Proapoptotic histone H1.2 induces CASP-3 and -7 activation by forming a protein complex with CYT c,...

FEBS Letters 581 (2007) 3422–3428

Proapoptotic histone H1.2 induces CASP-3 and -7 activationby forming a protein complex with CYT c, APAF-1 and CASP-9

Antonio Ruiz-Vela*, Stanley J. Korsmeyer

Howard Hughes Medical Institute, Department of Pathology and Medicine, Harvard Medical School, Boston, MA 02115, USA

Received 9 April 2007; revised 5 June 2007; accepted 18 June 2007

Available online 28 June 2007

Edited by Michael R. Bubb

Abstract Cytochrome c (CYT c) is a protein that employs thecaspase recruitment domain (CARD)-containing proteinsAPAF-1 and CASP-9 to activate effectors CASP-3 and -7. Byusing affinity labeling techniques and mass spectrometry analy-sis, we show that histone H1.2 is a regulator of caspases uponUV irradiation. We demonstrated that histone H1.2 forms a pro-tein complex with APAF-1, CASP-9 and CYT c upon UV irra-diation. In cell-free systems, we show that histone H1.2 triggersactivation of CASP-3 and -7 via APAF-1 and CASP-9. Wetherefore conclude that upon DNA damage histone H1.2 actsas a positive regulator of apoptosome formation.Published by Elsevier B.V. on behalf of the Federation ofEuropean Biochemical Societies.

Keywords: CASP-9; Histone H1.2; APAF-1; UV irradiation

1. Introduction

Apoptotic cells typically exhibit a large number of morpho-

logical and biochemical changes, including DNA condensation

and degradation, loss of mitochondrial potential (DWm) and

cell shrinkage [1]. Upon intrinsic cell death signaling a set of

cysteinyl aspartases (caspases, CASP) and calcium-activated

endopeptidases (calpains, CAPN) become activated [2–4].

Most caspases regulate the apoptotic program through a

caspase activation cascade involving initiator CASP-1, -2, -9,

-11, -12 and effector CASP-3 and -7 [3,5,6], which crosstalk

with CAPN-1 and -2 [4,7].

In response to DNA damage the proapoptotic protein cyto-

chrome c (CYT c) exits from the mitochondrial intermembrane

space to the cytoplasm and nucleus [8]. Fundamental and pio-

neer studies using cell-free systems have demonstrated that

CYT c is a regulatory protein that binds to the CED-4 like

adaptor protein APAF-1, generating a high molecular weight

protein complex termed apoptosome, which then activates the

initiator CASP-9 and CASP-3 and -7 [9,10]. CASP-9 requires

the physical interaction with APAF-1 to be activated, forming

thus a holoenzyme [11]. In vivo, the functional significance of

APAF-1 and CASP-9 was validated in Casp-9�/� and Apaf-1�/�

mouse embryonic fibroblasts (MEFs) [12,13].

*Corresponding author. Present address: Department of Pathology,Spanish National Cancer Centre (CNIO), Melchor Fernandez Alm-agro, E-28029, Madrid, Spain. Fax: +34 912 242 980.E-mail address: [email protected] (A. Ruiz-Vela).

0014-5793/$32.00 Published by Elsevier B.V. on behalf of the Federation of

doi:10.1016/j.febslet.2007.06.049

To identify unrecognized cell death regulators upon DNA

damage in the form of UV irradiation, we carried out a proteo-

mic analysis to search for regulatory proteins of caspases. We

employed affinity labeling techniques using the caspase inhibi-

tors (Ac-Tyr-Val-Lys-[biotinyl]-Asp-fmk) bound to an oligo-

meric irreversible streptavidin column. Mass spectrometry

analysis identified histone H1, H2A and H2B as regulators

of caspases. To further validate the role of these proteins in

the activation of caspases, we developed a novel non-hypo-

tonic cell-free system based on the zwitterionic detergent

CHAPS. Using this system we demonstrated that histone

H1.2, showed the capability to trigger CASP-3 and -7 in cell-

free systems. Biochemical analysis in Apaf-1�/� and Casp-9�/�

cell protein extracts demonstrated that histone H1.2 specifically

requires APAF-1 and CASP-9 to activate downstream CASP-3

and -7. Finally, we demonstrated that histone H1.2 forms a pro-

tein complex with APAF-1, CASP-9 and CYT c upon UV irra-

diation. Thus, we conclude that proapoptotic histone H1.2 acts

as a positive regulator of apoptosome formation upon DNA

damage.

