Degradation of (ADP-ribose)n in permeabilized HeLa cells

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 37-45

Vol. 139, No. 1, 1986 August 29, 1986

DEGRADATION

OF (ADP-RIBOSE) Jozsef

C. Gaal

n IN PERMFABILIZEDHELA

and Colin

CELLS

K. Pearson

Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen, AB9 IAS, Scotland, UK

Received July 3, 1986 SUMMARY Determination of (ADP-ribose) n degradation rates in permeabilized HeLa cells, measured as loss of acid-ifiseluble ~adioa£tivity from permeabilized cells previously incubated with [~H]NAD', showed bi-phasic kinetics. The majority of label was lost within 20 rain at pH 6.0 and 37°C and has a half-life of about 12-15 rain. The minor ADP-ribose cemponent was either removed very slowly, or appeared to be stable over an 80 rain incubation. The degradation rate of the labile cemponent was directly proportional to the initial amount of ADP-ribose present, and was independent of the experimental conditions used to create various elevated levels. The degradation rates of monomerie and oligo/polymeric ADP-ribose were the same, surprising since different enzymes catalyse the respective reactions. The more stable ADP-ribose component could be more inaccessible to degrading enzymes and/or might represent a different linkage to protein, the cleavage Of which is slow. ©

1986 Academic Press,

Inc.

The covalent established process

modification

post-translational

include

In order

limited turns

over

turnover vitro

rapidly,

rate

however,

usually

apparently including

where

a rapid

presumably

have

rate,

turnover

suggest

TI/2 about reports

observations

rate has been

reported

of the

[1-3].

in fine cellular

to be readily

reversible.

A

that poly(ADP-ribose)

of less than

with other

our own previous

functions

cell differentiation

to be important

with a half-life

contrasts

Suggested

activity,

out in vivo

turnover

is now a well

role has yet been defined

modification

carried

that has a slower

[7,8],

no precise

it would

of studies

event.

in DNA repair

for this protein

n~nber

component

although

processes

by ADP-ribosylation

modification

an involvement

and carcinogenesis,

regulatory

of proteins

1 rain, with

5 rain [4-6]. on work [9,10].

carried

a second

This rapid out in

In all cases,

protein

Abbreviations: (ADP-ribose)_, n=l, monomeric adenosine diphosphate ribose and h-~ ~ ~--q-~er; ADPRT, ADP-~ibosyl transferase; DNAase, pancreatic deoxyribonuclease I; DMS, dimethyl sulphate; PMSF, phenylmethylsulphonyl fluoride; PEI-eel lulose, polyethylenimine eellulose. 0006-291X/86 $1.50 37

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 139, No. 1, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ADP-ribosylation

stimulated

promoting

has been

ADPRT

activity,

The simplest

both

by treatments

in vivo

interpretation

[4-6,

of these

11-13]

n degradation

is dictated

present

[6].

tested

cell

We have

damage

and in vitro

observations

rate of (ADP-ribose) in the cell

which

to date

by the amount

this proposal

using

DNA,

thus

[14]. is that

the

of ADP-ribose a permeabilized

system.

MATERIALS

AND METHODS

Conditions of HeLa cell growth, cell are described in detail elsewhere [15].

permeabilization,

and ADPRT

assays

ADP-ribose degradation assay. ~ermea~ilized cells were initially incubated for 20 rain in the presence of [~H]NAD' (see legends) to label protein-bound ADP-r~bose residues (conditions as in reference 15). After 20 rain the cells (2x10) were pelleted and resuspended in 300#1 of 25mM Na~HP0a, 25ram NaH~P04 (pH 6.0) containing 5ram PMSF, 10ram MgCI~, imM dithiothrei~ol ~nd 6ram -- 3-aminobenzamide. Incubations were the~ continued at 37 C for up to 2 hours. The loss of acid-insoluble radioactivity with time was taken as a measure of (ADP-ribose) n degradation. Control experiments showed that the transferase was completely znhibited under the conditions used. Hydroxylapatite column chromatography. The sample preparation procedures were as previously described [16]. Briefly, the degradation assays were stopped by adding trichloroacetic acid. The insoluble material was collected and incubated at 37°C with 0.3M NaOH to cleave protein-ADP-ribose bonds and to degrade RNA. DNA and protein were degraded with deoxyribonuclease I and pronase respectively. Samples (500pl) were applied to iml hydroxylapatite coltmms (in a Pasteur pipette) which were then washed with 4.8mi of in~M potassium phosphate then eluted with a 25mi linear gradient of potassium phosphate buffer (l-500mM, pH 6.8). Fractions of 0.6ml were collected and counted for radioactivity. RESULTS

