Group Title: Molecular Pain 2005, 1:15
Title: Physical interaction and functional coupling between ACDP4 and the intracellular ion chaperone COX11, an implication of the role of ACDP4 in essential metal ion transport and homeostasis
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Title: Physical interaction and functional coupling between ACDP4 and the intracellular ion chaperone COX11, an implication of the role of ACDP4 in essential metal ion transport and homeostasis
Series Title: Molecular Pain 2005, 1:15
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Ling J
Wang MH
She JX
Gu J
Wang CY
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Molecular Pain BioMed



Research

Physical interaction and functional coupling between ACDP4 and
the intracellular ion chaperone COX I I, an implication of the role of
ACDP4 in essential metal ion transport and homeostasis
Dehuang Guol, Jennifer Ling2, Mong-Heng Wang3, Jin-Xiong She',
Jianguo Gu*2 and Cong-Yi Wang*


Address: 1Center for Biotechnology and Genomic Medicine, Medical College of Georgia, 1120 15th Street, CA4098, Augusta, GA 30912, USA,
2Department of Oral and Maxillofacial Surgery, Mcknight Brain Institute and College of Dentistry, University of Florida, Gainesville, Florida,
32610, USA and 3Department of Physiology, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA
Email: Dehuang Guo dguo@mcg.edu; Jennifer Ling jling@dental.ufl.edu; Mong-Heng Wang mwang@mcg.edu; Jin-
Xiong She jshe@mcg.edu; Jianguo Gu* jgu@dental.ufl.edu; Cong-Yi Wang* cwang@mcg.edu
* Corresponding authors


Published: 19 April 2005
Molecular Pain 2005, 1:15 doi:10.1 186/1744-8069-1-15


Received: 17 March 2005
Accepted: 19 April 2005


This article is available from: http://www.molecularpain.com/content/1/1/15
2005 Guo et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Divalent metal ions such as copper, manganese, and cobalt are essential for cell development,
differentiation, function and survival. These essential metal ions are delivered into intracellular
domains as cofactors for enzymes involved in neuropeptide and neurotransmitter synthesis,
superoxide metabolism, and other biological functions in a target specific fashion. Altering the
homeostasis of these essential metal ions is known to connect to a number of human diseases
including Alzheimer disease, amyotrophic lateral sclerosis, and pain. It remains unclear how these
essential metal ions are delivered to intracellular targets in mammalian cells. Here we report that
rat spinal cord dorsal horn neurons express ACDP4, a member of Ancient Conserved Domain
Protein family. By screening a pretransformed human fetal brain cDNA library in a yeast two-hybrid
system, we have identified that ACDP4 specifically interacts with COX I I, an intracellular metal ion
chaperone. Ectopic expression of ACDP4 in HEK293 cells resulted in enhanced toxicity to metal
ions including copper, manganese, and cobalt. The metal ion toxicity became more pronounced
when ACDP4 and COXI I were co-expressed ectopically in HEK293 cells, suggesting a functional
coupling between them. Our results indicate a role of ACDP4 in metal ion homeostasis and
toxicity. This is the first report revealing a functional aspect of this ancient conserved domain
protein family. We propose that ACDP is a family of transporter protein or chaperone proteins
for delivering essential metal ions in different mammalian tissues. The expression of ACDP4 on
spinal cord dorsal horn neurons may have implications in sensory neuron functions under
physiological and pathological conditions.


Background
Essential metal ions such as copper, manganese and
cobalt are vital elements involved in functions of numer-
ous enzymes and proteins in mammalian cells. Mamma-


lian cells not only possess efficient uptake mechanisms to
obtain these ions from their extracellular environment,
but also have intracellular delivery system to translocate
essential metal ions to specific enzymes and proteins.


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Deficiency in these essential metal ions affects normal cell
functions, but they are toxic when present in excess. For
example, they can damage DNA and proteins to induce
cell death. Therefore, proper delivery of these essential
metal ions into intracellular functional domains is vital. It
is known that alteration of essential metal ion homeosta-
sis is associated with diseases including Alzheimer's dis-
ease, amyotrophic lateral sclerosis, prion diseases,
cataracts, mitochondrial disorders and Parkinson's dis-
ease [1-8]. Essential metal ion homeostasis also has
important implications in sensory physiology, pathology
and pain [9-11]. For example, copper is a cofactor for pep-
tidylglycine a-amidating monooxygenase, an enzyme cat-
alyzes the formation of a number of biologically active
peptides including the pronociceptive peptide substance
P [9]. Copper and manganese are cofactors for copper/
zinc superoxide dismutase (Cu/ZnSOD) and Mn-superox-
ide dismutase (MnSOD), respectively [10,11]. These
metal ion-dependent enzymes are involved in superoxide
metabolism. Studies have shown that activity of these
SOD enzymes can become abnormal during inflamma-
tion, which is an important underlying mechanism of
pathological pain conditions and other neurological dis-
orders [10,11].