2. Materials and methods

2.1. Cell cultureMurine embryonic fibroblasts (MEFs) were derived from day 13.5

embryos of wild type (WT), Apaf-1�/� [12], and Casp-9�/� [14].

2.2. Western blot analysisTotal protein extracts were prepared by lysing cells in RIPA lysis

buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% sodium deoxy-cholate, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1 mM phenyl-methylsulfonyl fluoride (PMSF), 2 mg/ml aprotinin, 2 mg/mlleupeptin, 1 mg/ml pepstatin) for 10 min on ice. Cell debris was re-moved by centrifugation (10000 · g, 10 min). Protein concentrationwas measured using the BCA assay (Pierce). Samples (30 lg protein)were resolved on a 4–12% gradient NuPAGE gel, transferred to PVDFmembranes, and blocked overnight with 5% non-fat dry milk in TBSbuffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl). Subsequent antibodyincubations and membrane washes were performed in TBS-T buffer(20 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.2% Tween 20) with 5%non-fat dry milk. Antibodies were detected using enhanced chemolu-minescence (Western Lightning, Perkin–Elmer).

2.3. S100ZWI and S8ZWI preparationFor S100ZWI fraction preparation, SV40 immortalized murine

embryonic fibroblasts (MEFs) were collected, washed in PBS and thenresuspended in buffer A (100 mM HEPES, pH 7.2, 10% sucrose, 0.1%3-[(3-cholamido propyl)dimethyl ammonio]-1-propanesulfonate, and10 mM DTT) at 4 �C for 20 min. After incubation, cells were disruptedby Dounce homogenization, followed by 30 strokes through a 30-gauge needle and then incubated 40 min at 4 �C. Lysates were centri-fuged (8000 · g, 20 min, 4 �C), and the resulting supernatant further

European Biochemical Societies.

A. Ruiz-Vela, S.J. Korsmeyer / FEBS Letters 581 (2007) 3422–3428 3423

centrifuged (100000 · g, 1 h. 4 �C). For S8SWI fraction preparation,cells were resuspended in buffer A (20 min, 4 �C); after incubation, cellswere disrupted by 30 strokes through a 30-gauge needle. Lysates werecentrifuged (8000 · g, 20 min, 4 �C), and the supernatant further cen-trifuged (8000 · g, 20 min, 4 �C).

2.4. Affinity labeling and purificationS100SWI fractions were pre-cleared with streptavidin–agarose

(Novagene) (2 h, 4 �C), and then incubated with the caspase substrate[Ac-Tyr-Val-Lys(biotinyl)-Asp-fmk] (Calbiochem) for 1 h at 4 �C, afterthat the samples were applied to an oligomeric streptavidin–agarosecolumn (1 h, 4 �C). After extensive washing in buffer A, bound mate-rial was eluted in 10 mM Tris, pH 7.5, 1% SDS and 100 mM 2-mercap-toethanol at 95 �C for 10 min. Samples were recovered by spinningdown (12000 · g, 1 min) and the resulting eluate was resolved on a4–12% gradient NuPAGE gel, and then analyzed by silver staining(Bio-Rad). Bands corresponding to UV-treated S100SWI fractions wereexcised, and then analyzed by liquid chromatography and tandemmass.

2.5. ImmunoprecipitationS100SWI fractions were pre-cleared with protein A/G-agarose (Santa

Cruz) (2 h, 4 �C), incubated with 20 lg of anti-histone H1.2 antibodyovernight at 4 �C, then applied to an protein A/G-agarose column(2 h, 4 �C). After washing in buffer A, bound material was eluted in10 mM Tris, pH 7.5, 1% SDS and 100 mM 2-mercaptoethanol. Theresulting eluant was resolved on a 4–12% gradient NuPAGE gel, andanalyzed by silver staining (Bio-Rad). Western blots were revealedusing mouse TrueBlot� ULTRA (HRP) anti-mouse IgG (eBioscience)and TrueBlot� (HRP) anti-rabbit IgG (eBioscience).