AND DISCUSSION

Fig. residues,

1 shows

the bi-phasic

previously

synthesized

[3H]NAD +, are rendered 3-aminobenzamide. assay

conditions

by incubating

The ADPRT used.

that

re_in is proportional confirms over

that

the initial

In the experiment

level

cells

to the amount

Similar

shown

(not shown)

of (ADP-ribose) n was manipulated 38

were

of

the degradation

over

The inset

radioactvity

with

in the presence

concentrations

n degradation

initially.

acid-insoluble

under

ADP-ribose

cells

the transferase

with various

present

experiments

incubation

inhibited

the rate of (ADP-ribose)

radiolabelled

permeabilized

on continued

is completely

the rate at which

this period.

with which

by incubating

acid-soluble

the permeabilized

It is clear

kinetics

was activated of DNAase the first

I. 20

to the figure

is lost is linear

carried

by incubating

out in which cells

with

Vol. 139, No. 1, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

_.~ 25 0

E v 20

t-

"E 20 15

E

¢9 n"

10

>, > .i-, O

5

15

0

0

10

20

30

0 ¢-¢- 1 0

0

I

"0 0

O~

~

0

20

40

60

80

Time (rain) Figure I. Degradatio~ of A~P-ribose. Permeabilized HeLa cells were incubated for 20 rain with ImM [OH]NAD' (5#Ci/assay, sp. act. 16.6mCi/mmol) in the absence of pancreatic deoxyribonuclease I (@, control) or with DNAase added to 50~g (O), 75#g (•), lO0#g ( A ) or 150#g (•) per ml of assay medium. The cells were then pelleted and resuspended in ADP-ribose degradation mix at 37°C containing 6ram 3-aminobenzamide. The time 0 rain (degradation) on the figure refers to the acid-insoluble radioactivity present after the 20 min incubation with the i~H]N~ +. The inset shows that the rate of degradation was in fact linear Over the first 20 min (axes labelling is the same as the main figure).

different

concentrations

sulphate,

an alkylating

establish

that the initial

proportional

of the transferase agent.

is achieved;

creating

~A

damage

amount

6.0. various

Both acidic

to stimulate

Experiments

catalysing

the cleavage

about pH 7.0

2)

is directly

concentration

or by

ADPRT activity.

described

[17,18]

(Fig.

of how this initial

with high substrate

degradation

and alkaline

glycohydrolases

of all the experiments

and is independent

that is, either

The rate of (ADP-ribose)n than at pH 8.0.

NAD +, or with dimethyl

rate of (ADP-ribose) n degradation

to its starting

quantity

The results

substrate,

was greater

(about

5%) at pH 6.0

in this paper were carried

pH optima

have previously

and that of protein

of the protein-monomeric

[19,20]. 39

out at pH

been reported

lyase,

for

the enzyme

(ADP-ribose)

linkage,

is

Vol. 139, No. 1, 1986

~

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

30

o

o5

~

@n

~2o ~.om -k0 .~ lO

'S

z~ e cit

f 0.4

0.2

I 0.6

I 0,8

I 1.0

A D P - R i b o s e Degraded per min (nmoles)

Figure 2. Relationship between the rate of (ADP-ribese)_ degradation (loss of acid-insolubility over the first 20 rain) and the amount 8f radiolabelled ADP-ribose present at the beginning of the degradation assay. The following conditiQns we~'e used during the incubation of the permeabilized cells with [OH]N~D' (5#Ci/assa~) : + + (a) 0.1ram NAD" (b) ~.SmM NAD' (c) I,IM NAD
(f) 25~¥ D~S, i~ N~

(g) 100~gl~ ~N~so, 0.1ram ~

(h) i00~ ~S,

imM NAD (i) 50~g/ml DNAase, SmM NAD (j) 75#g/ml DNAase, I~M NAD (k) 100~g/ml DN~ase, 0.SmM NAD (I) 100~g/ml ~NAase, imM HAD (m) 100~g/ml + [NAase, 2ram NAD (n) 100#g/ml BNAase, 5him NAD (o) 150pg/ml DNAase, imM NAD .

