Download Phosphorylation and concomitant structural changes in human 2

FEBS Letters 580 (2006) 351–355
Phosphorylation and concomitant structural changes in human 2-Cys
peroxiredoxin isotype I differentially regulate its peroxidase and
molecular chaperone functions
Ho Hee Janga,b,1, Sun Young Kima,b,1, Soo Kwon Parka,b,1, Hye Sook Jeona,b, Young Mee Leea,b,
Ji Hyun Junga,b, Sun Yong Leea,b, Ho Byoung Chaea,b, Young Jun Junga,b, Kyun Oh Leea,b,
Chae Oh Lima,b, Woo Sik Chunga,b, Jeong Dong Bahka,b, Dae-Jin Yuna,b, Moo Je Chob,
Sang Yeol Leea,b,*
b
a
Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea
Division of Applied Life Sciences (BK21 Program), Environmental Biotechnology Research Center, Gyeongsang National University,
Jinju 660-701, Republic of Korea
Received 15 November 2005; accepted 8 December 2005
Available online 19 December 2005
Edited by David Lambeth
Abstract The H2O2-catabolizing peroxidase activity of human
peroxiredoxin I (hPrxI) was previously shown to be regulated by
phosphorylation of Thr90. Here, we show that hPrxI forms multiple oligomers with distinct secondary structures. HPrxI is a
dual function protein, since it can behave either as a peroxidase
or as a molecular chaperone. The effects of phosphorylation of
hPrxI on its protein structure and dual functions were determined using site-directed mutagenesis, in which the phosphorylation site was substituted with aspartate to mimic the
phosphorylated status of the protein (T90D-hPrxI). Phosphorylation of the protein induces significant changes in its protein
structure from low molecular weight (MW) protein species to
high MW protein complexes as well as its dual functions. In contrast to the wild type (WT)- and T90A-hPrxI, the T90D-hPrxI
exhibited a markedly reduced peroxidase activity, but showed
about sixfold higher chaperone activity than WT-hPrxI.
2005 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Keywords: 2-Cys peroxiredoxin; Chaperone; Dual function;
Phosphorylation; Structural change
1. Introduction
Peroxiredoxins (Prxs) are a ubiquitous peroxidase family
whose functions are associated with diverse cellular processes,
including cell proliferation, differentiation, immune response,
and apoptosis [1,2]. To regulate the intracellular levels of reactive oxygen species (ROS) generated in cells, Prxs decompose
H2O2, peroxinitrite, and organic peroxides using thioredoxin
(Trx) as an electron donor [3,4]. Based on the number of con-
served Cys residues in their sequences, Prxs are largely divided
into two groups, such as 1-Cys Prxs and 2-Cys Prxs.
In addition to their well-known peroxidase function, specific
isotypes of 2-Cys Prxs in yeast and human cells have been
shown to possess the additional function of molecular chaperone [5,6]. Upon oxidation of their peroxidatic Cys, eukaryotic
2-Cys Prxs undergo a structural conversion from a low molecular weight (LMW) species acting as a peroxidase to a high
molecular weight (HMW) complex functioning as a chaperone. The N-terminal peroxidatic Cys residue acting as a sensor
of H2O2 concentration in the cell and the C-terminal tail of
eukaryotic 2-Cys Prxs containing a ‘YF-motif’ play critical
roles in this structural and functional transition [6–8]. Also,
the 2-Cys Prxs was shown to interact with several regulatory
proteins including Trx, Srx1, and Sestrin [5,7,9]. Furthermore,
like many enzymes involved in intracellular metabolism, the
peroxidase activity of 2-Cys Prxs in humans, isotype I and II
(hPrxI and hPrxII), is regulated by proteolysis and by phosphorylation mediated by cyclin-dependent kinases (CDKs).
HPrxI is phosphorylated by Cdc2, a CDK, which is activated
during the mitotic phase of the cell cycle, but not in interphase.
The specific phosphorylation of hPrxI on Thr90 resulted in a
remarkable inhibition of its peroxidase activity [10,11].