2.6. Caspase assaysCaspase activity was measured as indicated [3].

2.7. Histone H1.2-induced caspase activationS8SWI fractions were incubated with 5 lM of histones and 1 mM

dATP (Sigma) at 37 �C for 45 min. Sample were resolved on a 4–12% gradient NuPAGE gel and transferred to PVDF membranes.Western blotting was then performed to detect active CASP-3,CASP-7 and CASP-9.

2.8. ReagentsRecombinant H1.0, H1.2, H1.3, H1.4, H1.5 proteins were purchased

from Alexis. H2A and H2B were purchased from Upstate. Purified his-tone H1 from calf thymus was purchased from Alexis. Purified CYT cand dATP were from Sigma–Aldrich. UV light was induced using aStratalinker� 2400 UV Crosslinker, 230 V (Stratagen).

2.9. AntibodiesAntibodies for immunoblot analysis included mouse anti-actin mAb

(clone AC-15, Sigma–Aldrich), polyclonal rabbit anti-cleaved CASP-3(#9661, Cell Signaling), rabbit anti-CASP-7 (#9492, Cell Signaling),and rabbit anti-CASP-9 (#9504, Cell Signaling), rabbit anti-histoneH1.2 (ab17677, Upstate), mouse anti-CYT c (clone 7H82C12, Pharm-ingen), anti-histone H3 (clone A35, Upstate), and rabbit anti-APAF-1(#4452, Cell Signaling). We used HRP-conjugated anti-rabbit andanti-mouse (DAKO).

3. Results

Analysis of UV irradiation-induced DEVD-ase activity

(CASP-3 and CASP-7 activity) [15] in SV40 transformed mur-

ine embryonic fibroblasts (SV40 MEFs) revealed a reproduc-

ible and marked peak �14 h post-treatment (Fig. 1A). To

identify unknown caspase regulators upon UV irradiation

(250 nm), we used affinity labeling techniques with the irrevers-

ible caspase inhibitor (Ac-Tyr-Val-Lys-[biotinyl]-Asp-fmk) in

S100 fractions extracted using the zwitterionic detergent

CHAPS (S100ZWI fractions) (see Section 2). After a screening

of reliable experimental conditions, we optimized several pro-

tein purification steps (Fig. 1B).

Fresh S100ZWI fractions from either untreated (negative con-

trol) or 14 h UV-irradiated cells (UV-14) were incubated with

the caspase inhibitor Ac-Tyr-Val-Lys-[biotinyl]-Asp-fmk, fol-

lowed by purification on an oligomeric streptavidin column

(Fig. 1B). Interestingly, S100ZWI fractions from 14 h UV-irra-

diated cells [(UV-14)-S-100ZWI fractions] were specifically en-

riched in two bands corresponding to �20 and �34 kDa

(Fig. 1C). To determine their amino acid sequences, we per-

formed liquid chromatography and mass spectrometry analy-

sis [16,17]. Two tryptic fragments identical to mouse histone

H1 were found in the 34 kDa band (Fig. 1B), whereas three

peptides corresponding to mouse histone H2A and four pep-

tides for mouse histone H2B were detected in the 20 kDa band

(Fig. 1B). As expected, none of the enriched proteins in the

purification were caspases, consistent with the irreversible

interaction of biotin with streptavidin [18] and active caspases

with the caspase inhibitor (Ac-Tyr-Val-Lys-[biotinyl]-Asp-

fmk) [5].

In our affinity labeling experiments using S100ZWI fractions

from 14 h UV-irradiated cells, histone H3 and H4 were not

identified by mass spectrometry analysis. These results suggest

that nucleosome contamination from apoptotic cells in the

purification step is thus unlikely. To test this hypothesis, we

analyzed the levels of histone H3 by Western blot in

S-100ZWI and (UV-14)-S-100ZWI fractions (Fig. 1D). As posi-

tive control, we checked the levels of histone H3 in total pro-

tein extracts (Fig. 1D). As histone H3 is not detected by

Western blot assay in both S-100ZWI and (UV-14)-S-100ZWI

fractions, we conclude that S-100ZWI and (UV-14)-S-100ZWI

fractions were not contaminated with nuclear proteins.

