egl-1

ced-9

ced-4

? ?

ced-3

APOPTOSIS

Cloning ced-4 and ced-3

ced-4 encodes a protein of 546 amino acids
no similarity to any other known protein at the time no known motifs
ICE

29% amino acid identity

ced-3 encodes a protein with significant similarity to mammalian ICE

ICE = interleukin-1-converting enzyme a cysteine protease
Cys
Yuan, J. et al. (1993). The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell, 75(4), 641-652. http://www.cell.com/retrieve/pii/0092867493904859

active site

ICE

Mammalian ICE
ICE cleaves the inactive precursor of interleukin-1 (IL-1)
cleavage yields the mature IL-1 protein cleavage is between aspartic acid (D) and alanine (A) residues

IL-1

IL-1

Mammalian ICE
ICE itself is also synthesized as a pro-enzyme (zymogen)
needs to be activated by cleavage of prodomain
Asp Asp

prodomain
Asp

p20
Asp

p10

Mammalian ICE
ICE itself is also synthesized as a pro-enzyme (zymogen)
needs to be activated by cleavage of prodomain cleavage occurs at four aspartic acid (Asp) residue sites
two of these are conserved within the CED-3 sequence CED-3 might also be activated through proteolytic cleavage

p20 p10 p10 p20

inactive

prodomains

active

cleavage at Asp sites by active ICE

C. elegans CED-3
CED-3 is a cysteine protease, like mammalian ICE
synthesized as an inactive zymogen activated by cleavage at aspartic acid residues
Asp Asp

prodomain

p17
Asp

p15 p13

Xue, D. et al. (1996). The Caenorhabditis elegans cell-death protein CED-3 is a cysteine protease with substrate specificities similar to those of the human CPP32 protease. Genes Dev., 10(9), 1073-1083. http://genesdev.cshlp.org/content/10/9/1073

C. elegans CED-3
CED-3 is a cysteine protease, like mammalian ICE
synthesized as an inactive zymogen activated by cleavage at aspartic acid residues inactive active p17 p13 or p15 p13 or p15 p17

prodomains

cleavage at Asp sites by active CED-3

Xue, D. et al. (1996). The Caenorhabditis elegans cell-death protein CED-3 is a cysteine protease with substrate specificities similar to those of the human CPP32 protease. Genes Dev., 10(9), 1073-1083. http://genesdev.cshlp.org/content/10/9/1073

inactive zymogens

?
? ?

point of no return?

mature protease

THIS WAS GREAT!

ced-3 encodes a protease

Can ICE or ICE-like proteases also commit cells to the apoptotic fate

CED-3

ICE

and induce apoptosis in mammalian cells?

J. Yuan et al.
generated a number of different ICE and CED-3 constructs

The morphology of cells expressing the various constructs was analyzed in rat fibroblasts

ced-3 construct
strong promoter strong promoter

Ice construct

ICE and CED-3 constructs

1 Expression of ICE can induce apoptosis
both p10 and p20 subunits are required also the active site cysteine residue p10 p10 p20 The apoptotic role of cysteine proteases is conserved p20 Cys

ICE and CED-3 constructs

2 Expression of CED-3 can induce apoptosis
the active site cysteine residue is required p17 p13 or p15 p13 or p15 p17 Cys

C. elegans protein CED-3 induces apoptosis in mammalian cells

ICE and CED-3 might have similar substrates

ICE and CED-3 constructs

3 Constructs were expressed in cells that overexpress bcl-2
egl-1 ced-9

ced-4

ced-3

Remember:

bcl-2 is homologous to ced-9 ced-9 acts upstream of ced-3

ICE and CED-3 constructs

3 Constructs were expressed in cells that overexpress bcl-2
ICE-induced apoptosis was blocked ced-3-induced apoptosis was blocked, but only weakly
The results in rat fibroblasts suggested that

Bcl-2 family members control the activities of ICE-like and CED-3-like cysteine proteases
Miura, M. et al. (1993). Induction of apoptosis in fibroblasts by IL-1-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Genes Dev., 75(4), 653-660. http://www.cell.com/retrieve/pii/009286749390486A

CED-3 and ICE

caspases
C. elegans humans
Yuan, J. et al. (1993). The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell, 75(4), 641-652. http://www.cell.com/retrieve/pii/0092867493904859 Miura, M. et al. (1993). Induction of apoptosis in fibroblasts by IL-1-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell, 75(4), 653-660. http://www.cell.com/retrieve/pii/009286749390486A

cysteine-dependent aspartate-directed proteases