In order represented carried

to confirm (ADP-ribose)n

degradation,

out a double-labelling

of 14C-labelled They were

then

subsequently

amino

acids

incubated

carried

residues

are rendered

provided

protease

of monomeric Hydroxylapatite permeabilized

cells

concentration

monomeric

ADP-ribose

ADP-ribose,

peak

considered

to label

3) that whilst

the faster

ADP-ribose, acceptors chains

with

cell proteins. assay

ADP-ribose

were used was present 6).

to elevate (Fig.

proteins,

in which

ADP-ribose

This predominance

over component catalysed

rate-limiting

synthesised

4a; monomer

4O

turning

the lyase

being

that in our experiments

at fraction

in the presence

is no loss of labelled

that

of radioactive

shows

substrate

there

from protein

analysis

(Fig.

we

are present.

of polymeric

ADP-ribose

were grown

truly

proteins,

[3H]NAD + for 20 rain and a degradation

acid-soluble

been

Cells

to permeabilization

It is clear

inhibitors

be degradation

prior

radioactivity

and not that of acceptor

experiment.

with

out.

It has previously might

that the loss of acid-insoluble

removal

[4].

in the increases

levels,

in

then mostly

= 87% of radiolabelled of monomer

under

Vol. 139, No. 1, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

150~

a

3

0Q'E10] " :~

400500

> .--> .¢,., 5 0¢1 ~-/100 ¢:: ¢: E (9 n"

~00

!00 "O

g

o

. L ~

b

75

>

' '°Fll

0 I~

? ooi

5O

(D

-6 t!

"10 0

25

<

® ooT

I

I

40

O

80

~

10

-

20

J

30

40

50

1

Fraction Number

Time (min)

Figure 3. Loss of acid-insoluble radioactivity is due to the degradation of (ADP-ribose) and not protein,. Heia cells were grown for 24h in the presence of a "~C-labnlled amino acid mixture (0.3~Ci/ml median; Amersham mixture CFB 104) to ra~iolab~l proteins. They were harvested, permeabilized and incubated with imM [~H]NAD- (sp. act. 16.6mCi/mmole). They were then resuspended and incubated in the degradation assay solution. The Fig. shows the loss of radioactivity when cells were incubated with 0.SmM PMSF throug.~t the permeabilization, transferase reaction and ~egradation period ( C, • ; 3H, • ) or without the protease inhibitor ( C, O; ~H, A ). Figure 4. Hydroxylapatite col~nn chromatography of ADP-ribose from permeabilized cells.q Expo~entially-growing HeLa cells were permeabilized and incubated with imM [UH]NAD-. They were then resuspended in degradation assay mix and incubated for 0 min (a) or 60 rain (b). ADP-ribose chains were subsequently cleaved from proteins with alkali before chromatography. In experiment (a) 259,881 c.p.m, were applied to the coltmm and recovery was 105%. In experiment (b) 45,198 c.p.m, were applied and recovery was 93%. Symbols: ( • ), radioactivity; ( O ) phosphate concentration.

conditions

in which

no DNA damage

consistent

with

situation

Thin

layer

of mono(ADP-ribose)

the

PEI-cellulose eluting

has

in vivo

been

introduced

is

[21,1].

chromatography from

intentionally

was

hydroxylapatite

41

used

to confirm

in all

the

experiments.

identity When

Vol. 139, No. 1, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ADP-ribose

content

additional

synthesis

(Fig.

