Supplementary information
Supplementary information includes verification of the applied cell fractionation technique, a
MS/MS spectrum used for identification of Cyp18, control CoIP experiments, a further
experiment for identification of the interaction of purified recombinant p53 and Cyp18
proteins by MS, p53 basic activation after temperature shift (37°C  32°C), p53 activation
and p53-target gene expression after Cyclosporin A (CsA) treatment and UVC irradiation and
localization of endogenous p53 in Cyp18-proficient (Cyp18+/+) and Cyp18-deficient (Cyp18-/-)
Jurkat T cells. A table lists molecular chaperones identified as potential interacting partners of
p53. Details of the molecular modeling are also included.
Figure 1: Verification of the applied cell fractionation technique. The efficiency of subcellular
protein fractionation was determined by immunoblotting using cytoplasmic and nuclear
marker proteins. Topoisomerase I (TopoI) was detected as a nuclear marker protein using an
anti-DNA-TopoI antibody (BD Pharmingen, Heidelberg, Germany) and the inhibitor of kappa
B as a cytosolic marker protein was detected using the anti IkB antibody (H-4, Santa Cruz
Biotech., Heidelberg, Germany).
CCRF-CEM
cyt
nuc
MCF-7
cyt
nuc
topoisomerase I
(nuclear marker)
inhibitor of kappa B
(cytoplasmic marker)
Figure 2: MS/MS spectrum of the peptide VSFELFADK of Cyp18 detected in the tryptic
digest of gel section a of Figure 1, panel A of the main section. The detectable eight ions of
the B- and Y-ion series are depicted.
Figure 3: Gel analysis of control precipitations, showing that neither the carrier protein ASepharose nor DO1 precipitated Cyp18 in the absence of p53.
Figure 4: A p53-Cyp18 complex also forms with purified recombinant p53 and Cyp18.
0.7 µM p53 and 5.7 µM Cyp18 were mixed and precipitated using an anti-Cyp18 serum (lane
1) or the anti-p53 antibody DO1 (lane 2), respectively. The protein samples were separated by
SDS-PAGE and stained with Coomassie. The upper rectangle indicates the gel sections where
p53 was detected by MS analysis and the lower rectangle indicates the region of Cyp18,
analysed by MS. P53- and Cyp18-relevant peptides identified by MS/MS are indicated.
Figure 5: p53 basic activation (phosphorylation of serine 15) after temperature shift (37°C to
32°C).
Subcellular cytosolic (cyt) and nuclear (nuc) protein fractions from H1299(p53-/-),
H1299(p5372P) and H1299(p5372R) cells, cultivated at either 37oC or 32oC as described in
Siddique & Sabapathy (2006), were subjected to protein electrophoresis with subsequent
immunoblotting. P53 was detected using the anti-p53 monoclonal antibody DO1.
Phosphorylated serine at position 15 was detected with the p-p53 (Ser15) antibody (Santa
Cruz Biotech., Heidelberg, Germany) and Cyp18 was detected using the anti-cyp18 antibody
described in the main manuscript.
immunoblot
32oC
H1299(p53-/) cyt
nuc
H1299(p5372P
)
cyt
nuc
37oC
H1299(p5372R
) cyt
nuc
H1299(p53-/)
cyt
H1299(p5372P
nuc ) cyt
nuc
H1299(p5372R
)
cyt
nuc
p53
p53_pSer15
Cyp18
Figure 6: P53 activation (phosphorylation of serine 15) and p53-target gene expression after
Cyclosporin A (CsA) treatment and UVC irradiation.
H1299(p53-/-), H1299(p5372P) and H1299(p5372R) cells were cultivated at 32oC and treated
with CsA (0.5 µM) or UVC irradiation (25 J/m2) for four hours, respectively. Total cell
lysates were prepared and subjected to protein electrophoresis with subsequent
immunoblotting. P53 was detected using the anti-p53 monoclonal antibody DO1.
Phosphorylated serine at position 15 was detected with the p-p53 (Ser15) antibody (Santa
Cruz Biotech., Heidelberg, Germany), MDM2 was detected with anti-MDM2 antibody (Ab-2,
Calbiochem., Darmstadt, Germany). Cyp18 was detected using the anti-cyp18 antibody
described in the main manuscript.
immunoblot
no
treatment
1
2
3
UVC
CsA
1
2
3
1
2
3
p53
p53_pSer15
MDM2
Cyp18
1: H1299(p53-/-) 2: H1299(p5372P) 3: H1299(p5372R)
Figure 7: Localization of endogenous p53 in Cyp18-proficient (Cyp18+/+) and Cyp18-deficient
(Cyp18-/-) Jurkat T cells.
Cells were attached to poly-L-lysine coated cover slips and fixed with paraformaldehyde (2%).
Endogenous p53 was immunostained with a monoclonal anti-p53 antibody (DO1) and secondary antimouse FITC labelled antibody (green). DNA was stained with DAPI (blue). Cells were analysed by
confocal microscopy with a Nikon confocal C1 microscope.
Cyp18+/+
p53
DAPI
merge
Cyp18-/-
Table I:
Molecular chaperones identified as potential interacting partners of p53.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
protein name
cyclophilin 18
cyclophilin 23
endoplasmic reticulum protein ERp29
FK506-binding protein 3
heat shock 70 kDa protein
heat shock 70 kDa protein 12A
heat shock protein 27 kDa beta
heat shock protein 60 kDa
heat shock protein HSP90-alpha
heat shock protein HSP90-beta
heat shock 70 kDa protein 6
prefoldin subunit 1
protein disulfide-isomerase
UDP-glucose ceramide glycosyltransferase
swissprot acc. No. mol. weight (kDa)
P62937
17.9
P23284
22.7
P30040
29.0
Q00688
25.2
P11142
70.8
O43301
75.0
P04792
22.8
P10809
61.1
P07900
84.6
P08238
83.1
P17066
70.9
O60925
14.2
P07237
57.1
Q9NYU2
175.0
Molecular modeling
Modeling was performed with the molecular modeling and drug design program WHAT IF
(1) on a Silicon Graphics workstation (SGI, Sunnyvale, USA). Backbone atom
superimpositions and figures were prepared with the program MOLMOL (2). 3D structures
for p53 are available for several p53 stretches and domains. However, a structure of the region
between amino acids 61 and 93 is missing, probably due to higher residual dynamics. Since
the results presented in this study favor the binding motif to Cyp18 to be localized exactly in
this stretch, the p53 chain was modeled on the basis of the available coordinates of Cyp18
bound to the amino-terminal domain of the HIV-1 capsid protein (PDB entry 1AK4; (3)).
Beside of the residues of the proposed binding motif no further sequence homology criterion
was taken into account. The sequence overlap of the HIV-1 capsid protein with p53
terminates at p53´s residue 127, which is within the N-terminus of the DNA-binding domain.
Therefore, the presented model was supplemented with full coordinates derived from the Xray structure of the DNA-binding domain (1TSR; (4)).
References:
1. Vriend G. (1990) WHAT IF: a molecular modeling and drug design program. J Mol Graph
8: 52-56.
2. Koradi R. et al. (1996) MOLMOL: a program for display and analysis of macromolecular
structures. J Mol Graph 14: 51-55.
3. Gamble T.R. et al. (1996) Crystal structure of human cyclophilin A bound to the aminoterminal domain of HIV-1 capsid. Cell 87: 1285-1294.
4. Cho Y. et al. (1994) Crystal structure of a p53 tumor suppressor-DNA complex:
Understanding tumorigenic mutations. Science 265: 346-355.