Metal ion homeostasis is maintained through highly reg-
ulated processes, including transport, translocation, stor-
age and secretion. Essential metal ions are transported
into cells and then translocated to intracellular organelles
to function as catalytic and structural cofactors for com-
partmentalized enzymes [12]. Although a number of ion
transporters have been identified and characterized bio-
chemically over the past several decades [13-18], the
molecular identities of transporters for many metal ions
are still elusive in mammalian cells. Unlike membrane
ion channels permeable for ions such as Ca2+ [19 essen-
tial metal ion transporters are coupled with intracellular
metal ion chaperones [12,20-22]. Metal ion chaperones
interact with transporters to receive metal ions, and then
carry metal ions to target enzymes or proteins and donate
metal ions to the targets. Several chaperones for copper
ions have been identified in mammalian cells including
COX17 and COX11 and they are shown to be essential for
cell survival [20-22].

We have recently cloned and characterized a novel gene
family named Ancient Conserved Domain Protein
(ACDP) [23]. ACDP encodes four protein members
(ACDP1-4) in both human and mouse [23,24]. The most
prominent feature of ACDP gene family is the ancient
conserved domain (ACD) found in evolutionarily diver-
gent species ranging from bacteria, yeast, C. elegans, and
D. melanogaster to mammals. ACDP proteins showed high
amino acid homology to bacteria CorC protein (e.g., 35%
AA identity with 55% homology), a protein believed to be


involved in metal ion toxicity in bacteria [25]. Based on
TopPred analysis it appears that ACDP can be a family of
membrane proteins [24]. Consistent with this analysis, a
recent study from our group has suggested that ACDP1,
the first member of ACDP family, is localized close to or
on the plasma membranes of hippocampus neurons [24].
Because ACDP4 was shown to have broader tissue distri-
butions, including in both neuronal and non-neuronal
tissues, we took ACDP4 as a start for exploring functions
ofACDP family.

Results
Expression of ACDP4 proteins on spinal cord dorsal horn
neurons
We performed immunostaining of ACDP4 on the spinal
cord dorsal horn neurons. ACDP1 immunostaining was
also performed as additional information ofACDP family
expression on dorsal horn neurons. Spinal cord dorsal
horn neurons are CNS (central nervous system) neurons
involved in somatosensory functions, including transmit-
ting signals ofnociceptive, mechanical, and thermal stim-
uli. Immunostaining was performed using polyclonal
antibodies specific for ACDP1 and ACDP4, respectively.
Both antibodies have been previously shown to have spe-
cific interactions with the corresponding target proteins
[24]. We used cultured neurons grown on a monolayer of
astracyte bedding for immunostaining. Therefore, cellular
and subcellular distribution ofACDP1 and ACDP4 can be
viewed clearly under a microscope. ACDP1 was expressed
on both soma and dendrites of neurons (Fig. 1A &1B).
Interestingly, ACDP1-ir on dendrites was shown to be
punctuated (Fig. 1A &1B). ACDP4-ir was also found on
the soma and dendrites of neurons, its distribution on
dendrites was not punctuated (Fig. 1C). Neither ACDP1-
ir nor ACDP4-ir was observed positively on astrocytes, the
bedding underneath neurons. Confocal images showed
that ACDP1-ir (Fig. 1D) and ACDP4-ir (Fig. 1E) were
strongest at the edge of cells, suggesting that most ACDP1
and ACDP4 proteins were located close to and some of
them maybe on the plasma membranes of spinal cord
dorsal horn neurons.