In this present study, we examined the effect of phosphorylation of hPrxI on its protein structure and dual functions of
peroxidase and molecular chaperone using site-directed mutagenesis, in which the phosphorylation site (Thr90) was substituted with Asp to mimic the phosphorylated status of the
protein [11]. Based on the report that hPrxI was more strongly
phosphorylated than hPrxII in vitro [11], we selected hPrxI to
study the effects of its phosphorylation.
2. Materials and methods
*
Corresponding author. Fax: +82 55 759 9363.
E-mail address: [email protected] (S.Y. Lee).
1
These authors equally contributed to this work.
Abbreviations: 2-Cys Prx, 2-cysteine peroxiredoxin; ROS, reactive oxygen species; HMW, high molecular weight; LMW, low molecular
weight
2.1. Materials
Malate dehydrogenase (MDH), H2O2 and NADPH were obtained
from Sigma (St. Louis, MO) and recombinant Cdc2-cyclin B complex
was purchased from New England Biolabs. 1,1 0 -Bis(4-anilino)naphthalene-5,5 0 -disulfonic acid (bis-ANS) was obtained from Molecular
Probes (Eugene, OR), and the high-performance liquid chromatography (HPLC) column, TSK G4000SWXL (7.8 · 300), was from Tosoh
(Tokyo, Japan).
0014-5793/$32.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2005.12.030
352
2.2. Cloning and mutation of the hPrxI gene, and expression in E. coli
The hPrxI gene was cloned from a human placental cDNA library
and point-mutated hPrxI (T90D- and T90A-hPrxI) DNAs were generated by polymerase chain reaction-mediated mutagenesis. Using the
pGEX expression vector, various forms of hPrxI proteins were expressed in Escherichia coli BL21 (DE3) and purified as described [12].
2.3. In vitro phosphorylation of hPrxI by Cdc2-cyclin B complex
Recombinant hPrxI was reacted with Cdc2-cyclin B complex in a solution containing 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 1 mM
EGTA, 2 mM DTT, and 200 lM ATP for 6 h at 30;C. The phosphorylated-hPrxI was separated from the unphosphorylated protein by applying the resulting solution to a Q-Sepharose column that had been
equilibrated with 50 mM Tris–HCl (pH 8.5) as described previously [11].
H.H. Jang et al. / FEBS Letters 580 (2006) 351–355
of phosphorylated-hPrxI (p-hPrxI), and T90A-hPrxI which
lost its phosphorylation site by the mutation [11]. After separating the p-hPrxI from the unphosphorylated protein by using
a Q-Sepharose column, various forms of the hPrxI were separated by 12% SDS–PAGE gel and subjected to Western blotting with an anti-hPrxI antibody. Then we found that all the
proteins showed similar apparent molecular mass of 21 kDa,
which corresponded to the monomeric size of hPrxI
(Fig. 1A, lower panel). However, when we analyzed their pro-
2.4. Assay of peroxidase and chaperone activities
The Trx-dependent, NADPH oxidation-linked peroxidase activity
of hPrxI was measured by the decrease in absorbance at 340 nm
(A340) and chaperone activity of the protein was measured by using
MDH as a substrate, as described [12,13]. Turbidity due to substrate
aggregation, indicating a lack of chaperone activity, was monitored
in a DU800 spectrophotometer equipped with a thermostatic cell
holder (Beckmann, Fullerton, CA, USA).
2.5. Size-exclusion chromatography (SEC) and polyacrylamide gel
electrophoresis (PAGE)
Protein structure of hPrxI was analyzed by SEC using a HPLC with
a TSK G4000SWXL column equilibrated with buffer containing
50 mM HEPES, pH 7.0, and 100 mM NaCl. SDS and native-PAGE
were performed as described previously [6].
2.6. Circular dichroism (CD) spectroscopy
Wild type (WT)-, T90A-, and T90D-hPrxI in 10 mM sodium phosphate buffer (pH 7.4) were used for the Far UV-CD spectral analysis
with a Jasco J-715 spectropolarimeter (Jasco, Great Dunmow, UK)
and the spectra were accumulated five times, as described [14].