By using affinity labeling pull-down experiments (Fig. 1C),

we show that histone H1 could form a protein complex with

active caspases, suggesting that histone H1 might regulate acti-

vation of caspases. To test this hypothesis, we analyzed

whether purified histone H1 from calf thymus could induce

caspase activation in cell-free systems. To this end, we em-

ployed two distinct cell-free systems; one based on S-100ZWI

fractions (soluble fractions after centrifugation at

100000 · g) and other based on S-8ZWI fractions (soluble frac-

tions after centrifugation at 8000 · g) (see Section 2). We first

checked the levels of CYT c, CASP-9 and APAF-1 in S-8ZWI

and in S-100ZWI fractions by Western blot analysis (Fig. 2A).

Notably, the incubation of purified histone H1 plus dATP with

S-8ZWI fractions resulted in a marked CASP-3 activation

(Fig. 2B), indicating thus a role for histone H1 in caspase acti-

vation.

We also observed that incubation of purified histone H1 plus

dATP with S-100ZWI fractions resulted in low levels of CASP-3

activation (Fig. 2B); likely due to the low levels of soluble CYT

c and APAF-1 detected in those S-100ZWI fractions (Fig. 2A).

These data are in accordance with others [19–21] showing that

the relative amount of endogenous monomeric CYT c, APAF-

1 or CASP-9 in cytosolic extracts regulate the intensity of

apoptosome assembly and levels of caspase activation.

Since histone H1 present distinct variants (such as H1.0,

H1.2, H1.3, H1.4 and H1.5), we tested the ability of different

recombinant histone H1 variants to induce caspase activity.

To this end, we analyzed CASP-3 and -7 activity upon incuba-

tion of recombinant histone H1.0, H1.2, H1.3, H1.4 or H1.5

with S-8ZWI fractions. As shown in the Fig. 2C, recombinant

SV40 MEFs

Solubilize 100 mM HEPES, pH 7.2, 10% sucrose,0.1 % zwitterionic, 10 mM DTT

Centrifugation at 100,000 g

S100ZWI protein extracts

Affinity labeling withAc-Tyr-Val-Lys-(biotinyl)-Asp-fmk

Oligomeric streptavidin-agarose column10 mM Tris, pH 7.5, 1% SDS,100 mM β-mercaptoethanol Elution

B

C

Histone H3

ACTIN

S-100ZWI

UV-14Control UV-14Control

Total protein extract

D

MwMw

14.3 kDa

20.1 kDa

30 kDa

45 kDa

66 kDa

Histone H2AHistone H2B

Histone H1

UV-14Control

ASGPPVSELITKALAAAGYDVEK

AGLQFPVGRHLQLAIRTIAQGGVLPNIQAVLLPK

KESYSVYVYKAMGIMNSFVNDIFERLAHYNKRHAVSEGTK

A

0

10

20

30

40

50

60

70

80

90

100

(

DEV

D-p

NA

hydr

olys

is)

(A

bs. 4

05nm

/ a.u

)

DEVD-ase activity(CASP-3 and -7 activity)

Time (h)0 2 4 6 8 10 12 14 16 18 20 22 24

Peak of CASP-3 and -7 activity

Fig. 1. Histone H1, H2A and H2B are candidate regulator of caspase activation. (A) SV40 transformed MEFs were UV-irradiated (250 nm) atdifferent time points. After that CASP-3 and CASP-7 activities were measured as indicated [3]. (B) Summary of purification steps. Briefly, SV40-MEFs are solubilized, thoroughly disrupted and agitated. Lysates are centrifuged to obtain S100ZWI fractions, then incubated with the caspasesubstrate [Ac-Tyr-Val-Lys(biotinyl)-Asp-fmk]. S100ZWI fractions from untreated SV40 MEFs (control) and 14 h UV-irradiated SV40 MEFs (UV-14)are then applied to an oligomeric streptavidin–agarose column and then the bound proteins eluted. (C) Samples from eluate in B was resolved on a 4–12% gradient NuPAGE gel and silver-stained (Bio-Rad). Bands are shown for S100ZWI fractions from control, untreated SV40 immortalized MEFs(left) and UV (250 nm)-treated SV40 immortalized MEFs (right), which were excised for liquid chromatography and tandem mass spectrometryanalysis. Mw indicates molecular weight. Sequence peptides are indicated in the figure. For complete experimental protocols, see Section 2. (D)Western blot for histone H3 in S-100ZWI fractions and total protein extracts (RIPA extraction) from untreated and UV-irradiated cells. Actin wasused as loading control.