Interactions between ACDP4 and intracellular metal ion
chaperone COXI I
To explore the potential functions of ACDP proteins, we
first searched whether any known proteins interact with
them by screening a pretransformed human fetal brain
cDNA library in a yeast two-hybrid system. This Match-
maker system is an advanced GAL4-based two-hybrid sys-
tem that provides a transcriptional assay for detecting
protein interactions in vivo in yeast. We used ACDP4 as a
model in this study because it has broader tissue distribu-
tion than ACDP1. In the Matchmaker system ACDP4 (bait
gene) was expressed as a fusion to the GAL4 DNA-binding
domain (DNA-BD), which was used to screen a


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Figure I
Immunoreactivity of ACDPI and ACDP4 on rat spinal cord dorsal horn neurons A. The Micrograph shows a cul-
tured dorsal horn neuron with its processes. The image was taken under a DIC (Nomarski differential interference contrast)
microscope. B. ACDPI immunoreactivity (ACDPI-ir) on the same neuron in A. Arrows indicate several punctuated sites of
ACDPI-ir along dendrites. C. ACDP4-ir on another dorsal horn neuron. Scale bars: 10 |m. Two-week neuron cultures were
used for the immunostaining. D. A confocal image of ACDP I -ir on a dorsal horn neuron. E. A confocal image of ACDP4-ir on
anther dorsal horn neuron. The images were taken at the meddle sections of the cells. Scale bars were 20 [im for A, B and C
and 10 [m for D and E.


pretransformed human fetal Matchmaker cDNA library.
When the bait and library fusion proteins interact, the
DNA-BD and activation domain (AD) are brought into
proximity, thus activating transcription of the reporter
gene. In order to prevent non-specific interactions, we cul-
tured the cells in the highest stringency condition. Among
2 x 106 transformants, only one clone was identified to
strongly interact with ACDP4. The DNA for this clone was
recovered from yeast and then transformed into XL-1 Blue
competent cells (Stratagene). Individual clones were
screened first by PCR and then sequenced using the prim-
ers from vector. We found that the yeast clone actually
contains two plasmids, one plasmid is corresponding to
the intracellular metal ion chaperone COX1 1, and the


other one is an RNA binding motif protein 30 (RBM30)
with unknown function.

To confirm the above results, we established full-length
constructs for the pGADT7-COX11 and pGADT7-RBM30
(as targets). Subsequently, the pGBKT7-ACDP4 plasmid
was co-transformed into an AH 109 yeast strain either with
the pGADT7-COXl 1 or the pGADT7-RBM30 plasmid. An
empty pGADT7 vector was used as a control. The cultures
were assayed for p-galactosidase to verify two-hybrid
interactions (Fig. 2). In response to GAL4 activation, yeast
containing the lacZ reporter gene secretes P-galactosidase,
which can be detected in the presence of the chromogenic
substrate ONPG (ortho-nitrophenyl p-D-galactopyrano-


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ACDP4-ir







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E
*3



0
r
vi
VS

I


10 f


ACDP4

COX-11
RBM30
Vector


+


+




+


Figure 2
Interactions of ACDP4 with COXI I or with RBM30 in a yeast two-hybrid system. Cultures were assayed for f-
galactosidase to verify two-hybrid interactions. P-galactosidase secreted from yeast containing the lacZ reporter gene after
GAL4 activation were detected in the presence of chromogenic substrate ONPG (ortho-nitrophenyl P-D-galactopyranoside).
Vector, empty pGADT7 vector.


side). When hydrolyzed by P-galactosidase, colorless
ONPG turns to ONP (ortho-nitrophenyl), a yellow prod-
uct. Therefore, enzyme activity of P-galactosidase can be
measured by the rate of appearance of yellow color using
a spectrophotometer. We found that ACDP4 strongly
interacts with both COX11 and RBM30 (Fig. 2). On the
other hand, there was no P-galactosidase activity detected
in the controls, suggesting that the interactions detected
are true. The interactions between ACDP4 and the metal
ion chaperone COX11 provided an initial clue for a
potential role of ACDP4 in metal ion homeostasis.

