Nicholson, Miller et al.

PARP
cleavage
enzyme
PARP

P
oly
D E V D
Asp Glu Val Asp

A
DP-

R
ICE
ibose IL-1β

F E A D
P
olymerase Phe Glu Ala Asp
 cancer
and genetic approaches
forward …based on the
identification of
mutants with specific
phenotypes, caused by
DNA mutations

biochemical approach
…based on the in vitro reconstitution of a process from its cellular components
Geneticist Biochemist

✔
 A good assay is important
specific easy fast

What was used as

an assay to
identify the PARP
cleavage enzyme?

Ced phenotype
Egl phenotype
 PARP cleavage assay

•  PARP was generated in vitro and labeled with 35S methionine

DEVD
cleavage site
kDa
150
100 113
75 89

50
113 kDa
37

25
24 apoptotic cell lysate
15

24 kDa 89 kDa
 PARP cleavage assay

•  Cell lysates contain thousands of different proteins

How can you find the PARP
cleavage enzyme?

apoptotic cell lysate

?
Finding the PARP cleavage enzyme

Chromatography
Separate the proteins!

size vs.

+ + – –
charge + + ++ vs. – – – –
+ + + – – –
 Finding the PARP cleavage enzyme
(anion-exchange chromatography)

elute
(–) proteins

flow-through
 Finding the PARP cleavage enzyme
(DEVD peptide)
PARP
cleavage
•  A DEVD peptide might block the activity enzyme
of the PARP cleavage enzyme PARP

D E V D
D E V D Asp Glu Val Asp

activity blocked

D E V D

PARP cleavage enzyme

 Finding the PARP cleavage enzyme
(DEVD peptide)

active site available

PARP cleaved

active site blocked

PARP not cleaved

 Finding the PARP cleavage enzyme
(affinity chromatography)

•  DEVD peptide was coupled to stationary resin

p17
~17kDa

elute
p11
proteins ~11kDa
bound to
DEVD
 Finding the PARP cleavage enzyme
(affinity chromatography)

•  DEVD peptide was coupled to stationary resin

p17
~17kDa

elute
p11
proteins ~11kDa
bound to
DEVD

caspase-3
(CPP32)
 Caspase-3

Caspase-3 is the mammalian homolog of CED-3

p17
~17kDa

p11
~11kDa
inactive active
prodomains

cleavage
 Caspase-3

•  The DEVD peptide could also block apoptosis

in mammalian cells grown in culture

The activity of this caspase

is necessary for apoptosis!

Nicholson, D.W. et al. (1995). Identification and inhibition of the ICE/CED-3

protease necessary for mammalian apoptosis. Nature, 376(6535), 37-43.
http://dx.doi.org/10.1038/376037a0
 C. elegans egl-1 ced-9 ced-4 ced-3   APOPTOSIS
Bcl-2 superfamily ? caspase

Sensors Guardians Effectors caspases   APOPTOSIS

mammals
?

The identification of the first caspases was an important

advance that really moved the field forward.
 What cleaves pro-caspases?

•  Xiaodong Wang et al. used a biochemical approach

–  identify factors required for cleavage/activation of pro-caspase-3
•  Labeled full-length pro-caspase-3 with 35S methionine
–  as an assay, they tested for presence of p17 and p11 cleavage products

labeled pro-caspase -3 HeLa cell lysate

 HeLa cells

•  Immortal human cell line

–  first human cell line isolated
–  most commonly used human cell line
•  1951 – cervical cancer cells

Henrietta Lacks

Bestselling book
The Immortal Life of
Henrietta Lacks
by Rebecca Skloot
 What cleaves pro-caspases?
•  Incubation of pro-caspase-3 with HeLa cell lysate resulted in cleavage

kDa

50 p17

37 HeLa cell lysate

p11
32
25

20

15 17

10 11
 What cleaves pro-caspases?
•  Incubation of pro-caspase-3 with HeLa cell lysate resulted in cleavage
p17

PARP
p11 PARP cleaved
kDa

50
p17
37
DNA
ladder
32 p11
25

20

15 17
This confirmed that the active caspase-3
10 11
(p17 + p11)
had been generated by HeLa cell lysate
 What cleaves pro-caspases?

•  Narrowed down to three different proteins from the HeLa cell lysate
apoptotic
1 Apaf-1
protease
all three required activation
together
for pro-caspase-3
2 Apaf-2 factor
cleavage

3 Apaf-3