3424 A. Ruiz-Vela, S.J. Korsmeyer / FEBS Letters 581 (2007) 3422–3428

H1.2, but not H1.0, H1.3, H1.4 or H1.5, showed the ability to

induce DEVD-ase activity in presence of dATP. Thus, all these

data together indicate that histone H1.2 specifically regulates

caspase activation.

We also analyzed the role of histone H2A and H2B in the

regulation of caspases, using histone H1.2 and CYT c as con-

trols. Addition of recombinant histone H1.2 plus dATP coop-

erated to induce CASP-3 processing in S8ZWI protein extracts,

whereas neither H2A nor H2B had effect (Fig. 2D). Moreover,

we analyzed whether recombinant histone H1.2 plus dATP

could induce CASP-7 and CASP-9 activation (Fig. 2E). Our

results show that histone H1.2 specifically exhibits the ability

to trigger CASP-3, -7 and -9 activation.

Biochemical studies in hypotonic S-100 fractions have dem-

onstrated that oligomerization of APAF-1 and CASP-9 is pos-

itively regulated by CYT c plus dATP, [10]. We therefore

tested whether recombinant histone H1.2, H2A and H2B could

initiate caspase activation in hypotonic S-100 fractions, using

purified CYT c as positive control. Incubation of the recombi-

nant proteins histone H1.2, H2A or H2B with hypotonic S100

fractions [9,10] in presence of dATP did not induced CASP-3

activation (Fig. 2F). These results imply that histone H1.2-in-

duced caspase activation requires putative cofactors that are

present in S-8ZWI fractions but not in hypotonic S-100 frac-

tions.

Next, we assessed the potential of histone H1.2 to induce

CASP-3, -7 and -9 in S8ZWI fractions from Apaf-1�/� and

Casp-9�/� SV40 immortalized MEFs, using wild type MEFs

as negative control. Addition of recombinant H1.2 plus dATP

to (WT)-S8ZWI extracts resulted in a simultaneous CASP-3, -7

and -9 proteolysis (Fig. 3). In contrast, histone H1.2 did not

promote CASP-3, -7 and -9 cleavage in absence of APAF-1

proCASP-3

p17p19

Cytc + dATP

H2A + dATPH1.2 + dATP-

-

+-

- ---

+

-

+-

-

-

-H2B + dATP-+---

H1.2----+

S-8ZWI

Cytc + dATP

H2A + dATPH1.2 + dATP-

-

-+

- +--

-

-

-+

-

-

-H2B + dATP----+

H1.2+----

proCASP-3

p17p19

Hypotonic S-100

+--+

H1.2 + dATPdATP

proCASP-7

p20

proCASP-9

p37

C DEVD-ase activity

0 10 20 30 40 50 60 70 80 90 100

Control

dATP

Histone H1.0

Histone H1.2

Histone H1.3

Histone H1.4

Histone H1.5

Histone H1.0 + dATP

Histone H1.2 + dATP

Histone H1.3 + dATP

Histone H1.4 + dATP

Histone H1.5 + dATP

DEVD-pNA hydrolysis (Abs. 405 nm/a.u)

(CASP-3 and -7 activity)

A

proCASP-3

p17 (Cleaved CASP-3)p19 (Cleaved CASP-3)

-++-

Histone H1 (Calf Thymus) + dATPdATP

S-8ZWI

-++-

CASP-9

S-8ZWI

CYT c

B

APAF-1

S-100ZWI S-100ZWI

D E F

Fig. 2. Histone H1.2 induces caspase activation in presence of the zwitterionic detergent CHAPS (3-[(3-cholamido propyl) dimethyl ammonio]-1-propanesulfonate). (A) Western blot for CYT c, APAF-1 and CASP-9 in S-8ZWI and S-100ZWI fractions. (B) S-100ZWI and S-8ZWI fractions wereincubated with purified histone H1 plus dATP or with dATP only, then samples were analyzed by Western blotting for anti-cleaved CASP-3. (C) S-8ZWI fractions were incubated with recombinant H1.0, H1.2, H1.3, H1.4 or H1.5 in presence or in absence of dATP, then samples were analyzed forCASP-3 and CASP-7 activities [3]. (D) S8SWI fractions were incubated with recombinant histones H1.2, H2A, H2B, or purified CYT c in presence ofdATP, then analyzed by Western blotting for active CASP-3. (E) S8SWI fractions were incubated with recombinant histones H1.2 only or in presenceof dATP, then analyzed by Western blotting for CASP-7 and CASP-9. (F) Hypotonic S100 fractions [9,10] were incubated with dATP and withrecombinant histones H1.2, H2A, H2B, or with CYT c (positive control), and then analyzed by Western blotting for active CASP-3.


or CASP-9 (Fig. 4). These results thus indicate that histone

H1.2-induced CASP-3 and -7 requires APAF-1 and CASP-9

in cell-free systems.