The effects of ectopic expression of ACDP4 on metal ion
toxicity in HEK293 cells
Essential metal ions can produce oxidative toxicity when
excessive amounts are accumulated inside cells. We first
determined metal ion toxicity using HEK293 cells that
were not transfected with ACDP4 plasmids. This serves as
baseline of metal ion toxicity. Five divalent ions including


Cu2+, Mn2+, Co2+, Mg2+ and Zn2+ were selected for the
study. To establish a killing curve for each of the selected
metal ions, HEK293 cells were plated at 3 x 105 per well in
6-well cultural plates and then cultured in medium con-
taining different concentrations of CuCl2, MnC12, CoC12,
MgCl2 and ZnC12 for 48 hrs, respectively. The cells were
then examined for viability by trypan blue staining. As
shown in Fig. 3, the concentrations responsible for 50% of
cell death (EC5s) for CuCl2, MnC12, CoC12 and ZnC12 were
0.9 mM, 1.4 mM, 0.8 mM and 0.6 mM, respectively (Fig.
3A). On the other hand, MgCl2, showed much less cell
toxicity at low minimolar concentrations (Fig. 3B).

Next, we tested whether ectopic ACDP4 expression may
enhance metal ion toxicity in HEK293 cells. pcDNA3.1-
ACDP4 plasmid was transfected into HEK293 cells using
the Effectene Transfection Reagent. Transfection of an
empty vector (pcDNA3.1) was used as a control. The cells
were then cultured in medium containing the above metal


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--ZnCI2
CoCI2
-- CuCI2
--MnCI2


0.5 1.0 1.5 2.0 2.5 3.

Metal ion concentration (mM)


0 8 16 32 64 128
MgCl2 concentration (mM)


Figure 3
Killing curve of HEK293 cells for selected divalent ions A. Killing curves for Cu2+, Mn2+, Co2+ and Zn2+. B. Killing curve
for Mg2+. Cell death was estimated by counting a total of 300 cells in each field with 2-3 fields under microscope. Apparent
EC50 values were 0.9 mM for Cu2+, 1.4 mM for Mn2+, 0.8 mM for Co2', 0.6 mM for Zn2+ and 64 mM for Mg2+.



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100-



, 75.
50
(-

50

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25 -



0
0.0


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Cu2+


=1~


Mn2+


o pcDNA3.1 vector
* ACDP4


Co2+ Zn2+


Mg2+


Figure 4
The effects of ectopic expression of ACDP4 on metal ion toxicity in HEK293 cells. ACDP4 is ectopically expressed
in HEK293 cells using pcDNA3. I-ACDP4 plasmid transfection. Transfection of an empty vector (pcDNA3.1) was used as a
control. Cells were cultured for 48 hrs in medium containing the 5 type of metal ions, and each type of metal ions was at its
concentration of the apparent EC50 established in Figure 3. For normalization, the viability of control cells was scaled to 100%,
and cell viabilities for cells transfected with ACDP4 were normalized in reference to the control cells. Data represent mean
SEM, P < 0.05, Student-t test.


ions each at its apparent EC5s identified above, and metal
toxicity was measured at 48 hrs after the addition of metal
ions. Ectopic ACDP4 expression significantly increased
metal ion toxicity for cells cultured with Cu2+, Mn2+ and
Co2+ (Fig. 4). The cell viabilities with ectopic ACDP4 in
medium containing Cu2+, Mn2+ and Co2+ was 12% (P <
0.04), 14% (P < 0.02) and 8% (P = 0.05) lower than that
of the cells transfected with empty vectors, respectively
(Fig. 4). We did not observe any significant difference of
cell viabilities between cells with ectopic ACDP4 and con-
trol cells when Mg2+ or Zn2+ were tested (Fig. 4). These
results suggest that ACDP4 is probably preferential for
Mn2+, Cu2+ and Co2+ over Mg2+ and Zn2+.

Enhanced metal ion toxicity by ectopic co-expression of
ACDP4 and COXI I
While we showed physical interaction between ACDP4
and COX11 in a yeast two-hybrid system, it is more


important to know whether ACDP4 may be functionally
coupled with COX11 in mammalian cells. To address this
question, we investigated metal ion toxicity in cells ectop-
ically co-expressing ACDP4 and COX11. For this purpose,
HEK293 cells were co-transfected with ACDP4-pcDNA3.1
plasmid along with COX11-pIRES-eGFP plasmid. The
transfected cells were cultured in medium with the five
metal ions at their apparent EC50 concentrations identi-
fied in Figure 3, and metal ion toxicity was examined after
48 hrs. Co-transfection of ACDP4 and COX11 signifi-
cantly increased HEK293 cell toxicity to Cu2+, Mn2+ and
Co2+ (Fig. 5A) but not to Zn2+ and Mg2+ (not shown). The
cell liabilities in medium with Cu2+, Mn2+ and Co2+ were
32% (P < 0.006), 34% (P < 0.001) and 23% (P < 0.002)
lower than the control cells, respectively (Fig. 5A). The cell
death for Cu2+, Mn2+ and Co2+ were increased by 2.8 fold,
2.5 fold 2.9 fold, respectively, compared to transfection of
ACDP4 alone (Fig. 4).