2.7. Fluorescence studies
Trp fluorescence spectra of 100 lg/ml hPrxI in 50 mM HEPES buffer
(pH 8.0) were recorded using a SFM25 spectrofluorometer (Kontron,
Germany) with an excitation wavelength of 295 nm. Hydrophobic domain exposure of hPrxI was examined by the binding of 10 ll of
10 mM bis-ANS to the 30 lg proteins of hPrxI with the spectrofluorometer. The excitation wavelength was set at 380 nm and emission
spectra were monitored from 400 to 600 nm as described [15].
2.8. In vivo analysis of the hPrxI protein structures in HeLa cells
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and
streptomycin (100 U/ml) at 37 C in a CO2 incubator. After the
WT-, T90A-, and T90D-hPrxI DNA constructs bearing a His6-tag in
their NH2-termini were cloned into pcDNA3.1 vector (Invitrogen),
they were individually transfected into HeLa cells and the cells overexpressing the proteins were selected at the presence of G418 (1 mg/
ml), as described [5]. The antibiotic-resistant clones were expanded
for further analysis in the medium containing G418. Crude cell lysates
were subjected on a native-PAGE and their structural properties were
analyzed by immunoblotting with the use of an anti-His-tag antibody.
3. Results
3.1. Effect of phosphorylation of hPrxI on its protein structure
It has been shown that hPrxI containing a consensus site
(Thr90-Pro-Lys-Lys) for phosphorylation by CDKs is specifically phosphorylated on Thr90 by several CDKs, including
Cdc2, in vitro [11]. To investigate the effect of hPrxI phosphorylation on its protein structure, we prepared several kinds of
hPrxI proteins including the phosphorylated form of hPrxI
by Cdc2-kinase in vitro, T90D-hPrxI acting as a mimic form
Fig. 1. Effects of phosphorylation of hPrxI protein structure analyzed
by Western blotting on a native-PAGE gel (A) and SEC (B). (A)
Purified proteins of WT-, T90A-, T90D-hPrxI, and hPrxI phosphorylated in vitro by Cdc2-cyclin B complex (p-hPrxI) were separated by
10% native-PAGE (upper panel) or 12% SDS–PAGE (lower panel)
gels and subjected to Western blotting with an anti-hPrxI antibody. (B)
Protein structures of WT-, T90A-, T90D-, and p-hPrxI were analyzed
using a TSK G4000SWXL HPLC-column.
H.H. Jang et al. / FEBS Letters 580 (2006) 351–355
tein structures by Western blotting on a native-PAGE gel, they
showed quite different patterns of protein structures (Fig. 1A,
upper panel). In the sample of WT-hPrxI, multiple forms of
low and oligomeric protein species were detected with a small
amount of HMW protein complexes on a native-PAGE gel.
Although the T90A-hPrxI produced a little more HMW complex than WT-hPrxI, the predominant form of its protein
structure consisted of oligomeric and LMW protein species.
On the other hand, most of the LMW protein species were
shifted to HMW complexes by the phosphorylation of Thr90
in hPrxI (p-hPrxI) or by the mutation of Thr90 to Asp
(T90D-hPrxI) (Fig. 1A). The elution profiles of p-hPrxI,
T90A-hPrxI and T90D-hPrxI in SEC were compared again
with that of WT-hPrxI (Fig. 1B). Like the protein patterns appeared in a native-PAGE gel, SEC chromatogram also showed
that the protein structures of WT- and T90A-hPrxI were
mostly consisted of LMW and oligomeric structures along
with a small amount of HMW complex, whereas predominant
forms of the p-hPrxI and T90D-hPrxI were HMW complexes
(Fig. 1B).
The results strongly suggest that the phosphorylation of
hPrxI induces a significant change in its protein structures
from LMW proteins to HMW complexes. To explore in vivo
structures of the mutant proteins, each DNA fused with a
His-tag at its N-terminus was cloned into pcDNA3.1 vector.
The DNA constructs were transfected into HeLa cells and
the cell lines that stably overexpressed the WT-, T90A-, and
T90D-hPrxI proteins were selected. Cytosolic fractions were
extracted from the cells and analyzed their protein structures
by using Western blotting on a native-PAGE gel. From the
experiment, we found that the in vivo structures of the proteins
were quite similar to those of the proteins obtained from
in vitro investigation (data not shown). Since the T90D-hPrxI
was shown to mimic the phosphorylation status of hPrxI at the
level of protein structures in vivo, the bacterially expressed
T90D-hPrxI was used to evaluate the phosphorylation effect
of hPrxI on its functional switching in the next studies.