To understand the mechanism by which histone H1.2 trig-

gers caspase activation, we tested the interaction of CYT c

and APAF-1 with histone H1.2 in pull-down experiments

proCASP-3

p17 (Cleaved CASP-3)

proCASP-7


proCASP-9p37 (Cleaved CASP-9)



Apaf-1-/-Casp-9-/-WT- - - +++ H1.2 + dATP

Fig. 3. Histone H1.2 uses APAF-1 and CASP-9 to induce CASP-3 and-7. WT, Apaf-1�/� or Casp-9�/� -S8SWI fractions were incubated withdATP and recombinant H1.2, followed by Western blotting for CASP-3, CASP-7 and CASP-9.


using affinity labeling techniques (Fig. 4A). To this end, fresh

S100ZWI fractions from either untreated (negative control) or

14 h UV-irradiated cells (UV-14) were incubated with the cas-

pase inhibitor Ac-Tyr-Val-Lys-[biotinyl]-Asp-fmk, followed

Histone H1.2

Affinity labeling withAc-Tyr-Val-Lys-(biotinyl)-Asp-fmk

UV-14Control

CYT c

APAF-1

INPUT

UV-14Control

ACTIN 14.3 kDa

20.1 kDa

30 kDa

45 kDa

66 kDa

Mw UV-Control

Immunoprecipitatio Anti-Histone H1.2

A B

Fig. 4. Histone H1.2 forms a protein complex with CYT c, APAF-1 and CASMEFs (UV-14) were solubilized to obtain S100ZWI fractions, then S100ZW

Lys(biotinyl)-Asp-fmk] followed by purification in an oligomeric streptavidieluate were analyzed by Western blotting for histone H1.2, CYT c and APAanalyzed by Western blotting for actin (loading control). (B) S100SWI fractioimmunoprecipitated using anti-histone H1.2 (for complete experimental prWestern blot analysis for APAF-1, CASP-9, cleaved CASP-3 and CYT c.

by purification on an oligomeric streptavidin column. The

resulting eluant was further analyzed by Western blot assay

using anti-histone H1.2, anti-CYT c and anti-APAF-1 anti-

bodies (Fig. 4A). Our data show that histone H1.2, CYT c

and APAF-1 interact with the caspase inhibitor Ac-Tyr-Val-

Lys-[biotinyl]-Asp-fmk upon UV irradiation, and suggest that

histone H1.2 might form a protein complex with APAF-1 and

CYT c.

To further corroborate the association of histone H1.2 with

APAF-1 and CYT c, we immunoprecipitated histone H1.2

proteins using anti-histone H1.2 antibodies in S-100ZWI frac-

tions from either untreated or 14 h UV-irradiated cells

(Fig. 4B). Upon immunoprecipitation, the resulting eluant

was evaluated by silver staining as well as by Western blot

analysis using anti-APAF-1, anti-CASP-9, anti-cleaved

CASP-3 and anti-CYT c antibodies (Fig. 4B). Our results show

that histone H1.2 interacts with components of the apopto-

some (such as APAF-1, CASP-9, CASP-3 and CYT c) upon

UV irradiation. Thus, by using two different approaches (affin-

ity labelling pull-down and immunoprecipitation techniques)

we have demonstrated that histone H1.2 forms a protein com-

plex with components of the apoptosome after UV irradiation.

These results are in agreement with the ability of histone H1.2

to trigger caspase activation in cell-free systems.