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100


80


60


40


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1 ACDP4
ACDP4+COX11


100
o
, 80-

S60
U
" 40-
N
S20-
20
I-
C


*i


Cu2+


I


*i


Mn2+ Co2+


I I pIRES vector


COX11


100


3 80


; 60


40
N
*
"~ 20
E


Cu2+


Mn2+


a-


Co2+


Figure 5
Functional coupling of ACDP4 with COX I I in metal ion toxicity A. The effects of ectopic co-expression of ACDP4
and COXI I on metal ion toxicity in HEK293 cells. Co-expression of ACDP4 and COXI I significantly enhanced metal ion tox-
icity. B. Metal ion toxicity for HEK293 cells ectopically expressed COXI I alone. There were no significant differences in cell
viability between control cells and cells ectopically expressed COXI I alone.


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While tranfection of ACDP4 increased cell toxicity to
Cu2+, Mn2+ and Co2+ (Fig. 4) and co-tranfection of ACDP4
with COX 11 further enhanced cell toxicity to these ions
(Figure 5A), transfection of COX11 alone had no signifi-
cant effect on baseline metal ion toxicity (Figure 5B).
These results together suggest a functional coupling
between ACDP4 and COX 11 for metal ion toxicity in
HEK239 cells.

Discussion
In the present study, we have demonstrated that ACDP4
physically interacts with COX11 and functionally coupled
with this metal ion chaperone. We have shown that
ectopic expression of ACDP4 enhances cell toxicity to sev-
eral essential metal ions including Cu2+, Mn2+ and Co2+,
and that co-expression of ACDP4 with COX11 produces
more pronounced increases of metal ion toxicity. This is
the first study to explore potential functions of a member
of ACDP protein family. The physical interaction and
functional coupling with a metal ion chaperone indicates
that ACDP4 is involved in the homeostasis and toxicity of
essential metal ions.

The ACDP gene family is highly conserved in both human
and mouse [23,24]. However, it has been complete
unknown for their potential functions before the present
study. Nevertheless, the sequence conservation and the
presence of multiple members within a species imply a
functional importance associated with this gene family.
While we were completing this work, Yang et al. reported
the involvement of a yeast ACDP homolog, Mam3p, in
manganese homeostasis and toxicity in yeast [26]. Their
results demonstrated that Mam3p operates independently
to the well-established manganese trafficking pathways in
yeast involving the manganese transporters, Pmrlp,
Smf2p and pho84p [26]. In our present study, we have
demonstrated the involvement of ACDP4 in copper and
cobalt toxicity in addition to manganese. Furthermore,
our functional results were obtained from mammalian
cells.

From our functional study using metal ion toxicity as a
measure, it appears that ACDP4 functions at a site
upstream to COX11. This idea is supported by three lines
of evidence. First, ectopic expression of ACDP4 alone
could enhance metal ion toxicity. Second, ectopic expres-
sion of COX11 alone did not affect metal ion toxicity.
Third, co-expression of ACDP4 with COX11 produced
more pronounced metal ion toxicity than ectopic expres-
sion of ACDP4 alone. Interactions between ACDP4 and
COX11 suggest that ACDP4 may be a metal ion trans-
porter in mammalian cells (Fig. 6). This is because a metal
ion chaperone normally interacts with upstream metal
ion transporters to receive metal ions and interacts with
downstream target proteins to deliver metal ions. How-


ever, a direct study on ACDP4-mediated metal ion
accumulation in cells is needed to confirm transport func-
tions of ACDP4. Alternatively, ACDP4 may be an interme-
diate metal ion chaperone upstream to COX11. Detailed
studies in the future on ACDP4 membrane localization
and its interactions with metal ion chaperones in mam-
malian cells will provide more insights into the mecha-
nisms by which ACDP4 is involved in metal ion
homeostasis and toxicity. It would also be interesting to
study whether other members of ACDP family may be
involved in essential metal ion homeostasis and toxicity
in mammalian cells. A particular interesting issue is the
potential neuronal functions of ACDP1 since it is almost
exclusively expressed in CNS neurons in the brain (24)
and the spinal cord neurons (Fig. 1).