5a; monomer During

of oligemeric

radioactivity

experiment

the radioactivity for degrading relationship

ADP-ribose

= 37% of total

assay,

of the transferase

therefore,

became

radiolabel). most

(90%) of the

lost in the first experiment

(Fig. 4b) was due to

eatalysed

by protein

(Fig. 5b) monomer

accounted

for 52% and polymer

Despite (probably

is obtained

this different

composition

lyase and glycohydrolase)

between

the initial

then

evident

prestm~bly

lost.

enzymes

activation

and polymeric

= 63% and polymer

of mono(ADP-ribose),

the DNAase

by E~Aase

a 60 rain degradation

acid-insoluble removal

was elevated

lyase,

whereas 48% of

of substrates a direct

amount of radiolabelled

2O.L-- / J

5OO

10":"

400 >'

300

5 -

200 ,oo

°II

m

"0

oJ eo

0

1 b

,eLI1 20

5OO X

4 0 0 ~3

?

200

100 1

10

20

30

40

50

Fraction Number Figure 5. Hydroxylapatite coltmm chromatography of ADP-ribose from permeabilized cells incubated with deoxyribonuclease I. The transferase reaction was as described for Fig. 4 except that DNAase was present at 100#g/ml. ADP-ribose was from cells in which degradation bad proceeded for 0 min (a) or 60 wan (b). In experiment (a) 322,809 c.p.m, were applied to the colomn and recovery was 105%. In experiment (b) 95,590 c.p.m, were applied and recovery was 99%. Symbols as for Fig. 4.

42

in

Vol. 139, No. 1, 1986

ADP-ribose

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and its rate of removal

this that monomeric rate,

at least Our

in HeLa cells

the notion

from

the slowly

turning

the majority

For example,

the non-enzymic

attachment

build

on polymeric

ADP-ribose

We note still

the TI/2

considerably

greater

[4-6].

This

could

permeabilized

than values

(nuclear

degradation

into

group

they showed

ADPRT-stimulated

putative

to

bonds

is

may be due to

by glycohydrolase for the observed

described

by Wielckens

in cell

nuclei.

over component

(12-15

enzymes

described in HeLa

could

rain) is

reported

by leakage

from the

t~

different

$3 cells.

Some of the

then be due to entry of a cytosolic

dictated

and control

require

dsa%aged,

cells

degradation

cells cannot inhibiting

a mechanism proposed

a "suicide"

is to deplete

cellular

under NAD. 43

cells

Therefore,

is very

the large

that we observe

between

be due to a higher

NAD +

degradation.

for rapidly

ADP-ribosylation,

glycohydrolase.

in permeabilized

by the experimenter.

of ADP-ribose

in the control

of enhanced

that 0.33ram NAD + inhibits

that the NAD concentration

Sims et al [13] previously

heavily

linkage

of 1 rain or less previously

recently

[25] reported

cells

Why do cells

function

turning

and cytoselic)

in the rates

concentration

[24]

we observed

low and is essentially difference

account

or on

the nuclei.

Berger's However,

enzymes,

cells.

glycohydrolases

enzyme

produced

could

be due to loss of degrading

Tant~na and colleagues

ADP-ribose

on differential

residues

were operative

of our faster

either

of these

of mono(ADP-ribose)

[4], if such a mechanism

at a common

has a different

ADP-ribose

This

from

degradation

[22] to degrading

of moieties

[23,1].

removal

that

be centred

and cleavage

some monomeric

We infer

conditions.

over component

to proteins

up and rate-limiting

and co-workers

must

2).

are r6moved

(ADP-ribose)n

residues

of residues

(Fig.

residues

our experimental

ADP-ribose

rate-limiting.

activity

under

and ourselves,

of various that

ADP-ribose

then of the biphasic

seen by others

accessibility

protein

and polymeric

interpretation

kinetics,

in a 20 rain period

degrading model

conditions

(ADP-ribose)n?

in which

the global

in which

The recycling

DNA is

of the

Vol. 139, No. 1, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

nicotinamide,

produced

pyrophosphate

and ATP such that the cell cannot maintain

processes

in the transferase

and consequently

aberrant

DNA repair

to multi-cellular

dies.

leading

organisms.

result from the elevated

reaction,

ATP-dependent

The purpose of this scenario

to the fixation of mutations, A rapid degradation

levels of ADP-ribose

this would ensure continued

ADPRT activity,

Without

all ADP-ribose

this rapid turnover

const~nes phosphoribosyl

occupied,

and existing

chains maximally

activity

and consequent

NAD + depletion

probably

deleterious

of (ADP-ribose)n

then would

in the DNA dsmnged required

acceptor

elongated.

is to prevent

to deplete

cells and cellular

NAD.

sites might become quickly Further

transferase

would then cease.