4. Discussion

We performed a proteomic analysis to uncover regulatory

proteins of caspases in UV-irradiated cells. We employed the

caspase inhibitor (Ac-Tyr-Val-Lys-[biotinyl]-Asp-fmk) bound



Immunoprecipitation Anti-Histone H1.2

UV-14Control

APAF-1

CASP-9

CYT c

IgH

IgL

14

Histone H1.2

n

P-9. (A) Untreated SV40 MEFs (control) and 14 h UV irradiated SV40I fractions were incubated with the caspase substrate [Ac-Tyr-Val-

n–agarose column and then the bound proteins eluted. Samples fromF-1. A fraction of the total input used the pull-down experiment was

ns from untreated SV40 MEFs and 14 h UV-treated SV40 MEFs wereotocols, see Section 2). Samples were silver-stained and analyzed by


to an oligomeric streptavidin column to purify potential cell

death regulators. Mass spectrometry analysis identified histone

H1, H2A and H2B as candidate regulators of caspases. As

shown in Fig. 1C, we found no active caspases in the elution

of the streptavidin column likely due to two irreversible inter-

actions that includes (1) the binding of active caspases with the

caspase inhibitor (Ac-Tyr-Val-Lys-[biotinyl]-Asp-fmk) [5], and

(2) the biotin interaction with the streptavidin column [18],

implying thus that elution of active caspases from the oligo-

meric streptavidin column is not viable. None of the enriched

proteins would thus be active caspases but rather regulatory

proteins, cofactors or associated proteins.

Human and mice have several histone H1 genes; all the H1

somatic histones (H1.1, H1.2, H1.3, H1.4 and H1.5) are ex-

pressed ubiquitously and are extremely similar in DNA se-

quence [22]. In a cell-free system based on cell solubilization

with the zwitterionic detergent CHAPS (3-[(3-cholamido pro-

pyl)dimethyl ammonio]-1-propanesulfonate) we demonstrated

that histone H1.2, but not other variants, induced CASP-3,

CASP-7 and CASP-9. Biochemical analysis in Apaf-1�/� and

Casp-9�/� cell extracts showed that histone H1.2 specifically

requires caspase recruitment domain (CARD)-containing pro-

teins APAF-1 and CASP-9 to initiate downstream CASP-3

and CASP-7.

We have compared the levels of CYT c in S-8ZWI fractions

(soluble fractions after centrifugation at 8000 · g) as well as

in S-100ZWI fractions (soluble fractions after centrifugation

at 100000 · g) by Western blot analysis (Fig. 2A). Our data

indicate that 0.1% of CHAPS can solubilize CYT c from mito-

chodria in SV40 transformed MEFs. Since CYT c was en-

riched in S-8ZWI fractions compared to S-100ZWI fractions

(Fig. 2A), these data suggest that most of the CYT c protein

is not present as monomeric protein in CHAPS-based buffers,

but forming a high molecular protein complex with other mito-

chondrial proteins [23]. In this regard, it has been shown that

CYT c interact with multiple mitochondrial components such

as the cytochrome bc1 complex [25], voltage-dependent anion-

selective channel (VDAC) [26], cytochrome b5 [27], NADPH

dependent diflavin oxidoreductase 1 [28] and cytochrome c oxi-

dase subunit IV isoform 2 [29].

Monomeric CYT c binds to APAF-1 in the absence of dATP

in hypotonic S-100 fractions [9,24]. Only when dATP is added

to hypotonic S-100 fractions the CYT c/APAF-1 protein com-

plex can assembly with CASP-9 to produce a higher molecular

weight protein complex called apoptosome [9,24]. Here, we

show that incubation of dATP only with S-8ZWI fractions is

not sufficient to trigger caspase activation (Fig. 2B). Therefore,

it is feasible that CYT c could be sequestered in a high molec-

ular mitochondrial protein complex impeding thus the correct

interaction with APAF-1 upon incubation with dATP only.

We show that incubation of dATP only with S-8ZWI frac-

tions (Fig. 2B, C, E) resulted in no discernible CASP-3,

CASP-7 and CASP-9 activation. Only when dATP was incu-

bated with purified histone H1 (Fig. 2B) or recombinant his-

tone H1.2 (Fig. 2C–E) we detected CASP-3, CASP-7 and

CASP-9 activation. Since histone H1.2 induces the release of

CYT c from isolated mitochondria [30], our results suggest

that histone H1.2-induced caspase activation in CHAPS-based

cell-free systems is the consequence of the exit of monomeric

CYT c from a putative mitochondrial protein complex, allow-

ing thus the formation of the apoptosome.

Acknowledgement: A.R.V. was supported by a postdoctoral fellowshipfrom the European Molecular Biology Organization (EMBO).

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Proapoptotic histone H1.2 induces CASP-3 and -7 activation by forming a protein complex with CYT c,...

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