egl-1 Bad

transcriptional integrator post-translational integrator

Also:
Bik
HRK
Bad BH3
Puma
Bim BH3
TM
domain
Noxa
Bid BH3 Bmf
 Li, H. et al. (1998). Cleavage of BID by caspase 8 mediates the mitochondrial
damage in the Fas pathway of apoptosis. Cell, 94(4), 491-501.
http://dx.doi.org/10.1016/S0092-8674(00)81590-1

Luo, X. et al. (1998). Bid, a Bcl2 interacting protein, mediates cytochrome c release from
mitochondria in response to activation of cell surface death receptors. Cell, 94(4), 481-490.
http://dx.doi.org/10.1016/S0092-8674(00)81589-5

The Wang Lab The Yuan Lab

Bad
Apaf proteins BH3 ced-3 and ICE

Bim BH3
TM
domain

Bid BH3
 Caspase-8

•  Caspase-8 is an initiator caspase (like caspase-9)

–  but it is not activated by the apoptosome
•  Caspase-8 is activated by the DISC complex Bcl-2
–  assembles at the plasma membrane superfamily
–  triggered by TNFα ligand family
•  tumor necrosis factor α

TNFα
ligand
family
 How is Bid activated?
Yuan Lab approach

•  Small pool in vitro expression cloning

x 17 x 17
reverse
transcription
00 in vitro
transcription
00
(RNA → DNA) (DNA → RNA)

divide into and translation

complete mRNA library 1700 pools cDNA library pool into peptides expression library pool
all mRNA present in tissue complimentary DNA polypeptides
(mouse spleen)

Li, H. et al. (1998). Cleavage of BID by caspase 8 mediates the mitochondrial

damage in the Fas pathway of apoptosis. Cell, 94(4), 491-501.
http://dx.doi.org/10.1016/S0092-8674(00)81590-1
 How is Bid activated?
Yuan Lab approach

•  Small pool in vitro expression cloning

–  Incubate 1700 expression pools with/without caspase-8

1700 pools incubated

±
Pool 814 Pool 815 with and without
no + no + caspase-8
kDa Casp8 Casp8 Casp8 Casp8
50 caspase-8
37
3400 reactions!

25
20
15
10
 How is Bid activated? mammals
BH3-only
Yuan Lab approach
Bcl-2/Bcl-xL

•  Small pool in vitro expression cloning Bax/Bak

–  Incubate 1700 expression pools with/without caspase-8 cyt c

Apaf-1
Pool 814
no + caspase-9
kDa Casp8 Casp8
50 caspase-3
37

25
20
15
Bid
is a substrate of caspase-8
10
 How is Bid activated?
Wang Lab approach

Bcl-2
superfamily
FasL

Fas ligand
cyt c release

caspase-8
?
 How is Bid activated?
Wang Lab approach

•  Identify factors required for caspase-8-induced cytochrome c release

–  HeLa cell lysate + mitochondria + caspase-8 ➞ cytochrome c release
–  use cytochrome c release as an assay to identify factors from HeLa lysate
What is causing the release of cytochrome c?

cyt c release
Luo, X. et al. (1998). Bid, a Bcl2 interacting protein, mediates cytochrome c release from
mitochondria in response to activation of cell surface death receptors. Cell, 94(4), 481-490.
http://dx.doi.org/10.1016/S0092-8674(00)81589-5
 How is Bid activated?
Wang Lab approach

•  Identify factors required for caspase-8-induced cytochrome c release

–  HeLa cell lysate + mitochondria + caspase-8 ➞ cytochrome c release
–  use cytochrome c release as an assay to identify factors from HeLa lysate

CCRF
24 kDa protein
cyt c release

caspase-8-dependent cytochrome c releasing factor

 How is Bid activated?
Wang Lab approach

•  Identify factors required for caspase-8-induced cytochrome c release

–  HeLa cell lysate + mitochondria + caspase-8 ➞ cytochrome c release
–  use cytochrome c release as an assay to identify factors from HeLa lysate

Why is
Bid
a caspase-8 cyt c release

substrate?
 Bid is a substrate of caspase-8
mammals C. elegans
Intrinsic
or
BH3-only
C P egl-1

Mitochondrial
Pathway
Bcl-2/Bcl-xL
E A ced-9

Bax/Bak
N T
cyt c
T H
Apaf-1
R W ced-4

caspase-9 A A
ced-3
caspase-3 L Y
 Bid is a substrate of caspase-8
mammals
vertebrates vertebrates C. elegans
Intrinsic
or
BH3-only
C P egl-1

Mitochondrial caspase-8ced-9
Pathway
Bcl-2/Bcl-xL
E A
Bax/Bak
N Bid
T Extrinsic
or
cyt c
T H Death Receptor
Pathway
Apaf-1
R W ced-4
caspase-9 A A
ced-3
caspase-3 L Y
 Death receptor pathway
vertebrates

TNFα-like
DISC ligand

Extrinsic
DISC or
complex Death Receptor
death-inducing signaling complex Pathway
 Death receptor pathway
vertebrates

TNFα-like
caspase-8 FADD DISC ligand

Extrinsic
or
Death Receptor
Pathway
The purpose of the DISC complex is to cause
FADD pro-caspase-8 activation…

pro-caspase-8 …it is therefore functionally equivalent to the

apoptosome
 All together now…
mammals
vertebrates vertebrates C. elegans
Intrinsic
or
BH3-only
C P egl-1

Mitochondrial
Pathway
Bcl-2/Bcl-xL
E caspase-8
A ced-9
FADD DISC
TNFα-like
ligand
Bax/Bak
N
tBid
T Bid
Extrinsic
or
cyt c
T H Death Receptor
Pathway
Apaf-1
R W ced-4
caspase-9 A A
ced-3
caspase-3 L Y
 Intrinsic Extrinsic

Mitochondrial Death-Receptor
Pathway Pathway
 many different predominantly in
types of cells cells of the
immune system
PREDOMINANT
APOPTOSIS PATHWAY

Mitochondrial Death-Receptor
Pathway Pathway

!
 Movie

Apoptosis and Signal Transduction

4m 39s

Berry D. (2007). Molecular animation of cell death mediated

by the Fas pathway. Sci. STKE 2007, tr1.

http://youtu.be/DR80Huxp4y8