ACENOWLEDGE~S We would like to thank the SERC for a studentship for J.C. Gaal, Professor H.M. Keir for providing research facilities and Mrs Fiona Mitchell for typing the manuscript. We are indebted to Mrs Mary Evans for technical assistance. REF~alENCES i. 2. 3. 4. 5. 6. 7. 8. 9.

GoA1, J.C. and Pearson, C.K. (1985) Biochem. J. 230, 1-18. GoAl, J.C. and Pearson, C.K. (1986) Trends in Biochem. Sci. ii, 171-175. Ueda, K. and Hayaishi, O. (1985) Ann. Rev. Biochem. 54, 78-100. Wielckens, K., Schmidt, A., George, E., Bredehorst, R. and Hilz, H. (1982) J. Biol. Chem. 257, 12872-12877. Jacobson, E.L., Antol, K.M., Juarez-Salinas, H. and Jacobson, M.K. (1983) J. Biol. Chem. 258, 103-107. Alvarez-Gonzalez, R., Eichenberger, R., UStscher, P. and Althaus, F.R. (1985) reported at the Third European Workshop on ADP-Ribosylation of Protein, University of Reading, UK. Levi, V., Juarez-Salinas, H. and Jaeobson, M.K. (1981) Fed. Proc. 40, 171. Tantlna, S., Arita, T., Kawashima, K. and Endo, H. (1982) Biochem. Biophys. Res. Commun. i04.., 483-490. Wallace, H.M., Gordon, A.M., Keir, H.M. and Pearson, C.K. (1984) Biochem.

j . 219, 211-221. 10. ii. 12. 13. 14. 15. 16. 17. 18.

GoAl, J.C. and P e a r s o n , C.K. (1986) Biochem. Biophys. Acta 88__~1, 196-209. Juarez-Salinas, H., Sims, J.L. and Jacobson, M.K. (1979) Nature 282, 740-741. Sims, J.L., Sikorski, G.W., Catino, D.M., Berger, S.J. and Berger, N.A. (1982) Biochemistry 21, 1813-1821. Sims, J.L., Berger, S.---J. and Berger, N.A. (1983) Biochemistry 22, 5188-5194. Benjamin, R.C. and Gill, D.M. (1980) J. Biol. Chem. 255, 10493-10501. Prise, K.M., Gaal, J.C. and Pearson, C.K. (1986) Bi~-h-em. Biolxhys. Acta 887, 13-22. Farzaneh, F. and Pearson, C.K. (1978) Biochem. Biophys. Res. Commun. 84, 537-543. Burzio, L.O., Riquelme, P.T., Ohtsuka, E. and Koide, S.S. (1976) Arch. Biochem. Biophys. 173, 306-319. Tanaka, M., Miwa, M., Matsushima, T., Sugimura, T. and Shall, S. (1976) Arch. Biochem. Biophys. 172, 224-229. 44

Vol. 139, No. 1, 1986

19. 20. 21. 22. 23. 24. 25.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Oka, J., Ueda, K. and Hayaishi, O. (1982) in ADP-Ribosylation Reactions (Biology and Medicine) (Hayaishi, 0 and Ueda, K., eds.), pp 279-285. Academic Press, London and New York. Oka, J., Ueda, K., Hayaishi, 0., Kemura, H. and Nakanishi (1984) J. Biol. Chem. 259, 986-995. Adamietz, P., Bredehorst, R. and Hilz, H. (1978) Eur. J. Biochem. 91, 317-326. Gaudreau, A., Menard, L., DeMurcia, G. and Poirier, G.G. (1986) Biochem. Cell Biol. 64, 146-153. Hilz, H., Koch, R., Fanick, W., Klapproth, K. and Adamietz, P. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3929-3933. Tan~na, S., Kawashima, K. and En---do, H. (1986) Biochem. Biophys. Res. Commun. 135, 979-986. Berger, N.A., Petzold, S.J. and Berger, S.J. (1979) Biochem. Biophys. Acta 5,64, 90-104.

45