3.2. Phosphorylation-mediated functional switching of hPrxI
It was shown that the dual functions of eukaryotic 2-Cys
Prxs were reversibly regulated by their protein structures
353
[5,6]. Based on the data of 2-Cys Prxs in yeast and human cells
(hPrxII) [5,6], we investigated the peroxidase and molecular
chaperone activities of the various forms of hPrxI as prepared
in Section 3.1. We found that WT-hPrxI exhibited not only a
H2O2 catabolic peroxidase activity (Fig. 2A) but also a chaperone activity that suppressed thermal aggregation of model substrate MDH at 43 C (Fig. 2B). However, no inhibition of
MDH aggregation was observed by the addition of excessive
amounts of bovine serum albumin alone. To estimate the influence of phosphorylation of hPrxI on the two activities, we
examined and compared the dual peroxidase and chaperone
activities using the mutant proteins of T90A- and T90D-hPrxI.
As reported elsewhere [11], the peroxidase activity of T90DhPrxI was markedly inhibited, but the chaperone activity
was significantly increased, about sixfold higher than that of
WT-hPrxI (Fig. 2C). In contrast, although the activity was significantly lower than that of T90D-hPrxI, the T90A-hPrxI
exhibited a slightly higher chaperone activity and a little lower
peroxidase activity than WT-hPrxI as shown in the other report [11]. These results, taken together with the structural assays presented in Fig. 1, strongly suggest that
phosphorylation of hPrxI switches the function of hPrxI from
a peroxidase to a molecular chaperone, accompanied by its
structural changes.
3.3. Phosphorylation-mediated changes in hydrophobicity and
secondary structures of hPrxI
To protect target substrates from stress-induced aggregation, chaperones bind to non-native states of substrate proteins
through hydrophobic interactions. Therefore, we compared
the extent of hydrophobicity of WT-, T90A-, and T90D-hPrxI
proteins by measuring bis-ANS binding, which has been
widely used as a probe for detection of hydrophobic regions
on the surface of proteins [15,16]. The fluorescence of bisANS bound to T90A-hPrxI and WT-hPrxI was significantly
lower than that of T90D-hPrxI and displayed broad spectra,
showing maximal emissions at around 490 520 nm, respectively (Fig. 3A). However, the fluorescence spectrum of bisANS bound to T90D-hPrxI showed a significant increase in
its intensity, accompanied by a shift of its emission maximum
to 487 nm. This result suggests that phosphorylation of hPrxI
Fig. 2. Effects of the replacement of Thr90 with Asp on the dual functions of hPrxI. (A) Peroxidase activity of WT-, T90A-, and T90D-hPrxI was
measured in the presence of 30 lg BSA (d–d), 10 lg WT-hPrxI (j–j), 10 lg T90A-hPrxI (e–e), 10 lg T90D-hPrxI (h–h), and 30 lg T90D-hPrxI
(n–n), of the reaction mixtures as described in Section 2. (B) Chaperone activity of the proteins used in panel A was measured using MDH as a
substrate. 1 lM MDH was incubated with 5 lM BSA (d–d), 0.5 lM WT-hPrxI (j–j), 3 lM WT-hPrxI (r–r), 0.5 lM T90A-hPrxI (e–e), and
0.5 lM T90D-hPrxI (h–h) at 43 C. (C) Relative activities of the two functions of WT-hPrxI (j), T90A-hPrxI (M) and T90DhPrxI (h) were
compared. The data shown are the means of at least three independent experiments.
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H.H. Jang et al. / FEBS Letters 580 (2006) 351–355
Fig. 3. Changes in surface hydrophobicity and secondary structure of hPrxI by phosphorylation. Fluorescence spectra of bis-ANS bound to 100 lg/
ml WT-hPrxI (solid line), T90A-hPrxI (dash dotted line) and T90D-hPrxI (dotted line) (A), and intrinsic Trp fluorescence spectra of the proteins (B)
were monitored using a fluorescence spectrofluorometer. Changes in the far UV-CD spectra of WT-hPrxI (solid line), T90A-hPrxI (dash dotted line)
and T90D-hPrxI (dotted line) (C) were measured using a protein concentration of 500 lg /ml. [h]M is the mean residue mass ellipticity.
greatly induces the exposure of hydrophobic domains, which
results in polymerization of the protein into HMW complexes
and provides binding sites for the partially-denatured substrate
proteins.