Interactions between ACDP4 and COX11 provide a struc-
tural and functional linkage to COX11. COX11 is an intra-
cellular copper chaperone originally identified in yeast as
essential for cytochrome c oxidase activity and heme asta-
bility in subunit I. [20]. A recent study suggested a role for
this protein in formation of the binuclear copper-heme
center [21]. Spectroscopic and mutagenesis studies indi-
cate that the copper ion in COX11 is ligated by three con-
served cysteine residues [22]. The C-terminal domain of
this protein forms a dimer that coordinates a single CuC
per monomer. While COX11 was found to be a dimer, it
remains possible that copper transfer to and from COX11
occurs through heterodimeric interaction with other pro-
teins [22]. IfACDP4 is confirmed to be a metal ion trans-
porter or an upstream metal ion chaperone, it would be
interesting to know how ACDP4 delivers metal ions to
COX11, whether ACDP4 and COX11 are co-localized on
or near the plasma membranes, whether ACDP4 and
COX11 are also co-localized on the membranes of other
intracellular organelles such as mitochondria. We have
shown that ACDP4 also interacts with RBM30, an RNA
binding motif protein 30. RBM30 contains a zinc knuckle,
raising a possibility that it may serve as an intracellular
zinc chaperone. However, we did not observe an
enhanced toxicity to zinc in cells transfected with RBM30
or co-transfected with both ACDP4 and RBM30. There-
fore, it remains to determine whether interaction of
ACDP4 with RBM30 may have any biological
significance.

The expression of ACDP4 and ACDP1 on dorsal horn
neurons of the spinal cord shown in the present study
may have implications in sensory physiology and pathol-
ogy. Both ACDP4-ir and ACDP 1-ir were found to be local-
ized close to the plasma membranes of dorsal horn
neurons. IfACDP4 and ACDP1 are metal ion transporters
or chaperones in these neurons, they should be involved
in regulating homeostasis of essential metal ions in the
spinal cord dorsal horn neurons. Essential metal ions are


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Figure 6
A postulated model of ACDP-mediated metal ion delivery in mammalian cells. An essential metal ion is first trans-
ferred through plasma membrane via an ACDP. An intracellular chaperone specifically interacts with the intracellular parts of
ACDP. The chaperone receives the metal ion from ACDP and then carries the ion to a specific target protein. Overload of
cells with essential metals results in oxidative cell death.


essential for activity of many enzymes, including those for
synthesis of neuropeptides (e.g. substance P) and neuro-
transmitters (e.g. monoamine) in the spinal cord [9], and
those for superoxide metabolism in peripheral sensory
nerves and CNS sensory neurons in the spinal cord
[10,11]. Abnormal superoxide metabolism has been
shown to be a critical factor in inflammation and patho-
logical pain conditions [10,11]. It is conceivable that
through regulating essential metal ion homeostasis,
ACDP4 and perhaps also other ACDP members can affect
sensory process including nociception. It is also predicta-


ble that the functions of ACDP family will go beyond
somatosensory system.

Methods
Neuronal cell preparation and immuostaining
Sprague-Dawley rats were used according to the Institu-
tional Animal Care and Use Committee guideline of the
University of Florida. Dorsal horn neuron cultures were
prepared as described previously. [27]. In brief, spinal
cord dorsal horns were dissected out from rat embryos at
the age of 16 days in utero (E16). Dorsal horns were incu-


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bated separately for 25 min at 370C in S-MEM medium
(Gibco, Grand Island, NY) with 2.5% trypsin (Gibco) and
then triturated to dissociate neurons. The neurons were
plated on glass coverslips previously prepared with a
monolayer of rat cortical astrocytes. Neurons were
maintained in MEM (Gibco) culture medium that con-
tained 5% heat-inactivated horse serum (JRH Biosciences,
Lenexa, KS), uridine/5-flouro-2'-deoxyuridine (10 [tM;
Sigma, St. Louis, MO), 8 mg/ml glucose and 1% vitamin
solution (Gibco). The cultures were maintained at 37C
in a humidified atmosphere of 95% air and 5% CO2, and
were fed weekly with fresh culture medium.

