/ICH-1, human CPP32/Yama and chicken S/M extract. All four proteases most likely cleaved at the same site, since their cleavage products co-migrated and since a D87A substitution in
p35 completely prevented the cleavage reactions. This result suggests that
p35 may in general prevent programmed cell death by directly inhibiting activities of such cysteine proteases. References 1. A. Sugimoto, P. D. Friesen, J. H. Rothman, EMBO J 13, 2023-8 (1994). 2. H. Steller, Science 267, 1445-1449 (1995). 3. M. O. Hengartner, H. R. Horvitz, Philos Trans R Soc Lond B Biol Sci 345, 243-246 (1994). 4. D. Xue, H. R. Horvitz, 10th C. elegans Meeting Abstract, 554 (1995). 5. M. Miura, H. Zhu, R. Rotello, E. A. Hartwieg, J. Yuan, Cell 75, 653-60 (1993).