Since the intrinsic spectrum of Trp fluorescence is shown to
provide comparative information characterizing the environment around Trp residues of a protein [14], we examined the
fluorescence spectra of WT-, T90A-, and T90D-hPrxI proteins.
The spectrum of T90D-hPrxI was shifted to longer wavelengths compared to the spectra of WT- and T90A-hPrxI
and the intensity of fluorescence was significantly decreased
(Fig. 3B). This result suggests that the environment surrounding Trp residue in the WT-hPrxI is quite different from that of
T90DhPrxI and that the hydrophobic domains of the T90DhPrxI are greatly increased. In addition, we analyzed the secondary structure of hPrxI using far UV-CD (Fig. 3C). Analysis
of the CD spectra of WT- T90A- and T90D-hPrxI using Jasco
J-715 software shows that the secondary structures of hPrxI
were significantly changed by the phosphorylation of hPrxI.
4. Discussion
It was recently reported that Cdks inactivate the peroxidase
activity of hPrxI and hPrxII by phosphorylation specifically on
Thr90 at a certain stage of mammalian cell cycle [11]. Because
we identified a second, novel function of eukaryotic 2-Cys
Prxs, that of molecular chaperone, we examined the effects
of hPrxI phosphorylation on its protein structure and functions by replacing the target residue, Thr90, with an Asp, which
mimics the phosphorylation status of the protein (T90DhPrxI). In contrast to the effect of hPrxI phosphorylation on
peroxidase activity [11], the mutation of T90D in hPrxI causes
a significant increase in its chaperone function, and is accompanied by HMW complex formation (Figs. 1 and 2). The results of intrinsic Trp fluorescence, bis-ANS binding, and the
far UV-CD spectra of T90D-hPrxI suggest that the phosphorylation of hPrxI causes significant differences in the environment of aromatic amino acids and the extent of surface
hydrophobicity. These changes result in global changes in its
protein structure and induce formation of HMW complexes.
When we consider the crystal structure of hPrxI [2], the introduction of a negative charge by phosphorylation of Thr90,
which is located close to the active site Cys52, might disturb
the charge-charge or charge-dipole interaction of hPrxI, ren-
dering Cys52 insensitive to H2O2 and exposing hydrophobic
domains to induce structural changes [17,18].
In contrast to hPrxI, chaperone activities of aB-crystallin
[19] and HSP27 [20] were negatively regulated by phosphorylation, causing dissociation of large oligomers into small molecules and reducing their chaperone activity. Notably, protein
structure of HSP33 in E. coli is regulated in a manner similar
to the 2-Cys Prxs [6,21]. Under oxidative stress, HSP33 causes
conformational changes and allows the formation of oxidized
dimers of HSP33. Oxidized form of HSP33 dimers shows a
highly efficient chaperone activity, whereas under reducing
condition, HSP33 existed as a monomer is inactive as a chaperone [21,22]. These results suggest that the structural changes
caused by phosphorylation or redox state may be an important
mechanism of functional switching in molecular chaperones.
The Cdk-mediated phosphorylation and inactivation of
hPrxI as a peroxidase resulted in the intracellular accumulation
of H2O2, which may play important roles in the regulation of
cell cycle or in diverse cellular signaling processes. Even though
major new studies are required to elucidate the biological significance of phosphorylation-dependent structural and functional switching of hPrxI, one possible assumption is that the
p-hPrxI acts as a molecular chaperone to protect against protein unfolding or aggregation due to toxicity from ROS, which
play a causative role in many degenerative diseases [23] and are
generated from a variety of stress conditions in cells.
Acknowledgements: This work was supported by grants from the EBNCRC (Grant #: R15-2003-012-01001-0) supported by the KOSEF/
MOST, and by the Regional Technology Innovation Program of the
MOCIE (Grant #: RTI04-03-07), Korea. The first three authors were
supported by scholarships from the BK21 program.
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