Neurons were used for immunostaining of ACDP1 and
ACDP4 at two weeks in culture. For immunostaining,
neuronal cells on the coverslips were first fixed in PBS
containing 4% paraformaldehyde (PFA) for 12 hrs at 4 C
and then incubated in a solution containing 4% PFA and
0.4% Triton X-100 at 4 oC for 1 hr. After washing with PBS
three times, the cells were incubated with a blocking solu-
tion containing 1:30 normal goat serum, and subse-
quently incubated with a rabbit polyclonal anti-ACDP
antibody (1:3000) overnight at 4o C. After extensive wash-
ing with 1% goat serum PBS solution, the cells were incu-
bated with an Alex 488 conjugated secondary antibody
(1:100 in 1% goat serum PBS solution, Molecular Probes)
for 3 hrs at room temperature. Following final washes
with 1% goat serum PBS solution, the neuronal cells on
the coverslips were cover-slipped with a glycerol-based
anti-photobleach medium. The cells were viewed under a
fluorescence microscope (Olympus) with a 40X oil-
immersion objective or a confocal fluorescence micro-
scope (Carl Zeiss) with a 60X objective.

Establishment of killing curve
HEK293 cells were plated at 3 x 105 per well of 6 well
plates. The cells were cultured for 48 hrs in medium con-
taining different concentrations of MgCl2, CuC12, ZnCl2,
MnC12 and ECoCl2, respectively. Dead cells were then
stained with trypan blue solution at a ratio of 4:6 (Gibco).
The percentage of cell death was determined by counting
a total of 300 cells in each field with 2-3 fields under
microscope.

Plasmid construction
Full-length ACDP4 gene was cloned into the pGBKT7 and
pcDNA3.1 vectors using the EcoR I cutting site. COX11
and RBM30 were amplified from fetal brain cDNA and
then cloned into pIRES-eGFP vector with EcoR I and
BamH I, and Xho I and BamH I cutting sites, respectively.

Yeast two-hybrid analysis
The Matchmaker Galt4 two-hybrid system 3 kit (Clon-
tech) was used for two-hybrid analyses. The ACDP4 cod-
ing sequence was PCR engineered and cloned into the


pGBKT7 vector, which was used as a bait to screen a pre-
transformed human fetal brain Matchmaker cDNA library
at high stringency culture condition. To confirm the inter-
action between ACDP4 and COX11 or RBM30, the full-
length COX11 and RBM30 cDNA were cloned into the
pGADT7 vector (as targets), which was then co-trans-
fected into an AH109 yeast strain along with the pGBKT7-
ACDP4 plasmid, respectively. An empty pGADT7 vector
was used as a control. The cultures were assayed for p-
galactosidase to verify two-hybrid interaction according to
the manufacturer's instruction.

Cell culture and transfection
HEK293 cells were cultured in Dulbecco's modified
Eagles's medium (Mediatech, Inc.) supplemented with
10% fetal bovine serum and 100 units/ml antibiotic-
antimycotic (Invitrogen). Transfections were carried out
using 2 ug of plasmid DNA/100-mm dish and Effectene
Transfection Reagent according to the instructions of the
manufacturer (Qiagen).

Metal toxicity assay
We used an in vitro toxicology assay kit (Sigma) for meas-
urement of metal toxicity according to the manufacturer's
instruction. Cell viability estimated by this in vitro toxicol-
ogy assay determines cell number spectrophotometrically
as a function of mitochondrial activity in living cells.
Briefly, the cells were cultured with indicated metal ion for
44 hrs and then supplemented with 20 pl MTT per well.
The cells were cultured for additional 4 hrs. Subsequently,
the cultural plates were spun for 5 min at 1000 rpm to
remove the supernatant. The plates were dried in an incu-
bator for about 4 hrs. 200 pl of MTT solubilization solu-
tion was then used to dissolve the resulting formazan
crystals. The results were read at 570 nm after 6 hrs incu-
bation with a Synergy HT plate reader (Bio-Tek).

Data analysis and statistics
Unless otherwise indicated, data represent mean + SEM, p
< 0.05, student-t test.

Competing interests
The authors) declare that they have no competing
interests.

Acknowledgements
We thank Drs. Junyan Han and Jinlei Xi and Daniel Eisenman for their help
for preparation of figures of the manuscript. This work was supported by
the Juvenile Diabetes Research Foundation International (1-2004-235), the
American Diabetes Association (1-05-JF-47) to CYW and a National Insti-
tute of Health Grant NS38254 to JGG.

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